Congenital Endocrinopathies

Congenital Endocrinopathies
New Insights into Endocrine Diseases and Diabetes

Endocrine Development
Vol. 11
Series Editor
Martin O. Savage London

Congenital
Endocrinopathies
New Insights into Endocrine Diseases
and Diabetes
Volume Editors
Renata Lorini Genova

Mohamad Maghnie Genova
Giuseppe D’Annunzio Genova
Sandro Loche Cagliari
Martin O. Savage London
24 figures and 12 tables, 2007
Basel · Freiburg · Paris · London · New York ·

Bangalore · Bangkok · Singapore · Tokyo · Sydney
Renata Lorini, MD Martin O. Savage, MD
Mohamad Maghnie, MD, PhD Paediatric Endocrinology Section
Giuseppe D’Annunzio, MD Department of Endocrinology
Department of Paediatrics St Bartholomew's Hospital
IRCCS, Giannina Gaslini West Smithfield, London, UK
University of Genova
Genova, Italy
Sandro Loche, MD
Service of Paediatric Endocrinology
Regional Hospital for Microcytaemia
Cagliari, Italy
Library of Congress Cataloging-in-Publication Data
Congenital endocrinopathies : new insights into endocrine diseases and

diabetes / volume editors, Renata Lorini . . . [et al.].
p. ; cm. – (Endocrine development, ISSN 1421-7082 ; v. 11)
Includes bibliographical references and indexes.
ISBN 978–3–8055–8347–3 (hard cover : alk. paper)
1. Endocrine genetics–Congresses. 2. Endocrine glands–Diseases–Genetic
aspects–Congresses. 3. Diabetes–Genetic aspects–Congresses. I. Lorini,
Renata. II. Series.
[DNLM: 1. Endocrine System Diseases–congenital–Congresses. 2. Child.
3. Diabetes Mellitus, Type 1–congenital–Congresses. 4. Growth
Hormone–metabolism–Congresses. 5. Infant. W1 EN3635 v.11 2007 / WS 330
C749 2007]
QP187.5.C66 2007
616.4⬘042–dc22
2007033860
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and
Index Medicus.
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© Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)

www.karger.com
Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel
ISSN 1421–7082
ISBN 978–3–8055–8347–3
Contents
VII Foreword
Savage, M.O. (London)
VIII Preface
Lorini, R.; Maghnie, M. (Genova)
1 Genomic Approaches in Genetic Research for Endocrine Diseases

Ravazzolo, R. (Genova)
6 Genetic Defects of the Growth-Hormone-IGF Axis Associated with

Growth Hormone Insensitivity
Woods, K. (Portland, Oreg.)
16 Late Effects of Disturbed IGF Signaling in Congenital Diseases

Intrauterine and Postnatal Growth Retardation
Cianfarani, S.; Geremia, C.; Puglianiello, A.; Maiorana, A.; Germani, D. (Rome)
28 Growth Hormone Receptor Polymorphisms

Controversies and Outcome of Growth Hormone Treatment
Buzi, F.; Mella, P.; Pilotta, A.; Prandi, E.; Lanfranchi, F.; Carapella, T. (Brescia)
36 Genetic Disorders Involving Adrenal Development

Lin, L.; Ferraz-de-Souza, B.; Achermann, J.C. (London)
47 Early Management and Gender Assignment in Disorders of Sexual

Differentiation
Hughes, I.A. (Cambridge)
V
58 Prenatal and Early Postnatal Treatment of Congenital Adrenal
Hyperplasia
Ghizzoni, L.; Cesari, S.; Cremonini, G.; Melandri, L. (Parma)
70 Neonatal Diabetes
The Role of KCNJ11 (Kir6.2)
Tammaro, P. (Oxford)
83 Diagnosis of Neonatal and Infancy-Onset Diabetes

Barbetti, F. (Rome)
94 Management of Neonatal and Infancy-Onset Diabetes Mellitus

Søvik, O. (Bergen); Tansek, M.Z. (Ljubljana); Sagen, J.V.; Njølstad, P.R. (Bergen)
106 Insights in Congenital Hyperinsulinism

Hussain, K. (London)
122 A Clinical Approach to Severe Insulin Resistance

Savage, D.B.; Semple, R.K.; Chatterjee, V.K.K. (Cambridge); Wales, J.K.H.; Ross,
R.J.M. (Sheffield); O’Rahilly, S. (Cambridge)
133 Inherited Endocrine Diseases Involving G Proteins and

G Protein-Coupled Receptors
Spiegel, A.M. (Bronx, N.Y.)
145 Stem Cells

From Animal Research to Clinical Applications
Redi, C.A.; Monti, M.; Merico, V.; Neri, T.; Zanoni, M. (Pavia);
Zuccotti, M. (Parma); Garagna, S. (Pavia)
152 Author Index
153 Subject Index
This book has been printed with financial support from Pfizer Italia.
Contents VI
Foreword
This volume reports the proceedings of an outstanding symposium, held in

Genoa in January 2007, organised by Professor Renata Lorini and Professor
Mohamad Maghnie, on the very imaginative subject of congenital endocrino-
pathies. This subject is broad by definition and the resulting volume is conse-
quently interesting and informative and directly relevant to the care of the
patient.
The volume contains reviews of normal and abnormal hypothalamic-
pituitary development affecting growth hormone (GH) secretion and defects of
the GH-IGF-I axis influencing GH and IGF-I action. Abnormalities of the
pituitary-gonadal axis affecting normal puberty are covered together with
defects of steroidogenesis involving both adrenal and gonadal development.
The effect of glucocorticoid hormone programming in early life and defects of
G proteins and their receptors are also described. Key chapters are included on
neonatal diabetes, congenital insulin resistance syndromes and the pathogenesis
and management of persisting hyperinsulism of infancy. Finally, the application
of stem cell research from animal studies to human diagnosis and therapy has
been reviewed.
Overall this is an excellent volume. It provides information directly useful
to the clinician and stimulates thought and future research opportunities with
cutting-edge scientific results in the broad and very important field of congeni-
tal endocrinopathies.
Martin O. Savage, London
VII
Preface
In recent years, tremendous progress has been made in the field of genetics
and congenital diseases both in endocrinology and in diabetology. The opportu-
nity to provide an exceptional updated and prospective view of this field was
given by the meeting on ‘Congenital endocrinopathies: New insights into
endocrine diseases and diabetes’ held in Genoa, Italy, on January 18–19, 2007.
The scientific programme of the meeting was designed to focus on the
most recent breakthrough advances relevant to endocrinology and diabetes. The
impressive advances in gene technology have greatly improved our diagnostic
and therapeutic skills as well as our understanding of the pathogenesis of paedi-
atric endocrine diseases and diabetes.
This book provides an elucidation of the molecular aspects of various
endocrine diseases but of course cannot be exhaustive in covering all the
aspects of this complex field. Renowned and dedicated experts have covered
the current evidence and future directions on these topics, and we believe that
their contributions will ensure the exchange of valuable new information and
ideas. We are confident that a synthesis of modern concepts of basic and clini-
cal science within the broad field of molecular endocrinology and diabetology
is represented here, and will provide a state-of-the-art book that is of value to
physicians, non-clinical scientists and students from many disciplines.
Renata Lorini, Mohamad Maghnie, Genova

Lorini R, Maghnie M, D’Annunzio G, Loche S, Savage MO (eds):
Congenital Endocrinopathies. New Insights into Endocrine Diseases and Diabetes.
Endocr Dev. Basel, Karger, 2007, vol 11, pp 1–5
Genomic Approaches in Genetic Research

for Endocrine Diseases
Roberto Ravazzolo
Laboratory of Molecular Genetics, G. Gaslini Institute, and Department of Pediatrics
and CEBR, University of Genova, Genova, Italy
Abstract
Genomic research has made great progress to understanding functional roles in non-
coding DNA sequences. In particular, approaches to identify regulatory elements with
enhancer/silencer function based on the synergism between computational and experimental
techniques are discussed. Such approaches have been applied to gene-directed as well as
genome-wide investigations.
Copyright © 2007 S. Karger AG, Basel
Sequencing of genomes has enabled the ‘genomic’ approach to research,

by which new hormones, receptors and signaling molecules will be discovered,
and integrated understanding of mechanisms of intercellular communication
will be clarified. Furthermore, genomic knowledge provides information about
research on genes and their variation in individuals, families and populations,
which will extend the knowledge on monogenic and multifactorial disorders.
One of the fascinating issues in genomic research is trying to discover
functional roles in noncoding sequences, in particular related to gene regula-
tion. The Human Genome Project has provided a huge amount of information
on gene location and annotation and, basing on defined position immediately
upstream of each gene, significant progress has been made in the identification
of core promoter elements. However, although distant-acting gene regulatory
sequences play established roles in development, phenotypic diversity and
human disease, their identification has been limited up to now.
Research to identify such elements is strongly based on the synergism
between computational and experimental techniques. Methods of in silico
analysis include comparison of DNA and protein sequences and databases of
expression profiles (microarray) that can be analyzed by means of appropriate
algorithms. Great effort has been put in computational sciences to provide pow-
erful means of genetic and genomic analysis by taking advantage of the com-
pletion of a number of vertebrate genome sequences as well as the concurrent
development of genomic alignment, visualization and analytical bioinformatics
tools [1].
Identification of common features that may indicate functional signifi-
cance, such as the discovery of putative cis-regulatory elements, can be
achieved by comparison of DNA sequences from different organisms.
Bioinformatic algorithms have been optimized to derive information from
sequence alignments that can highlight genomic regions which underwent slow
evolution versus rapidly evolving ones. Pairwise and multiple-species sequence
comparisons have been carried out to identify novel regulatory elements in
mammalian genomes and the power of such comparative analyses was
increased by performing multispecies alignments that combine both closely
related and highly divergent organisms. Since noncoding sequences generally
lack sequence conservation between highly divergent species, finding highly
conserved noncoding sequences when comparing species with wide evolution-
ary distance, such as human and pufferfish, implies that sequences conserved
between these 2 species are likely to be fundamental to vertebrate life.
Examples of noncoding conservation between these species have been associ-
ated with genes that play critical roles in development, suggesting that common
mechanisms of developmental regulation take place in vertebrates [2].
Finding indications of putative function by bioinformatic means that high-
light sequence conservation has to be followed by functional tests that can con-
firm and validate the hypothesis. This step is often difficult to carry out but is
crucial and unavoidable.
Sometimes, chromosome rearrangements in patients who show peculiar
phenotypes provide evidence of function at particular genomic districts. Our
recent results obtained by a detailed molecular study of a balanced translocation
[3] have shown how regulatory elements located distant from gene promoter
regions can modify gene expression up to causing a malformative phenotype.
The distant regulatory elements are those controlling the COL1A2 gene tran-
scription in chromosome 7. It was interesting to discover, by analysis of non-
coding sequences conserved across species, that the genomic region upstream
of COL1A2 in chromosome 7 contains 5 of these conserved elements (fig. 1).
Part of them, in particular the 4 most proximal ones, were brought by the
translocation to a genomic region in which the NPPC gene, encoding the
C-type natriuretic peptide, is located (chromosome 2). It is already known that the
most proximal of the conserved elements [4], located around 20 kb upstream
of COL1A2, acts as enhancer. This rearrangement induced overexpression of
Ravazzolo 2
Breakpoint
COL1A2
Fig. 1. Scheme of the genomic region upstream of COL1A2, in which 5 conserved

noncoding sequences are located. The arrow indicates the translocation breakpoint in chro-
mosome 7.
C-type natriuretic peptide, which in turn caused a marfanoid phenotype in the

patient. The most distal one is separated by the translocation breakpoint from
COL1A2 and we found that the allele corresponding to this interrupted chromo-
some was underexpressing COL1A2. This finding suggests that this very distant
conserved sequence could act as putative enhancer.
Thus, the analysis of this single case allowed us to understand how the
COL1A2 gene is controlled by regulatory elements present in the far upstream
noncoding region, which, when translocated, exert their enhancer function also
in the context of a different chromosomal region.
Genomic sequences that include regulatory elements are usually part of
chromatin regions subjected to activating or silencing modification, which in
turn affect accessibility to transcription factors, polymerase, etc. [5]. Histone
acetylation/deacetylation state is one of the most important chromatin modifi-
cation processes by which gene silencing due to repressive chromatin structures
corresponds to low or absent histone acetylation at the functional regulatory
element. Multiprotein complexes in which proteins that display histone
deacetylase activity are involved are found associated to such DNA sequences.
In contrast, permissive chromatin structure that allows gene expression is asso-
ciated with recruitment of protein complexes in which proteins that display his-
tone acetyl transferase activity are involved, resulting in a high level of histone
acetylation. For distant-acting regulatory elements, we can hypothesize that
they are brought close to promoter elements by DNA looping, which allows
interaction at the level of chromatin regions that have a conformation likely to
cause silencing or expression (fig. 2).
Basing on the knowledge of these chromatin-modifying processes, our
group has described an approach to validate sequence comparison data [6]. A
wide genomic region, including the 5⬘ untranslated region and the first intron of
the RET gene, known to be mutated in multiple endocrine neoplasia type 2 A and
Hirschsprung disease, was analyzed for sequence conservation across species.
This bioinformatic analysis was combined with a functional test based on the
level of histone acetylation at each conserved element, assayed by the chromatin
Genomic Research in Noncoding Regions 3

Fig. 2. Chromatin region (gray area) subjected to activating or silencing modification,
i.e. accessibility to transcription factors, polymerase, etc.
Histone 4
Cell lines
acetylation level High RET expression
Low RET expression
Fig. 3. Example of differential histone acetylation level at 1 specific conserved ele-

ment in chromatin extracted from cells that express different RET mRNA levels.
immunoprecipitation method. The rationale for such an approach was that com-
parison of chromatin from cells that express high or low levels of RET could
show different levels of histone acetylation. The histone acetylation level was
quantified in each selected region by collecting DNA fragments bound to tetra-
acetylated histone H4 and performing real-time PCR with specific pairs of
primers. Our hypothesis proved to be correct in a number of the detected con-
served sequences, in which we found that the level of histone acetylation was in
accordance with the level of RET expression (fig. 3). The functional significance
was further confirmed by a second functional test in which we assessed the abil-
ity of enhancing transcription by a reporter gene assay.
In our hands, combined analysis of sequence conservation and chromatin
conformation assessed by chromatin immunoprecipitation, applied to a large
Ravazzolo 4
genomic region, has proved to be very useful to highlight and characterize
potential regulatory elements in noncoding regions.
Search for noncoding enhancers/silencers can be carried out by gene-
directed strategies like the ones described above, or by genome-wide approaches.
A recent article [7] described a well-designed analysis of conservation in
noncoding sequences between organisms separated by varying evolutionary dis-
tances at the genome-wide level. A number of human DNA fragments fulfilled
the criteria for conservation and were tested by a transgenic mouse enhancer
assay in which the human conserved fragments were inserted close to a minimal
mouse heat shock promoter fused to a lacZ reporter gene. Enhancer activity was
verified by whole-mount staining and whole-embryo visualization. Several of
these putative regulatory elements resulted positive for this type of assay and
were found in gene-poor genomic regions at distance from the regulated genes.
In this case combined computational and experimental methods were
applied to identify functional noncoding sequences at the genome-wide level.
References
1 Wasserman WW, Sandelin A: Applied bioinformatics for the identification of regulatory elements.
Nat Rev Genet 2004;5:276–287.
2 Woolfe A, Goodson M, Goode DK, Snell P, McEwen GK, Vavouri T, Smith SF, North P, Callaway H,
Kelly K, Walter K, Abnizova I, Gilks W, Edwards YJK, Cooke JE, Elgar G: Highly conserved non-
coding sequences are associated with vertebrate development. PLoS Biol 2005;3:e7.
3 Bocciardi R, Giorda R, Buttgereit J, Gimelli S, Divizia MT, Beri S, Garofalo S, Tavella S, Lerone M,
Zuffardi O, Bader M, Ravazzolo R, Gimelli G: Overexpression of the C-type natriuretic peptide
(CNP) is associated with overgrowth and bone anomalies in an individual with balanced t(2;7)
translocation. Hum Mutat 2007;28:724–731.
4 Antoniv TT, De Val S, Wells D, Denton CP, Rabe C, de Crombrugghe B, Ramirez F, Bou-Gharios G:
Characterization of an evolutionarily conserved far-upstream enhancer in the human alpha 2(I)
collagen (COL1A2) gene. J Biol Chem 2001;276:21754–21764.
5 Backs J, Olson EN: Control of cardiac growth by histone acetylation/deacetylation. Circ Res
2006;98:15–24.
6 Puppo F, Musso M, Pirulli D, Griseri P, Bachetti T, Crovella S, Patrone G, Ceccherini I, Ravazzolo R:
Comparative genomic sequence analysis coupled to chromatin immunoprecipitation: a screening
procedure applied to search for regulatory elements at the RET locus. Physiol Genomics
2005;23:269–274.
7 Pennacchio LA, Ahituv N, Moses AM, Prabhakar S, Nobrega MA, Shoukry M, Minovitsky S,
Dubchak I, Holt A, Lewis KD, Plajzer-Frick I, Akiyama J, De Val S, Afzal V, Black BL, Couronne O,
Eisen MB, Visel A, Rubin EM: In vivo enhancer analysis of human conserved non-coding
sequences. Nature 2006;444:499–502.
Prof. Roberto Ravazzolo

Lab. Genetica Molecolare, Istituto G. Gaslini
Largo G. Gaslini, 5
IT–16147 Genova (Italy)
Tel. ⫹39 010 5636400, Fax ⫹39 010 3779797, E-Mail rravazzo@unige.it
Genomic Research in Noncoding Regions 5

Genetic Defects of the Growth-Hormone-IGF

Axis Associated with Growth Hormone
Insensitivity
Katie Woods
Pediatric Endocrinology, Department of Pediatrics, Doernbecher Children’s Hospital,
Portland, Oreg., USA
Abstract
The central feature of growth hormone (GH) insensitivity is deficiency of insulin-like
growth factor-1 (IGF-1) in association with elevated GH secretion. This condition is also
known as primary IGF deficiency. There are currently four known genetic causes of GH
insensitivity/primary IGF deficiency: GH receptor deficiency (also known as Laron syndrome
or GH insensitivity syndrome), IGF-1 deficiency, signal transducer and activator of transcrip-
tion 5b (STAT5b) deficiency and acid labile subunit (ALS) deficiency. Despite sharing the
classical biochemical features of GH insensitivity, the phenotype in each of these conditions is
quite distinct. This review will discuss each of these causes in turn, highlighting the insights
these rare causes of growth failure afford into the functioning of the human GH-IGF-1 axis.
Introduction
Following binding to and activation of the cell-surface-bound growth hor-

mone (GH) receptor, GH mediates its effects by activating an intracellular sig-
naling cascade which ultimately leads to the synthesis of insulin-like growth
factor (IGF)-1, the main effector hormone of growth. GH not only stimulates
the production of circulating IGF-1 (mainly derived from stimulation of hepatic
GH receptors) but also acts directly on GH receptors on local tissues, such as
muscle and chondrocyte, to induce local IGF-1 synthesis. Thus, as originally
suggested in the ‘somatomedin hypothesis’, IGF-1 can act both as a classical
endocrine hormone and in an autocrine/paracrine manner [1] (fig. 1).
Furthermore, GH has effects which are independent of IGF-1 (‘direct’ GH
Pituitary
GH
Liver: ‘endocrine’
effect Local effects
GH GH
Negative
GH receptor feedback
Intracellular
signaling pathway
IGFBP-3
Local Direct effects
IGF-1
Circulating ALS
IGF-1
Circulating LINEAR
ternary GROWTH
complex
IGF-1 receptor
Fig. 1. Schematic diagram of the GH-IGF-1 axis, from secretion of GH to the initia-
tion of growth through the binding of IGF-1 to the IGF-1 receptor. Defects leading to GH
insensitivity have been identified in the GH receptor gene, the gene encoding the signaling
molecule STAT5, the IGF-1 gene and the ALS gene. IGFBP-3 ⫽ IGF-binding protein-3;
ALS ⫽ acid labile subunit.
effects). The relative contributions of circulating IGF-1, local IGF-1, and

‘direct’ GH effects on longitudinal growth remain the subject of debate. As dis-
cussed below, genetic defects at several points in the GH-IGF axis have now
been identified in subjects with biochemical GH insensitivity, allowing unique
insight into the complex and interrelated actions of GH and IGF-1.
Growth Hormone Receptor Gene Deficiency
In 1966, Laron et al. [2] reported 2 siblings with the classical clinical fea-
tures of congenital GH deficiency, yet elevated circulating GH. It was not until
over 20 years later, however, in 1989, that mutations in the gene encoding the
GH receptor were identified as the cause of this syndrome [3, 4]. This condition
is now known variably as Laron syndrome, GH receptor deficiency or GH
insensitivity syndrome. Since then, over 300 individuals with GH receptor defi-
ciency have been described worldwide.
Genetic Defects of the GH-IGF Axis 7

The mature GH receptor in humans spans 620 amino acid residues with 3
functional domains: an extracellular domain that binds GH, a single transmem-
brane domain, which anchors the receptor in the cell membrane, and a 350-
amino-acid-residue cytoplasmic domain responsible for intracellular signaling
[3]. The GH receptor gene contains 10 exons, with exons 2–7 encoding the sig-
nal peptide and extracellular domain, exon 8 primarily the transmembrane
domain, and the remaining exon 8 segment and exons 9 and 10 the intracellular
domain. Over 60 distinct mutations of the GH receptor have been described to
date, 95% of which are located in the extracellular domain of the receptor
(fig. 1) [5]. Only 2 of the GH receptor gene mutations so far described act in a
dominant manner: these 2 mutations, although affecting different nucleotides,
both result in the ‘skipping’ (or exclusion) of exon 9 from the GH receptor
gene mRNA, producing a truncated GH receptor which retains the ability to
bind GH and anchor in the cell membrane but not to transmit an intracellular
signal [6, 7].
The clinical phenotype of GH receptor deficiency is indistinguishable from
that of severe congenital GH deficiency: namely a relatively normal birth
weight, followed by profound postnatal growth failure (mean height SDS
⫺6.5 SD), central obesity, hypoglycemia in infancy, and a typical facial appear-
ance of a prominent forehead and relatively small midface. IGF-1 levels are very
low (SDS typically less than ⫺4) and do not increase after exogenous GH
administration. The levels of IGF-binding protein-3 (IGFBP-3) and acid labile
subunit (ALS), which bind IGF-1 in the circulation to form the ‘ternary com-
plex’, are also extremely low, demonstrating the GH-dependent nature of these
peptides, and do not increase after GH. GH levels are elevated and show an
exaggerated rise after pharmacological stimulation. Another useful biochemical
marker for GH receptor deficiency is GH-binding protein, low or absent in
around 75% of subjects with GH receptor deficiency [8]. GH-binding protein is
a circulating form of the extracellular domain of the GH receptor, formed by
proteolytic cleavage at the cell surface, and is measured using assays which
determine the GH-binding ability of serum. Low GH-binding protein suggests a
mutation of the GH receptor which either results in reduced receptor binding
activity or reduced receptor expression. However, it should be noted that normal
or even elevated GH-binding protein levels do not exclude a GH receptor defect.
Recombinant IGF-1 therapy has been used to treat GH receptor deficiency.
Although it does induce an increase in growth velocity in treated subjects, it does
not induce the sustained catch-up growth seen in subjects with severe GH defi-
ciency treated with GH [9]. However, it must be remembered that, unlike GH
treatment of GH deficiency, which results in the increase in both circulating and
local IGF-1, recombinant IGF-1 treatment of GH receptor deficiency replaces
only circulating IGF-1. Furthermore, IGFBP-3 and ALS levels remain low in
Woods 8
GH-receptor-deficient subjects treated with recombinant IGF-1, and any ‘direct’
(non-IGF-1) mediated effects of GH on linear growth remain unreplaced.
IGF-1 Gene Deficiency
In 1996, we described a 15-year-old boy with severe growth failure (height

SDS ⫺6.8) and biochemical features of severe GH insensitivity [10]. However,
unlike subjects with GH receptor deficiency, this patient had severe intrauterine
growth retardation (birth weight 1.7 kg at term), congenital sensorineural hearing
loss and mental retardation. He also lacked the typical facial appearance (rela-
tively large head with prominent forehead and midface hypoplasia) characteristic
of GH receptor deficiency. Despite elevated GH secretion and virtually unde-
tectable IGF-1 levels, pathognomic of GH insensitivity, IGFBP-3 and ALS levels
were normal, suggesting a molecular defect in the GH-IGF-1 axis beyond the GH
receptor. This patient was found to have a homozygous partial deletion of the
IGF-1 gene, resulting in a mature IGF-1 peptide truncated from 70 to 25 amino
acids, followed by an additional out-of-frame nonsense sequence of 8 residues
and a premature stop codon. There are 3 further human cases of IGF-1 gene
mutations now reported, all of which are homozygous point mutations of the
IGF-1 gene [11–13]. The affected patients share similar clinical features: namely
pre- and postnatal growth failure (all 3 cases), mental retardation (all 3 cases) and
sensorineural hearing loss (2 of 3 cases). All have normal IGBP-3 levels, but
IGF-1 levels were consistently low in only 1 case [12]. The most well-characterized
of these reports is the case of a 55-year-old male with severe pre- and postnatal
growth failure (height SDS ⫺8.8), mental retardation and sensorineural deaf-
ness, and a homozygous missense mutation in the IGF-1 gene which leads to a
valine to methionine substitution at residue 44 of the mature IGF-1 molecule
(G274A), resulting in almost complete loss of binding affinity for the IGF-1
receptor [11]. This patient had elevated levels of IGF-1, suggesting that the
mutant IGF-1 is still immunologically detectable and, under feedback from
increased GH secretion, expressed at increased levels (‘bioinactive’ IGF-1).
The severe postnatal growth failure of the subjects with disruption of IGF-1
gene function supports the central role of IGF-1 in mediating the effects of GH
on postnatal growth. However, the phenotypic differences between IGF-1 gene
deficiency and GH receptor gene deficiency outline the fact that, despite consid-
erable overlap, GH and IGF-1 have functions independent of each other. For
example, both GH and GHR deficiency are associated with normal, to mildly
reduced, birth weight suggesting that GH action is not necessary for the majority
of prenatal growth. In contrast, IGF-1 (and IGF-1 receptor) gene deficiency is
associated with substantial prenatal growth failure, indicating that IGF-1 is an

important prenatal growth factor. Furthermore, the sensorineural deafness and
mental retardation unique to IGF-1 gene deficiency suggest an important role
for non-GH-induced IGF-1 in central nervous system functioning. Finally, the
normal levels of IGFBP-3 and ALS, and the lack of hypoglycemia in IGF-1 gene
deficiency, most likely reflect the direct, non-IGF-1-mediated effects of GH.
STAT-5b Gene Deficiency
The GH receptor activates 3 main signaling pathways: the signal transducer

and activators of transcription (STAT) pathway, the mitogen-activated kinase
kinase pathway and the phosphatidlyl-inositol-3 kinase pathway. Until recently,
the relative contribution of each pathway to the growth-promoting, and IGF-1-
generating, effects of GH was unclear. However, the identification, in 2003, of
the first case of STAT 5b gene deficiency helped to clarify this issue. Kofoed
et al. [14] described a 16-year-old female from Argentina with a homozygous
missense mutation (A630P) in STAT 5b, located within the critical src homology
2 domain of this signaling molecule. Functional studies of this mutation have
indicated that the A630P is functionally null, unable either to be phosphorylated
by GH or activate GH-responsive genes (both key functions of STAT 5b). The
phenotype of this patient is remarkable for its similarity with severe GHR defi-
ciency, providing strong evidence that the effects of GH on growth and IGF-
1/IGFBP-3 levels are largely mediated through STAT 5b signaling. The subject
was mildly growth retarded at birth, with a birth weight of 1.4 kg at 33 weeks
(⫺2.0 SD). Birth length was not known. Postnatally, she grew extremely slowly,
and by 16.5 years had a height SDS of ⫺7.5 (117.8 cm). Clinical exam revealed
a prominent forehead and high-pitched voice. GH secretion was exaggerated
after insulin-induced hypoglycemia (peak 53.8 ng/ml or 107.2 U/l), and both
IGF-1 and IGBP-3 levels were very low (IGF-1: 38 ng/ml, IGFBP-3 levels:
874 ng/ml) and failed to increase after exogenous GH administration.
In addition to the above findings, all typical of classical GH insensitivity,
this patient also had a clinical picture suggesting a defect in T cell immunity.
She had recurrent pulmonary infections from an early age, including an episode
of Pneumocystis carinii pneumonia, and a lung biopsy was consistent with
lymphoid interstitial pneumonia. In addition, she had several serious viral
infections, including hemorrhagic varicella, and herpes zoster. Detailed studies
of her immune function, recently reported, indicate a defect in T cell regulation,
most likely as a consequence of impaired interleukin-2 signaling, as STAT 5b is
a key signaling molecule for the interleukin-2 receptor [15].
Three other patients (2 females, 1 male) with homozygous molecular
defects in STAT 5b have now been described (table 1) [16–18]. In all 3 patients,
Woods 10
Table 1. Details of the 4 reported cases of mutations in the STAT 5b gene described to date
Genetic Defects of the GH-IGF Axis
Author Height SDS Age Sex IGF-1 IGFBP-3 Immunodeficiency and

(mutation) years symptoms/signs
Kofoed et al. [14], ⫺7.5 15.8 female ↓↓↓ ↓↓↓ ⫹

2003 lymphoid interstitial pneumonia
(A630P) hemorrhagic varicella
recurrent herpes zoster
Pneumocystis carinii
pneumonia
Hwa et al. [16], ⫺7.8 16 female female ↓↓↓ ↓↓↓ ⫹
2005 recurrent pulmonary infections
(N398fs413) pulmonary fibrosis
pruritic skin lesions
Bernasconi et al. ⫺9.9 16 female ↓↓↓ ↓↓↓ ⫹⫹
[18], 2006 chronic diarrhea, eczema and
(R152X) recurrent skin infections
recurrent viral infections
chronic lung disease
Vidarsdottir et al. ⫺5.8 30 male ↓↓↓ ↓↓↓ ⫹/⫺
[17], 2006 hemorrhagic chicken pox
(Q368fsX376) as an adult
11
DNA binding SH2
domain domain
pY699
STAT 5b
protein
A630P: Kofoed et al. [14], 2003
N398fsX413: Hwa et al. [16], 2005
Q368fsX376: Vidarsottir et al. [17], 2006
R152X: Bernasconi et al. [18], 2006
Fig. 2. Domain structure of STAT5b protein and positions of the 4 currently reported
mutations. STAT 5b is phosphorylated by the activated GH receptor, which triggers dimer-
ization through the src homology 2 (SH2) domain, translocation of the STAT 5b dimer into
the nucleus and binding to STAT 5b-responsive genes (including the IGF-1 gene) through
residues in the DNA-binding domain. Of the 4 currently published mutations, 3 are predicted
to result in a severely truncated, nonfunctional STAT 5b protein. One mutation, the first to be
described to cause human STAT 5b deficiency (A630P), leads to an amino acid substitution
within the critical SH2 domain and has been demonstrated to result in a mutant STAT 5b
which cannot be phosphorylated or translocate to the nucleus.
the STAT 5b gene defect results in an extremely truncated and likely nonfunc-
tional STAT 5b molecule (fig. 2). These subjects have a very similar phenotype
to the original patient, with severe postnatal growth failure, very low IGF-1 and
IGFBP-3 levels, and increased GH secretion. All but 1 subject had recurrent
infections, particularly of the pulmonary system. The most recent subject to be
described, a 30-year-old Dutch male, had no immunodeficiency symptoms as a
child, although he did develop hemorrhagic chicken pox as an adult, suggesting
that the lack of recurrent infections in a patient with severe GH insensitivity
should not exclude the possibility of STAT 5b deficiency [18].
Acid Labile Subunit Deficiency
ALS is a GH-dependent glycoprotein which stabilizes the IGF-IGFBP-3

complex, forming the so-called ‘ternary’ complex. This 150-kD complex
reduces the passage of IGF-1 to the extravascular compartment and extends its
Woods 12
half-life. Thus, ALS is important for maintaining circulating IGF-1 levels but
does not impact local IGF-1 production. In 2004, the first case of human ALS
gene deficiency was described in an Argentinian male [19]. This patient has a
homozygous frameshift mutation of the ALS gene (1338delG) predicted to pro-
duce a severely truncated, functionally null ALS protein, and circulating ALS
levels were undetectable. Biochemical evaluation was consistent with severe
GH insensitivity: elevated overnight GH secretion and very low circulating lev-
els of IGF-1 (31 ng/ml, ⫺5.3 SDS) and IGFBP-3 (220 ng/ml, ⫺9.7 SDS), which
did not increase after exogenous GH administration. Somewhat surprisingly,
however, the patient exhibited only a mild degree of growth retardation, with a
height of 145.2 cm (⫺2.05 SDS) at 14.6 years of age, when first reported, and a
final adult height of 166.4 cm (⫺0.94 SDS).
A second case of ALS gene deficiency was described in 2006, a 14-year-
old Turkish female with an almost identical phenotype to the initial patient [20].
The ALS gene mutation in this case, D440N, is a point mutation, but circulating
ALS levels were undetectable, suggesting that the mutation produces an unsta-
ble or rapidly degraded mutant ALS protein molecule. Again, the height deficit
in this subject was relatively minor (height 144.6 cm at 14 years, ⫺2.12 SD),
despite profoundly reduced IGF-1 and IGFBP-3 levels.
These 2 cases of ALS gene deficiency provide important insights into the
function of ALS in man. Firstly, the severe reductions in IGF-1 and IGFBP-3 in
both cases underscore the important role played by ALS in maintaining circulat-
ing IGF levels. Secondly, the relatively mild growth failure of the subjects sug-
gests that lack of circulating IGF-1 has a relatively minor effect on linear growth,
when local IGF-1 production is preserved (or may even be increased, due to
increased GH secretion acting locally to increase local GH receptor signaling).
It has also been proposed that preserved free IGF-1 may explain the rela-
tively normal growth of these individuals: however, free IGF-1 levels were also
reduced in the case described by Hwa et al. [20].
Of note, inactivation of the ALS gene in the mouse produces a very similar
phenotype to the human cases: a severe reduction in IGF-1 levels (33% of wild
type) and IGFBP-3 levels (22% of wild type), yet only a minor effect on growth
(adult weight 87% of wild type) [21].
Conclusion
In recent years, the causes of genetic GH insensitivity in man have broad-

ened beyond molecular defects in the GH receptor gene, to include defects in
the genes encoding IGF-1, the signaling molecule and transcription factor
STAT 5b, and the IGF-binding molecule ALS. Phenotypic comparisons of the

subjects affected with these different forms of GH insensitivity have allowed
insights into the mechanisms of GH signaling, cross-talk of GH signaling mol-
ecules and other receptors, and a better understanding of the mechanisms by
which the GH-IGF-1 pathway promotes human linear growth.
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Katie Woods, MBBS, MRCP, MD

Pediatric Endocrinology, Department of Pediatrics, Doernbecher Children’s Hospital
707 SW Gaines Road
Portland, OR 97239-3098 (USA)
Tel. ⫹1 503 494 1926, Fax ⫹1 503 494 8311, E-Mail woodsk@ohsu.edu

Late Effects of Disturbed IGF Signaling

in Congenital Diseases
Intrauterine and Postnatal Growth Retardation
Stefano Cianfarani, Caterina Geremia, Antonella Puglianiello,

Arianna Maiorana, Daniela Germani
Rina Balducci Center of Pediatric Endocrinology, Department of Public Health and
Cell Biology, Tor Vergata University, Rome, Italy
Abstract
The biologic effects of insulin-like growth factor-1 (IGF-1) are mediated by specific
cell surface receptors. IGF-1 binding to the extracellular ␣-subunits activates the tyrosine
kinase intrinsic to the cytoplasmic portion of the IGF-1 receptor, leading to autophosphory-
lation of specific tyrosine residues in the receptor ␤-subunit. One early molecular event that
links the receptor kinase to the biologic actions of IGF-1 is tyrosine phosphorylation of the
insulin receptor substrate family (IRS-1 to -4). IRS acts as a multisite ‘docking’ protein by
binding to downstream signal-transducing molecules. Phosphorylation of multiple tyrosine
residues results in the association of IRS-1 with the Src homology 2 (SH2) domains of other
cytoplasmic signaling proteins, including phosphatidylinositol 3⬘ kinase, Syp, Grb2 and
Nck. By binding to Grb2, IRS proteins couple the IGF-1 receptor to the Ras/mitogen-
activated protein kinase pathway. This pathway regulates cell growth, differentiation and
proliferation. Severe pre- and postnatal growth retardation may arise from abnormalities of
IGF-1 signaling such as IGF-1-binding alterations and IGF-1 receptor mutations. Knockout
studies have shown severe growth impairment in mice lacking IRS family components or
Akt. Finally, in human placentas from pregnancies complicated by intrauterine growth
retardation, multiple alterations of IGF-1-signaling molecules have recently been described.
Structure of the Insulin and IGF-1 Receptors
Insulin and insulin-like growth factor-1 (IGF-1) are peptide hormones that
are homologous in primary structure but differ in their physiological effects.
Insulin and IGF-1 exert their biological effects by binding to their respective
Insulin IGF-1
␣ ss ␣ ␣ ss ␣ ␣ ss ␣
Insulin-binding Cysteine-rich
domains domain
ss ss ss
ss ss ss ss ss ss
Juxtamembrane
␤ domain ␤ ␤ β
Tyrosine kinase
Catalytic domain
C-terminal
domain
Insulin/IGF-1R
Insulin receptor IGF-1 receptor
hybrid receptor
Fig. 1. The IGF family of ligands and receptors (IGF-1R). Modified from Dupont et al. [1].
receptors, the insulin receptor (IR) and the IGF-1 receptor (IGF-1R). The IR and
IGF-1R have similar molecular weights, and both have tyrosine kinase activity.
The IR and IGF-1R are both comprised of 2 extracellular ␣-subunits containing
ligand-binding sites and 2 transmembrane ␤-subunits transmitting the ligand-
induced signal [1, 2]. More specifically, IGF-1R and IR ␤-subunits consist of 3
domains: (1) a juxtamembrane domain, with motifs required for recruiting the
major signaling adapter proteins; (2) a tyrosine kinase domain, essential for cat-
alytic activity of the receptor, and (3) the carboxyl-terminal domain, which has
several important residues for IGF-1R and IR signaling (fig. 1).
Despite the structural similarities between IGF-1 and insulin, the IR and
IGF-1R have a 100- to 1,000-fold higher binding affinity for their cognate lig-
ands. The ␣-subunits have been shown to confer ligand-binding specificity [3].
Structural differences in the cytoplasmic domain of the ␤-subunits of the
IR and IGF-1R may contribute to the divergence of these 2 signaling pathways.
The highest degree of homology between the 2 receptors is found within the
tyrosine kinase domain (about 84%), whereas the region of greatest divergence
between the IR and IGF-1R is found within the juxtamembrane domain (about
61%) and the carboxyl-terminal domain (about 56%) [4, 5].
IGF Signaling and Growth Retardation 17

IGF-1 receptor
␣ ␣
PDK-2 PDK-1 PI-3,4-P2 PI-3,4,5-P3 PI-4,5-P 2 Plasma membrane

PH PTEN P P GRB2 Ras
␤ ␤ Ras
P P
P P P SHC P
P
P Raf P
P P P P P Sos GDP GTP
CT IRS P
Kinase SHP-2
p110 p85 1-4 Or
P
Akt inactive PI 3’-kinase

P
P IRS
P
Fyn P Nck 1-4 P MEK1/2 P
P Syn P
P Kinase
CT PH
P
p38
Akt active ERK-2 P
Cell survival
Apoptosis JNK P
Cell proliferation
Glucose
transport Cell proliferation
Cell survival
Protein Cell differentiation
Glycogen synthesis
Apoptosis
synthesis
Fig. 2. Multiple signaling pathways for the IGF-1R. ERK ⫽ Extracellular signal-
regulated kinase; MEK ⫽ mitogen extracellular kinase; JNK ⫽ Jun kinase; CT ⫽ carboxy-
terminal; GDP ⫽ guanosine diphosphate; GTP ⫽ guanosine triphosphate; PDK ⫽
phosphoinositide-dependent kinase; PH ⫽ pleckstrin homology domain; PI ⫽ phos-
phatidylinositol; PTEN ⫽ phosphatase and tensin homologue; SHC ⫽ Src homology colla-
gen; SHP ⫽ Src homology phosphatase. Modified from Dupont et al. [1].
Signal Transduction via Insulin Receptor and IGF-1 Receptor
Many of the intracellular signaling events mediated by activation of the IR

and IGF-1R are remarkably similar [6–8]. Some of the shared substrates that
become phosphorylated by the IGF-1R and IR include members of the insulin
receptor substrate (IRS) family of proteins (IRS-1, -2, -3 and -4) [9–12], Gab-1
[13], and Shc [14]. The ability of phosphorylated soluble proteins, such as the
IRS family, to bind Src-homology-2-containing proteins may provide a way to
relay a signal from a receptor anchored in the plasma membrane to other cellu-
lar compartments. Upon stimulation by insulin or IGF-1, tyrosine-phosphorylated
IRS and Shc proteins form signaling complexes between phosphotyrosine-
containing binding motifs and Src homology 2 domains found in molecules
such as growth factor receptor binding-2 protein (Grb2) [15, 16] and the p85
regulatory subunit of the phosphatidylinositol 3⬘ kinase [17] and Syp (a tyrosine
phosphatase) [18] (fig. 2). Binding of phosphatidylinositol 3⬘ kinase to
Cianfarani/Geremia/Puglianiello/Maiorana/Germani 18
phosphorylated IRS leads to a 10-fold stimulation of its activity, accounting for
the rapid rise in phosphorylated phosphatidylinositols in stimulated cells.
Binding of Grb2 to IRS and the subsequent binding of Grb2 (an adapter pro-
tein) to Sos protein may account for the increase in the proportion of the active
Ras-GTP complex, which in turn leads to activation of mitogen-activated pro-
tein kinase (MAPK) cascade (fig. 2). Binding of Syp to IRS causes a marked
increase in its tyrosine phosphatase activity. Activated Syp may dephosphory-
late IRS, thereby terminating signaling. The phosphotyrosine residues on IRS-1
also form docking sites for other signaling molecules, including Fyn [19], Nck
[20] and Crk [21]. By binding to Grb2, the Ras/MAPK pathway regulates cell
growth, differentiation and proliferation in response to insulin and IGF-1 [22,
23]. Various protein tyrosine phosphatases can regulate the activities of the IR
and IGF-1R signaling systems. The specificity of signaling may be explained
by the preferential use of different substrates by the IR and IGF-1R [24]. In par-
ticular, the IR couples preferentially to IRS-2, whereas the IGF-1R couples
preferentially to IRS-1. This conclusion has been confirmed by ablation of the
IRS-1 and IRS-2 genes in mice [25–27].
Altered Ligand-Receptor Interaction:

Missense Mutation in the IGF-1 Gene
In mice, the growth-hormone-IGF-1 system plays a key role in intrauter-

ine development and postnatal growth and metabolism [28–30]. Knockout
models of the growth hormone receptor and IGF-1 have indicated that in
utero IGF-1, but not growth hormone, is required for normal fetal growth
[28, 30, 31].
Walenkamp et al. [32] recently described a 55-year-old patient, the first
child of consanguineous parents, presenting with severe intrauterine and post-
natal growth retardation, microcephaly and sensorineural deafness. A homozy-
gous G to A nucleotide substitution in the IGF-1 gene changing valine 44 into
methionine was found. The inactivating nature of the mutation was proven by
functional analysis. Proof for the inactivating nature of V44M was provided by
demonstrating a 90-fold lower binding affinity for the IGF-1R in receptor-
binding assays using recombinantly produced protein. Additional investigations
revealed osteoporosis, a partial gonadal dysfunction and a relatively well-
preserved cardiac function. The phenotype of this patient was caused by a com-
plete lack of bioactive IGF-1. IGF-2, although in the upper normal range, was
not able to compensate for IGF-1 deficiency in utero, in childhood and neither
in adulthood. Nine of the 24 relatives studied carried the mutation. They had a

significantly lower birth weight, final height and head circumference than
noncarriers.
IGF-1 Receptor Mutations
Since deletion of the murine IGF-1R gene causes marked prenatal growth
failure (birth weight, 45% of normal weight), with the affected neonates dying
from respiratory depression, the complete absence of IGF-1Rs in humans
would be expected to cause severe disease and perhaps be lethal. However, less
severe perturbations might attenuate the phenotype, as do naturally occurring
missense mutations in the IR gene that cause moderate insulin resistance.
Abuzzahab et al. [33] screened 2 groups of children for abnormalities in
the IGF-1R gene: (a) a group of 42 patients with unexplained intrauterine
growth retardation and subsequent short stature, and (b) a second cohort con-
sisting of 50 children with short stature who had elevated circulating IGF-1
concentrations. In the first cohort, 1 girl who was a compound heterozygote for
point mutations in exon 2 of the IGF-1R gene that altered the amino acid
sequence to Arg108Gln in one allele and Lys115Asn in the other was found.
Fibroblasts cultured from the patient had decreased IGF-1R function, as com-
pared with that in control fibroblasts. In the second group, 1 boy with a non-
sense mutation (Arg59stop) that reduced the number of IGF-1R on fibroblasts
was identified. Both children had intrauterine growth retardation and poor post-
natal growth.
Kawashima et al. [34] identified a heterozygous mutation (R709Q) chang-
ing the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Gln-Arg in a 6-year-old
Japanese girl (case 1) and her mother, who also showed intrauterine growth
restriction (IUGR) with short stature (case 2). Furthermore, (a) fibroblasts from
case 2 contained more IGF-1R proreceptor protein and less mature ␤-subunit
protein; (b) [125I]IGF-1 binding to fibroblasts from case 2 was reduced, com-
pared with normal controls, and (c) both IGF-1-stimulated [3H]thymidine
incorporation and IGF-1R ␤-subunit autophosphorylation were low in fibrob-
lasts from case 2, compared with those of controls. These findings strongly
suggest that this mutation leads to failure of processing of the IGF-1R prore-
ceptor to mature IGF-1R, causing short stature and IUGR.
More recently, Walenkamp et al. [35] described a 35-year-old female with
mild intrauterine growth failure, progressive postnatal growth retardation,
severe failure to thrive and microcephaly. Her daughter was born with severe
intrauterine growth retardation and also showed postnatal failure to thrive and
microcephaly. A heterozygous G31483A nucleotide substitution in the IGF-1R
gene, changing a negatively charged glutamic acid at position 1050 into a
Table 1. Clinical features of the 4 families with heterozygous IGF-1R mutations, modified from Walenkamp
et al. [35]
No. Subject Mutation Birth Birth Head Last

weight length circumference reported
height
1A index case R108Q ⫺3.5 ⫺4.8

K115N
1B mother K115N ⫺2.0 ⫺1.6
1C father R108Q ⫺2.0 ⫺2.8
2A index case R59stop ⫺3.5 ⫺5.8 ⫺4.6 at birth ⫺2.6
2B brother R59stop ⫺2.7 ⫺2.1
2C mother R59stop ⫺2.4 ⫺1.6 ⫺2.6
3A index case R709Q ⫺1.5 ⫺1.0 ⫺2.1
3B mother R709Q ⫺1.6 ⫺2.9
4A mother E1050K ⫺2.1 ⫺0.3 ⫺3.0 at 35 years ⫺4.0
4B daughter E1050K ⫺3.3 ⫺4.2 ⫺5.6 at 2 months ⫺2.3
Families 1 and 2 were described by Abuzzahab et al. [33], family 3 by Kawashima et al. [34] and family 4 by
Walenkamp et al. [35]. Data are expressed as SDS.
positively charged lysine residue (E1050K) was found. E1050 is a conserved

residue in the intracellular kinase domain. The patients had a mutation in exon
16, coding for the intracellular tyrosine kinase domain of the receptor. The tyro-
sine kinase (catalytic) domain is part of the cytoplasmic portion of the ␤-chain
of the IGF-1R. Binding of IGF-1 to the extracellular ␣-chain induces a confor-
mational change in the structure of the receptor, leading to autophosphorylation
of 3 tyrosines in the activation loop of the catalytic domain of the ␤-chain.
Phosphorylation of the tyrosine residues results in a dramatic conformational
change. Dermal fibroblasts of the mother showed normal binding of iodinated
IGF-1, but autophosphorylation and activation of downstream signaling cas-
cades upon challenging with IGF-1 was markedly reduced. The mutation affect-
ing the intracellular portion of IGF-1R caused IGF-1 insensitivity as shown by
the dramatic reduction of the fibrobast [3H]thymidine incorporation upon chal-
lenge with a dose range of IGF-1.
It is noteworthy that these patients with defects in the IGF-1R gene were
not phenotypically identical (table 1). The reasons for these differences are not
evident but could reflect differences in the intensity of IGF-1 signaling among
these patients, since the IGF-1R mutations blunt but do not abrogate IGF-1 sig-
naling. The phenotypic differences may also be explained by tissue-specific
imprinting of the expression of the IGF-1R alleles.

In the human, the IGF-1R gene is located on the distal long arm of chromo-
some 15 (15q26.3). The receptor is synthesized as a large precursor protein that
undergoes extensive posttranslational modifications including cleavage and glyco-
sylation. Monoallelic loss of chromosome 15q and loss of 1 copy of the IGF-1R
gene due to deletions of the distal long arm of chromosome 15 have been found in
patients with intrauterine growth retardation and postnatal growth deficit [36, 37].
In a tall child with 3 copies of the IGF-1R gene, accelerated growth
was ascribed to overactivation of the receptor kinase resulting from increased
binding of the ligand [38]. It was concluded that hemizygosity for IGF-1R can
cause primary IGF-1 resistance. Patients with loss of material from the distal
arm of chromosome 15 show intrauterine growth retardation, postnatal growth
deficits, occasionally craniofacial and skeletal abnormalities and mild to mod-
erate mental retardation [36].
IGF-1 Signaling Alterations
The activated receptors for insulin and IGF-1 phosphorylate various cellu-
lar substrates, including IRS-1 and IRS-2, which integrate the pleiotropic
effects of insulin, IGF-1 and other cytokines on cellular function. Deletion of
Irs1 produces small, insulin-resistant mice with nearly normal glucose home-
ostasis due to compensatory ␤-cell expansion [39]. In contrast, mice lacking
IRS-2 display nearly normal growth but develop diabetes 8–10 weeks after birth
accompanied by reduced ␤-cell mass and impaired function [40].
IRS-1 and IRS-2 mediate the effects of insulin and IGF-1 on embryonic
development, postnatal somatic growth and glucose homeostasis. IRS-1 has a
predominant role in somatic growth, as deletion of Irs1 reduces embryonic and
neonatal growth by 40%, whereas deletion of Irs2 reduces growth by 10%.
Irs1⫹/⫺Irs2⫺/⫺ mice are approximately 60% the size of wild-type animals,
whereas Irs1⫺/⫺Irs2⫹/⫺ mice are only 30% the size of controls, implicating
IRS-1 as the principal element by which IGF-1 mediates somatic growth [41].
The serine-threonine kinase Akt, also known as protein kinase B (PKB), is
an important effector for phosphatidylinositol 3⬘ kinase signaling initiated by
numerous growth factors and hormones. Akt2/PKB␤, 1 of 3 known mammalian
isoforms of Akt/PKB, was recently demonstrated to be required for at least some
of the metabolic actions of insulin. Cho et al. [42] showed that mice deficient in
another closely related isoform of the kinase, Akt1/PKB␣, display a conspicuous
impairment in growth. Akt1⫺/⫺ mice demonstrated defects in both fetal and
postnatal growth, and these persisted into adulthood. Akt1⫺/⫺ animals were dis-
tinguishable from wild-type animals because of their smaller size. Examination
of Akt1/PKB␣-deficient mice at birth revealed an ⬃20% reduction in body
weight in comparison with wild-type mice, suggesting that reduction in size
occurs during embryonic development. The decrease in body weight was evident
throughout postnatal development, regardless of sex, and persisted into adulthood.
However, in striking contrast to Akt2/PKB␤ null mice, Akt1/PKB␣-deficient
mice are normal with regard to glucose tolerance and insulin-stimulated disposal
of blood glucose. Thus, the characterization of the Akt1 knockout mice and its
comparison to the previously reported Akt2-deficiency phenotype revealed the
nonredundant functions of Akt1 and Akt2 genes with respect to growth and
insulin-regulated glucose metabolism [42].
IGF-1 Signaling Alterations in Human Placentas with

Intrauterine Growth Restriction
IGFs promote growth and development of the fetoplacental unit during

gestation [43], and impairment of their placental actions may result in altered
intrauterine growth of the fetus. Laviola et al. [44] investigated IGF-1 signaling
in human placentas from pregnancies complicated by IUGR. Placental tissue
was removed immediately after delivery and analyzed by immunoprecipitation
and immunoblotting techniques to study multiple signaling molecules involved
in IGF-1 regulation of growth and differentiation.
IUGR placentas exhibited a 33% reduction in the protein content of IGF-
1Rs but no changes in IR protein levels. In addition, IRS-2 protein levels were
reduced in IUGR placentas, with no changes in IRS-1 or Shc protein content,
and this was associated with a parallel decrease in IRS-2-associated phos-
phatidylinositol 3⬘ kinase.
Akt protein expression was also reduced in IUGR, whereas phosphoryla-
tion of Akt and its substrate glycogen synthase kinase-3 was unchanged.
Finally, in IUGR placentas there was impaired activation of multiple members
of the MAPK family, because phosphorylation of p38 and c-Jun N-terminal
kinase was reduced by 70%.
Targeted disruption of the p38 MAPK gene results in homozygous embry-
onic lethality because of severe defects in placental development. In particular,
p38 mutant placentas display impaired vascularization and insufficient oxygen
and nutrient transport as well as increased rates of apoptosis, consistent with a
defect in placental angiogenesis [45, 46]. In primary human trophoblast, spe-
cific activation of Jun kinase in response to placental growth factor protects
from serum-withdrawal-induced apoptosis [47]. Reduced activation of Jun
kinase has also been observed in placental tissue from women with pre-eclampsia
[48], which features a defective vascular development of the fetoplacental unit,
similar to IUGR pregnancies [49].

1.4 1.4
1.2 1.2
IGF-IR mRNA
INSR mRNA
1 1
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 0
Control IUGR Control IUGR
a (n ⫽ 4) (n ⫽ 3) b (n⫽4) (n ⫽3)
Fig. 3. RT-PCR analysis of IGF-1 (a) and IR (b) mRNA expression in human cytotro-
phoblasts of placentas from pregnancies complicated by intrauterine growth retardation and
controls. mRNA expression indicated as fold change.
Control IUGR
A 115.5 KDa
1 2 3 4 1 2 3 HepG2
Fig. 4. Western immunoblotting for IR protein in human cytotrophoblasts of placentas

from pregnancies complicated by intrauterine growth retardation and controls.
Together, these findings strongly support the hypothesis that the impair-
ment in the integrated activation of the MAPKs observed in IUGR placentas
may play an important role in altering placental angiogenesis, ultimately lead-
ing to reduced fetal growth. The human syncytial trophoblast is known to serve
several roles in pregnancy. It mediates the transport of nutrients and
immunoglobulins from the maternal to the fetal circulation and also functions
as an endocrine organ, secreting steroid and protein hormones [50]. Syncytial
trophoblast has been proposed to derive from mononuclear cytotrophoblasts
undergoing a process of differentiation and fusion, or, alternatively, endomito-
sis (i.e. nuclear division without cytokinesis).
We recently applied a method to generate purified human cytotrophoblasts
from human term placentas by adding a Percoll gradient centrifugation step to a
standard trypsin-DNase dispersion method [50]. Viability was greater than
90%. We investigated the expression of IGF-1 and IRs in pregnancies compli-
cated by intrauterine growth retardation. Preliminary data suggest that whilst
IGF-1R expression is unaltered, IR mRNA and protein expression seems to be
impaired in cytotrophoblasts from IUGR placentas (figs 3, 4).
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Stefano Cianfarani, MD
Rina Balducci Center of Pediatric Endocrinology
Department of Public Health and Cell Biology, Room E-178, Tor Vergata University,
Via Montpellier 1
IT–00133 Rome (Italy)
Tel. ⫹39 06 51002314, Fax ⫹39 06 5917415, E-Mail stefano.cianfarani@uniroma2.it

Growth Hormone Receptor Polymorphisms

Controversies and Outcome of Growth Hormone Treatment
Fabio Buzia, Patrizia Mellab, Alba Pilottaa, Elena Prandia,

Fabiana Lanfranchia, Teresa Carapellaa
a
Centro di Auxoendocrinologia, Department of Paediatrics, and bIstituto di Medicina
Molecolare A. Nocivelli, University of Brescia, Brescia, Italy
Abstract
Many variables influence the outcome of growth hormone (GH) therapy (GH dose and
duration, height – SDS at treatment start or at puberty onset, bone age, mid parental height,
growth velocity, age, etc.). Nevertheless, all these factors only partially explain the inter-
individual variability in response to GH in GH deficiency (GHD) and in short non-GHD
subjects. To this regard, genes coding for factors involved in GH action could play an impor-
tant role. GH acts through the GH receptor (GHR), and therefore the GHR gene could be the
first candidate to influence the response to GH. Polymorphisms of the GHR have been
described in exons 3, 6 and 10. The first one consists in the deletion (d3) or retention (fl) of
the entire exon 3. The d3 polymorphism has been recently associated with a better growth
response to GH in idiopathic short stature subjects and in short children born small for
gestational age. Subsequent studies on the same and other categories of short children
(idiopathic short stature, small for gestational age, GHD, Turner syndrome) have reported
controversial results, with some confirming the role of d3 and others showing no effect.
This review analyses these studies trying to explain the apparent discrepancies, mainly due
to different selection criteria and different dose regimens in treating GHD and non-GHD
short subjects.
Pharmacogenetics is the study of how a person’s genes can influence

her/his response to medication [1]. In the second half of the 20th century, it
became clear that genetic variation may explain why different people respond to
the same medicine in different ways. Since then, progress in pharmacogenetic
research has suggested that there is real potential for translation from the labo-
ratory into patient care [2]. There are several examples of genetic polymorphisms
that influence the outcome of drug therapy [1]. However, individual reaction to
a drug is not governed by genetic profile alone: several other variables are known
to play a role in response to drugs, like age, nutritional status, sex, smoking,
drinking, organ (dys)-function, other medications, infections and underlying
disease. With regard to growth hormone (GH) treatment, pharmacogenetics
may also play a role in the individual response to treatment, at least in some cat-
egories of patients. Today GH is used in different conditions, according to local
health administration rules, including: ‘classic’ GH deficiency (GHD), neu-
rosecretory dysfunction, biologically inactive/hypoactive GH, Turner syndrome
(TS), short children born small for gestational age (SGA), idiopathic short
stature (ISS), chronic renal failure, Prader-Willi syndrome and other possible
conditions where GH has been tried, such as achondro-hypochondroplasia,
Down syndrome, Noonan syndrome, etc. [3]. As far as GHD is concerned, not
all the GHD subjects will respond to GH treatment in the same way. This might
depend on several factors: first: does one deal with a real GHD? It is known that
different diagnostic criteria are used in different clinical settings, and this can
make a significant difference in defining GHD [4]. Moreover, GHD may either
be an isolated condition or be part of a multiple pituitary hormone deficiency or
even of a panhypopituitarism. Finally, many variables are known to influence
the outcome of GH therapy, including: treatment duration [5, 6]; height – SDS
at treatment start [5, 7]; bone age delay [6]; height at onset of puberty [7, 8];
mid parental height [5]; growth velocity (GV) in the first treatment year [5] and
GH dose [9]. All these variables correlate positively with growth outcome. On
the other hand, age at treatment start [5] and maximum GH peak following
provocation tests [6] have been reported to be negatively correlated.
Nevertheless, all these factors only partially explain the inter-individual vari-
ability in response to GH treatment in GHD children; as a consequence, current
prediction models might lack further parameters, among which genes coding
for factors involved in GH action could play an important role. GH acts through
the GH receptor (GHR), stimulating a cascade of events that eventually lead to
insulin-like growth factor (IGF) production [10]. GH binding to GHR leads to
the dimerisation of the receptor, resulting in the recruitment of JAK2 and acti-
vation of the MAPK-ERK 1/2, P13K and STAT pathways. Virtually each of the
genes coding for these post-receptor factors could be candidate to influence the
response to GH treatment, and in primis the GHR gene. The GHR gene is
located in the short arm of chromosome 5 (5p13.1-p12) and includes 9 coding
exons and several non-coding exons that undergo alternative splicing in the
untranslated 5⬘ region [11]. Defects of the GHR gene are responsible for the so-
called GH insensitivity or Laron syndrome [12] and have also been described in
subjects with ISS [13, 14]. While a mutation can change the amino acid
sequence and influence the transcript function, a polymorphism is not expected
to cause major changes in protein function. A polymorphism can be defined as
GH Receptor Polymorphisms and GH Treatment 29

a DNA sequence variant that occurs in at least 1% of the population [15]. We are
used to thinking of polymorphisms as single-nucleotide variations. Those
occurring in genes coding for drug-metabolising enzymes, drug transporters,
drug targets and DNA-repairing enzymes may be expected to influence drug
toxicity or efficacy, as already mentioned [1]. Polymorphisms of the GHR have
been reported in the general population and have been described in exons 3, 6
and 10 [14]. While the 2 latter are classical single-nucleotide polymorphisms,
the first one is an unusual genetic polymorphism, consisting in the deletion or
retention of an entire exon. This leads to the expression of the respective GHR
isoforms in humans, generated by retention (full-length GHR; ‘fl’) or exclusion
of exon 3 (exon 3-deleted GHR, ‘d3’) [16]. There appears to be a wide distrib-
ution in humans, the frequency of each allele ranging from 68–75% for fl to
25–32% for d3 [16, 17], with possible geographic differences. Exon 3 consists
of 22 amino acid residues; this peptide is located far from the binding inter-
faces. The S-S bond of the extracellular domain is apparently not influenced by
the loss of exon 3, and so the global folding of the extracellular domain is sup-
posed not to be altered. Experiments in the 1990s showed that fl and d3 retain
similar binding properties [18–20] and a single (fl or d3) allele appears to be
sufficient for normal growth in humans [21]. Pantel et al. [21] reported the case
of the first child of unrelated parents, whose parental heights were totally
normal. His birth weight and birth length were in the normal range, but his
growth rate declined rapidly within the first months of life, reaching ⫺5.5 SDS
at 6 months. His clinical appearance showed the typical features of GHD, with
episodes of hypoglycaemia in the first months. GH levels were high, IGF-1 and
IGF-binding protein 3 low and resistant to IGF generation, and GH-binding
protein undetectable. Therefore, the clinical diagnosis was that of a GH insensi-
tivity. He inherited 2 different GHR mutations, each from a single parent. These
were both nonsense mutations, leading to a stop codon. From the father he
received a mutation in exon 4, already described, and from his mother a muta-
tion in exon 3, where no mutation had been described before. The 2 heterozy-
gous parents had a normal phenotype, both carrying a null GHR allele in
combination with either a normal GHRfl or GHRd3 allele (in father and mother,
respectively). Therefore, the authors could conclude that a single copy of either
GHRfl or GHRd3 is sufficient for normal growth. A number of studies has
appeared since: the first, in 2004 [17], keeps a central position, both because it
was the first to be published and because it includes an in vitro experiment sup-
porting its clinical assumptions. Indeed, these authors reported that short chil-
dren with ISS or born SGA carrying at least 1 d3 allele show a better response
to GH in the first and second year of treatment compared to those homozygous
for the fl receptor. Afterwards, a series of clinical studies were published, all in
the Journal of Clinical Endocrinology and Metabolism and all in 2006 [22–26].
Buzi/Mella/Pilotta/Prandi/Lanfranchi/Carapella 30
Starting from those on GHD, we [22] were analysing the possible influence of
the main GHR polymorphisms on the growth response to GH, not limiting our
analysis to the sole d3, when the study by Dos Santos et al. [17] was published.
Therefore, we focused our analysis also on possible correlations between
growth response to GH and the fl and d3 polymorphisms. Our sample included
54 GHD children, ranging from severe to mild GHD, treated with a standard
European dose of GH. All the subjects responded to GH treatment doubling the
first-year GV both in terms of absolute velocity and of SDS. The prevalence of
the main polymorphisms found by us among the subjects (fl/fl: 51.9%; fl/d3:
38.8%; d3/d3: 9.3%) shows that there was an even distribution of exon 3 poly-
morphisms, with about 50% of homozygosity for the fl and about 50% of the
presence of d3 in our sample, either in heterozygosity or in homozygosity. No
difference in response to GH was found between patients with d3/d3 or d3/fl
and those with the fl GHR, both in terms of GV gain and GVSDS gain, nor did
we find significant differences in GV and GVSDS increment between the
groups defined by the single polymorphic genotypes or by the main genotype
associations. Moreover, no difference was observed with regards to the
response to GH between patients with d3 alone or in combination with other
polymorphisms and the remaining subjects with polymorphisms other than d3.
Therefore, we concluded that the most frequent polymorphisms of the GHR do
not appear to affect the growth response to exogenous GH in subjects with
GHD, at variance with what had been observed in other categories of children
with short stature. This study was presented as a poster at the joint
ESPE/LWPES (European Society for Paediatric Endocrinology/Lawson
Wilkins Pediatric Endocrine Society) meeting in Lyon, 2005, prior to publica-
tion in the Journal of Clinical Endocrinology and Metabolism, and, at that time,
it was in agreement with those by Blum et al. [23] and by Ito et al. [27], both
presented as communication and poster, respectively, at the same meeting. Our
results were, however, at variance with those by Dos Santos et al. [17], regard-
ing different categories of short stature, and with those by Jorge et al. [24],
regarding GHD subjects (also presented at the Lyon meeting as a communica-
tion). The study by Ito et al. [27], to our knowledge, has not been published so
far in any international journals in English. Their sample was not numerically
different from ours (51 GHD subjects), although the percentage of the d3 sub-
jects (16.3%) was significantly lower than ours and possibly too small to draw
statistical inferences. Again, the study by Blum et al. [23] published a little later
in the Journal of Clinical Endocrinology and Metabolism reported results very
similar to ours on a cohort of GHD subjects about twice as numerous as ours.
On the other hand, a study in favour of a positive role of d3 in growth response
to GH is that by Jorge et al. [24]. They analysed the growth response to GH in
75 severe GHD (⫺4.2 ⫾ 1.5 height SDS; peak GH range: 0.1–3.8 ng/ml). Of

these, 58 were pre-pubertal and served the analysis of growth response during
the first year of treatment (19 isolated GH deficiency, 39 multiple pituitary hor-
mone deficiency); in 44 the final stature was available. The distribution of
genotypes was about 50% for the fl and about 50% for the deleted isoform.
Height velocity was significantly greater in d3 than in fl, and multiple regres-
sion analysis revealed that the genotype was the one variable correlated with
GV, explaining 21% of the variability. Final stature was also bigger in the d3
group than in the fl, and height SDS at treatment start together with genotype
could explain 40% of the variability.
As in GHD, also in short stature not due to GHD has the response to GH
been shown to be variable and not accurately predictable for the individual
patient [28–30]. To this regard, the most important variables correlated with the
growth response to GH [31, 32] resulted to be age, BMI and GH dose. Since
prediction models explain about half the response to GH, again genetic factors
might play a role. As mentioned before, the study by Dos Santos et al. [17]
holds a central position in this issue. The study population consisted of 76 SGA
and 96 ISS treated with GH. The first 2 years of treatment were considered for
the analysis of growth response. The results revealed that children having 1 or 2
d3 GHR alleles showed a 75% higher increment in GV in comparison to those
homozygous for fl GHR. The study was supported by an in vitro demonstration
of the clinical observations. In human embryo kidney fibroblasts co-transfected
with vectors expressing either d3/d3 or fl/fl or d3/fl, by means of a lactogenic-
hormone-responsive-element-containing luciferase as reporter, exposed to
incremental GH concentrations as stimulus, d3 induced a more elevated
reporter transcriptional activity compared to the fl GHR. Still in short children
born SGA, the Spanish group led by Carrascosa et al. [26] published a study
with different results. These authors studied 86 SGA with short stature treated
with GH (1.4 U/kg/w) out of a cohort of 170 short SGA subjects, in a double-
blind randomised study. The subjects had equal genotype frequency distribu-
tion, and the treated children, who had an increment in GV in the first and
second year of therapy, showed no differences between d3 and fl as regards:
height velocity, height gain, delta height and height prediction. Similar results
on SGA but not in TS were found by Binder et al. [25]: these authors studied 2
cohorts of short subjects, 53 TS and 60 short SGA, in a retrospective way. The
distribution of the GHR genotypes was not significantly different between con-
trols, short children born SGA and TS girls. With regard to the efficacy of GH
treatment, in TS the increment in GV in the first year of GH therapy was signif-
icantly higher in both the d3/d3 group as compared with the d3/fl ⫹ fl/fl group
and in the pooled d3 group compared to the sole homozygous fl subjects. In
addition, the d3 group showed a better height prediction. In contrast, in the
SGA cohort there was no significant difference in GV increment between the
d3 and the fl subjects, but only a better height prediction in the first ones.
Among all these controversial results, what can the explanation for the
apparent discrepancies between the different studies be? As far as GHD is
concerned, the only study showing a significant influence of the d3 isoform on
growth response to GH is the Brazilian one [24]. In this study, the subjects
were severely GH deficient and most of them had multiple pituitary hormone
deficiency. Unfortunately then, no pre-treatment GV was available, and there-
fore no evaluation of differential increment in GV could be made. The Italian
and German studies [22, 23] (as well as the Japanese poster [27]) were very
similar with regard to the subjects studied, with a wide range going from
severe to mild GHD. These differences in selection criteria may explain the
contrasting results. Moving to SGA and ISS subjects, the study by Dos Santos
et al. [17] evaluated the SGA and ISS subjects as pooled with regard to growth
response, although the 2 groups were treated with different GH doses;
Carrascosa et al. [26] studied SGA only, homogeneously treated with higher
GH doses; Binder et al. [25], who used GH doses similar to those of
Carrascosa A et al. [26], had similar results. Therefore, one can speculate
whether a high GH dose might mask the genotype effect. On TS, only 1 study
has been published so far [25], with apparently promising results. It remains to
be seen what the role of GH pharmacogenetics can be in other categories of
short children, such as neurosecretory dysfunction, Prader-Willi syndrome
chronic renal failure, etc. In conclusion, once again the different criteria in the
selection of patients appear to be responsible for some mess in the results of
the studies; it is likely that non-GHD subjects represent the best category
where to study GH pharmacogenetics, which might though have a role only in
severe forms of GHD. Nevertheless, the differences in outcome measures,
even when statistically significant, show an overlapping of data that may result
difficult to be translated into practice.
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adolescent height with growth hormone therapy in very short children without growth hormone
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Fabio Buzi, MD
Department of Paediatrics, University of Brescia
P. le Spedali Civili 1
IT–25123 Brescia (Italy)
Tel. ⫹39 030 3996284, Fax ⫹39 030 3996059, E-Mail fbuzi@med.unibs.it

Genetic Disorders Involving Adrenal

Development
Lin Lin, Bruno Ferraz-de-Souza, John C. Achermann
Developmental Endocrinology Research Group, Clinical and Molecular Genetics
Unit, UCL Institute of Child Health, University College London, London, UK
Abstract
The past decade has seen significant advances in our understanding of the genetic aeti-
ology of several forms of adrenal failure that present in infancy or childhood. Several of
these disorders affect adrenal development and are termed ‘adrenal hypoplasia’. These con-
ditions can be broadly divided into: (1) secondary forms of adrenal hypoplasia due to panhy-
popituitarism (e.g. HESX1, LHX4, SOX3) or abnormalities in ACTH synthesis (TPIT) or
processing (e.g. POMC or PC1); (2) adrenal hypoplasia as part of an ACTH resistance syn-
drome [MC2R/ACTH receptor, MRAP, AAAS (triple A syndrome)], and (3) primary defects
in the development of the adrenal gland itself (primary adrenal hypoplasia). Primary adrenal
hypoplasia most commonly occurs in an X-linked form due to mutations in the nuclear
receptor DAX1 (NR0B1) but can occur in a poorly understood recessive form or as part of
the IMAGe (intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia, gen-
itourinary anomalies) syndrome. Defining the molecular basis of these conditions can have
significant clinical implications for management, counselling and presymptomatic diagno-
sis, as well as providing fascinating insight into normal and abnormal mechanisms of adrenal
development in humans.
Disorders of adrenal development generally result in small, hypofunctional

glands and a clinical condition termed ‘adrenal hypoplasia’ [1, 2]. Significant
progress in our understanding of factors involved in adrenal development and
function now mean that a genetic diagnosis can be reached in approximately
half of children with this condition, with important implications for monitoring
associated features, focusing long-term management, and for counselling the
family about risks of further children being affected. Here we provide a brief
overview of recent advances in our understanding of the genetic basis of
adrenal hypoplasia by considering: (1) secondary adrenal hypoplasia due to
2y adrenal hypoplasia
CRF
• Panhypopituitarism
• Abnormal ACTH synthesis
• Abnormal ACTH processing
ACTH
ACTH resistance
ATII DHEA • FGD1 (ACTH receptor)
• FGD2 (MRAP)
DHEA -S • Triple A syndrome
1y adrenal hypoplasia
Cortisol
Aldosterone • X-linked AHC (DAX1)
• Autosomal AHC (?/SF1)
• Syndromes (e.g. IMAGe)
Fig. 1. Overview of the hypothalamic-pituitary-adrenal axis showing the different

types of adrenal hypoplasia. ATII ⫽ Angiotensin II; DHEA ⫽ dehydroepiandrosterone;
FGD ⫽ familial glucocorticoid deficiency; AHC ⫽ adrenal hypoplasia congenita.
(Reproduced from Achermann and Silvermann [3]; copyright 2004, with permission from
Elsevier).
defects in adrenocorticotropin (ACTH) synthesis and release; (2) ACTH resis-

tance syndromes, and (3) primary adrenal hypoplasia due to defects in the
development of the adrenal gland itself (see fig. 1; table 1).
Secondary Adrenal Hypoplasia
ACTH is an important tropic stimulus to the adrenal gland during devel-

opment. Consequently, congenital defects in ACTH synthesis, processing or
release can result in a secondary form of adrenal hypoplasia. Most children
with this condition present with signs and symptoms of glucocorticoid insuf-
ficiency such as hypoglycaemia, which can be especially severe if concomi-
tant growth hormone deficiency is present as part of a multiple pituitary
hormone deficiency (MPHD). Children with secondary adrenal hypoplasia
do not tend to present with mineralocorticoid insufficiency or salt loss, as the
main drive to adrenal aldosterone production is angiotensin II. The low
serum ACTH levels, the absence of hyperpigmentation and the presence of
Adrenal Development 37
Table 1. Overview of some of the more common genetic causes of adrenal hypoplasia
Condition Protein No. ACTH Cortisol Aldo Features
MPHD HESX1 8 MPHD ⫹/⫺ SOD

LHX4 2 MPHD, cerebellar
SOX3 3 ↓ ↓ N MPHD
PROP1 ?30 MPHD
ACTH regulation Tpit 31 –
POMC 6 ↓ ↓ N Obesity, red hair
PC1 3 Obesity, hypoglycaemia, HH
FGD1 ACTHR 42 ↑ ↓ N1 ? tall stature
FGD2 MRAP 23 ↑ ↓ N ?
Triple A AAAS 90 ↑ ↓ N1 Achalasia, alacrima, neurological
X-linked AHC DAX1 240 ↑ ↓ ↓ HH, spermatogenesis
‘Recessive’ SF1 3 ↑ ↓ ↓ 46XY female, uterus
IMAGe ? 6 ↑ ↓ ↓ IUGR, metaphyseal,
genital hypoplasia
The approximate number of individuals or families reported with each condition is shown. The clinical
presentation, biochemical profile and association of specific features can sometimes help to focus the diag-
nosis or direct genetic analysis in individual cases. Modified with permission from Lin and Achermann [2];
copyright 2004, Blackwell Publishing Ltd.
No. ⫽ Number; Aldo ⫽ aldosterone; MPHD ⫽ multiple pituitary hormone deficiency; SOD ⫽ septo-
optic dysplasia; HH ⫽ hypogonadotropic hypogonadism; N ⫽ within the normal range; AHC ⫽ adrenal
hypoplasia congenita; IUGR ⫽ intrauterine growth restriction.
1
Mineralocorticoid insufficiency can occur in a number of cases of triple A syndrome, and apparent
hyponatraemia is seen rarely in FGD1.
associated features (see below and table 1) can all help to point to the diagno-
sis of secondary adrenal hypoplasia rather than ACTH resistance or a primary
adrenal defect.
Multiple Pituitary Hormone Deficiencies

Congenital defects in ACTH synthesis often occur as part of an MPHD. In
most cases, growth hormone, thyroid-stimulating hormone and gonadotropin
(LH, FSH) release will also be affected so that the child may have hypogly-
caemia, signs of congenital hypogonadotropic hypogonadism (micropenis,
undescended testes) or post-natal growth failure. Additional neurodevelopmental
Lin/Ferraz-de-Souza/Achermann 38
␣-MSH/␤-MSH
␤-endorphin Fig. 2. Diagrammatic representation of
the processes involved in POMC synthesis
POMC ACTH
and cleavage in the corticotrope. PC1 ⫽
Tpit
Pitx1
Prohormone convertase-1. (Modified with
PC1 permission from Lin and Achermann [2];
copyright 2004, Blackwell Publishing Ltd).
defects such as absent septum pellucidum or optic nerve hypoplasia may be

present.
A number of genetic causes of congenital hypopituitarism have been
reported in the past decade. Deletions, mutations or copy number changes in
transcription factors HESX1, LHX4 and SOX3 can all cause ACTH insuffi-
ciency as part of a defect in pituitary development. In some cases, ACTH insuf-
ficiency may not be present at the original time of diagnosis but may develop
progressively with time, and additional features may be present which can help
to focus the diagnosis or approach to molecular analysis (table 1).
Mutations in PROP1 are one of the best-established causes of MPHD. In
general, PROP1 is not thought to play a major role in corticotrope development
or function, and children with PROP1 mutations typically present with progres-
sive growth hormone, thyroid-stimulating hormone and gonadotropin insuffi-
ciencies. However, it is emerging that a significant proportion of individuals
with this form of MPHD may go on to develop ACTH insufficiency with time –
often in adulthood [4]. The molecular pathophysiology of this is unclear, but it
is certainly important that patients with MPHD are followed up into adulthood
and careful vigilance is kept for emerging defects in ACTH release or impaired
stress response.
Isolated ACTH Deficiency

Isolated ACTH insufficiency is a rare condition that can be caused by
recessively inherited mutations in TPIT (TBX19) [5]. TPIT encodes a T box
factor that regulates transcription of the pro-opiomelanocortin (POMC) pro-
moter specifically in corticotropes (fig. 2). Loss of TPIT action will result in
impaired synthesis of POMC and ACTH in the pituitary, but the regulation of
POMC synthesis in other cells (e.g. skin, hypothalamus) is unaffected (see
below). Patients with TPIT mutations usually present with severe, early-onset
ACTH insufficiency. Hypoglycaemia and prolonged jaundice are common, and
sudden neonatal death is reported [6]. TPIT mutations are less common when
isolated ACTH deficiency first presents in childhood. The molecular basis of
this later-onset form of the condition is not currently known.
Disorders in POMC Synthesis and Release
As shown in figure 2, the mature ACTH peptide is cleaved from POMC
together with other small peptides such as ␣- and ␤-melanocyte stimulating
hormone and ␤-endorphin. These peptides have an important role in regulating
pigmentation of skin and hair, and in appetite regulation and weight control.
Part of the processing of ACTH involves the actions of a cleavage enzyme pro-
hormone convertase-1 (PC1, also known as proprotein convertase, subtilisin/
kexin type 1).
A number of defects in POMC regulation have now been described.
Mutations or deletions in POMC itself can cause secondary adrenal hypoplasia
due to ACTH insufficiency [7]. However, as other POMC-derived peptides are
affected in all cells of the body, associated features include obesity, pale skin
and red hair. These cutaneous features are sometimes striking but may be less
apparent in individuals with dark hair and may diminish with age.
Abnormalities in ACTH processing due to defects in PC1 have been described
in rare cases [8]. As the processing of several other peptide hormones is dis-
rupted, associated features include hypoglycaemia, obesity, hypogonadism and
persistent malabsorptive diarrhoea.
Adrenocorticotropin Resistance Syndromes
Resistance to ACTH can occur in a number of well-defined conditions,

such as defects in the ACTH receptor (MC2R, familial glucocorticoid deficiency
type 1); MC2R accessory protein (MRAP, familial glucocorticoid deficiency
type 2); or as part of the triple A syndrome (Alacrima, Achalasia, Addison; also
known as Allgrove syndrome and due to defects in ALADIN/AAAS) [9]. These
conditions tend to present with isolated glucocorticoid deficiency, hyperpigmen-
tation and markedly elevated ACTH. Mild defects in mineralocorticoid release
or salt balance can occur in a proportion of individuals with triple A syndrome,
and children with severe disruptive changes in the ACTH receptor may also have
mild hyponatraemia for a variety of reasons. As these children can be misdiag-
nosed as having a salt-losing form of primary adrenal hypoplasia, making the
correct diagnosis has important implications for long-term management and
counselling [10].
Primary Adrenal Hypoplasia
Adrenal hypoplasia congenita (AHC), also known as congenital adrenal

hypoplasia, is a disorder of adrenal development resulting in primary adrenal
insufficiency. This condition can occur with several different inheritance pat-
terns and with a variety of associated or syndromic features.
X-linked Adrenal Hypoplasia

X-linked AHC is caused by mutations in the nuclear receptor DAX1
(NR0B1). This condition is the most prevalent form of primary adrenal
hypoplasia reported to date [11, 12]. X-linked AHC was probably first
described by the pathologist Sikl in 1948 in a male infant who died at 33 days of
age. The boy was noted to have ‘coal-black’ pigmentation and to have small
adrenal glands at post mortem. These adrenals contained ‘cytomegalic’ cells
typical of fetal adrenal tissue, which led to this condition being termed
‘cytomegalic adrenal hypoplasia’. In the 1960s the X-linked pattern of inheri-
tance of AHC became apparent and, soon afterwards, an association with
hypogonadotropic hypogonadism was described as boys who received steroid
treatment survived to adolescence but did not enter puberty.
The gene for X-linked AHC, DAX1 (NR0B1) was identified in 1994 and
localized to the short arm of the X chromosome (Xp21.3) [13, 14]. This discovery
was aided by many reports of X-linked AHC as part of a contiguous gene deletion
syndrome involving the loci for glycerol kinase deficiency, ornithine transcar-
bamylase deficiency and Duchenne muscular dystrophy centromeric to DAX1
(NR0B1), as well as rare cases of X-linked AHC together with an X-linked form
of developmental delay due to deletion of a telomeric gene, IL1RAPL1.
DAX1 is an ‘orphan’ member of the nuclear receptor superfamily. The car-
boxyl-terminal region of DAX1 has homology to the ligand-binding domain of
nuclear receptors but no naturally occurring ligand has been identified (fig. 3).
The amino-terminus of DAX1 contains an atypical repeat motif structure with
multiple LXXLL domains involved in nuclear receptor/co-factor interaction.
DAX1 is expressed in the developing adrenal gland, gonad and gonadotropes,
consistent with its role in the development of these tissues.
The clinical syndrome associated with DAX1 mutations includes (1) pri-
mary adrenal insufficiency; (2) hypogonadotropic hypogonadism, and (3) a
likely primary defect in spermatogenesis. Boys tend to present with salt-losing
adrenal failure in the first 2 months of life (60–70%) or more insidiously with
adrenal failure throughout childhood (30–40%) [12, 15]. Isolated mineralocor-
ticoid deficiency may be the presenting feature in some cases, but glucocorti-
coid deficiency usually develops with time [16]. Absent or arrested puberty due
to a combined hypothalamic and pituitary defect is seen in adolescence,
although a number of cases of limited testicular enlargement or signs of prema-
ture sexual maturation in childhood have been reported [17, 18]. The extent of
the spermatogenic defect in humans is still unclear. Insight into the intrinsic tes-
ticular defect arose from work in mice following targeted deletion of the gene
1 putative LBD 470
E38X W171X C255X Q395X

W171X Y399X
Q404X
151-152delAG 702delC
405delT 793-794insGC
501delA 986-987delGGinsA
501delA 1132delT
510-525del16nt 1251-1252insGATG
a 543delA 1301delT
L262Q L295P C368W

L262Q L297P E377K
R267P L297P E377K R425G
d269V A300V Y380D R425T
d269V A300V L381H R425T
L278P A300P L381V d430N
V287G A300P K382N I439S
W291C V385G N440I
1 putative LBD 470
b C200W L466R
Fig. 3. DAX1 (NR0B1) is an orphan nuclear receptor with an atypical amino-terminal

repeat motif structure and a carboxyl-terminal region that resembles a ligand-binding
domain. a Selection of frameshift (open arrows) and nonsense (filled arrows) mutations in
individuals with X-linked AHC. Those mutations identified in our laboratories are indicated.
b Missense mutations associated with this condition. Mutations Y380D and I439S are asso-
ciated with late-onset forms of X-linked AHC. (Modified with permission from Lin et al.
[12]; copyright 2006, The Endocrine Society).
encoding Dax1 [19]. Consistent with these findings, spontaneous fertility is

extremely rare in men with X-linked AHC, and the results of using standard
gonadotropin regimen to try to induce fertility are so far disappointing [20]. It
is not yet known whether techniques such as intracytoplasmic sperm injection
will be successful.
Although initial reports of X-linked AHC were biased to cases of contigu-
ous gene deletion syndrome, the identification of DAX1 as the gene responsible
for this condition has furthered our understanding of X-linked AHC consider-
ably. To date, more than 100 different mutations have been reported in DAX1 in
more than 200 individuals and families with this condition (fig. 3). Analysis of
37 cases of X-linked AHC from our centres over the past 10 years has shown
isolated DAX1 gene deletions in 8 cases (22%), contiguous gene deletions in 2
cases (5%) and point mutations in the rest [nonsense, 7 (19%); frameshift, 12
(32%); missense, 8 (22%)] (fig. 3) [12]. Nonsense and frameshift mutations are
located throughout the DAX1 gene and loss of just the carboxyl-terminal region
of the protein (containing the AF2 domain) is sufficient for complete loss of
protein function. Missense mutations tend to cluster within certain regions of
the ligand-like binding domain, but rare amino-terminal missense mutations
have now been reported (fig. 3) [21].
Recently, an adult-onset form of X-linked AHC has been described in men
who presented between 20 and 30 years of age with mild primary adrenal insuf-
ficiency or partial hypogonadism. In some cases, missense mutations have been
identified (I439S, Y380D) that retain limited DAX1 function as a transcrip-
tional repressor (fig. 3) [22, 23]. In other individuals, nonsense mutations at the
extreme amino-terminal region of the protein are associated with the translation
of an alternate in-frame DAX1 isoform from methionine at codon 83 [24]. This
amino-terminally truncated protein retains one functional LXXLL domain and
has partial activity, consistent with the milder phenotype seen in the patient.
Genetic analysis of DAX1 for individuals with X-linked AHC is now avail-
able as a clinical test. In our experience, DAX1 mutations were found in all indi-
viduals with primary adrenal failure, abnormal puberty and a family history of
adrenal disease in males (8/8, 100%) [12]. In addition, DAX1 mutations were
found in approximately 40% of a cohort of prepubertal boys with no family his-
tory of note, in whom other diagnoses such as congenital adrenal hyperplasia
(e.g. 21-hydroxylase deficiency) and metabolic defects (e.g. adrenoleukodystro-
phy) had been excluded. Making the genetic diagnosis of a DAX1 mutation has
significant implications for planning future management and the likely need for
puberty induction. Furthermore, female carriers of DAX1 mutations or deletions
are unaffected, but half of their sons will be affected. Close monitoring and
genetic counselling can help to prevent life-threatening adrenal crises in other
family members or future pregnancies [25]. Close liaison between clinical
geneticists and endocrinologists is needed to identify and counsel those individ-
uals at potential risk.
Autosomal Adrenal Hypoplasia

Autosomal forms of adrenal hypoplasia exist but the underlying basis for
these conditions is poorly understood. Heterozygous or homozygous mutations
in the nuclear receptor steroidogenic factor-1 (SF1, NR5A1) have been reported
in 46,XY phenotypic females with either spontaneous or recessively inherited
primary adrenal failure, and a heterozygous SF1 mutation has been described in
a 46,XX girl with adrenal dysfunction [26]. However, SF1 mutations have not
been found in phenotypic males with adrenal hypoplasia [12]. It is likely that
other autosomal genes involved in adrenal development exist, which could
cause adrenal hypoplasia when disrupted.
Syndromic Forms of Adrenal Hypoplasia

Primary adrenal failure has been reported rarely with syndromes such as
Pena-Shokeir syndrome type I, pseudotrisomy 13 and Meckel syndrome [1].
Primary adrenal hypoplasia also appears to be part of the IMAGe syndrome
(intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia,
genitourinary anomalies) [27]. A number of individuals and families with this
syndrome have now been reported, but the underlying aetiology of this condi-
tion remains unknown [28]. It is hoped that a better understanding of human
adrenal development and analysis of pedigrees where adrenal hypoplasia is a
feature will provide candidate genes for syndromes such as IMAGe, as well as
for the remaining individuals with adrenal hypoplasia in whom the aetiology is
currently unknown.
Acknowledgements
J.C.A. holds a Wellcome Trust Senior Research Fellowship in Clinical Science (079666).
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Dr. John C. Achermann

Developmental Endocrinology Research Group
Clinical and Molecular Genetics Unit
UCL Institute of Child Health, University College London
30 Guilford Street, London WC1N 1EH (UK)
Tel. ⫹44 207 905 2887, Fax ⫹44 207 404 6191, E-Mail j.achermann@ich.ucl.ac.uk
Early Management and Gender Assignment

in Disorders of Sexual Differentiation
Hughes, I.A.
Department of Paediatrics, Addenbrooke’s Hospital, University of Cambridge,
Cambridge, UK
Abstract
Ambiguous genitalia, sine qua non, defines a congenital endocrinopathy. The problem
is immediately apparent at birth and prompts an urgent response in management which
requires input from a multi-disciplinary team of experts. Assignment to a male or female
gender is instantaneous when a baby is born. That this may not be possible in rare instances
is immensely distressing to affected families. Indeed, abnormalities of the external genitalia
sufficient to warrant genetic and endocrine studies occur in 1 in 4,500 births. There has been
considerable progress in improved diagnosis and early management in recent decades, par-
ticularly with respect to congenital adrenal hyperplasia, the commonest cause of ambiguous
genitalia of the newborn. For the purposes of this chapter, attention is focussed on the new-
born with ambiguous genitalia and subsequent management in infancy and early childhood.
Ambiguous genitalia, sine qua non, defines a congenital endocrinopathy.

The problem is immediately apparent at birth and prompts an urgent response
in management which requires input from a multi-disciplinary team of experts.
Assignment to a male or female gender is instantaneous when a baby is born.
That this may not be possible in rare instances is immensely distressing to
affected families. Indeed, abnormalities of the external genitalia sufficient to
warrant genetic and endocrine studies occur in 1 in 4,500 births [1]. There has
been considerable progress in improved diagnosis and early management in
recent decades, particularly with respect to congenital adrenal hyperplasia, the
commonest cause of ambiguous genitalia of the newborn [2]. Nevertheless,
there remains considerable dissatisfaction with the overall management of the
newborn with ambiguous genitalia, including decisions on gender assignment,
the need for and timing of surgical intervention, aspects of disclosure and
consent, and the lack of evidence from outcome studies. Much of the demand
for improving care for this congenital endocrinopathy has come from affected
families and patient advocacy groups. A Clinical Guidelines and Handbook for
Parents is available on the Internet [3]. The professional societies representing
paediatric endocrinology responded by organizing a consensus meeting on the
management of intersex. This resulted in a consensus statement encompassing
many aspects of management extending from birth to adulthood [4, 5]. For the
purposes of this chapter, attention is focussed on the newborn with ambiguous
genitalia and subsequent management in infancy and early childhood.
Aims of the Consensus Meeting
A consensus is a general agreement or an accord, recognizing that the

objections of a minority of participants in the process should be resolved as far
as is practicable. It is about ‘splitting the difference’ and reaching a solution
which is workable and represents positive developments. In the case of man-
agement of intersex, there was recognition that a precise diagnosis was often
uncertain, early management was confusing and compounded by the use of
complex nomenclature, dissatisfaction with health professionals was rife and
there was a profound lack of knowledge of what happens to intersex individuals
in adulthood. The term, ‘intersex’ was highlighted as a problem which needed
addressing for seeking an alternative choice, especially as many affected fami-
lies regarded the current term as pejorative. The decision to address this ques-
tion spawned a subsequent major revamp of a swathe of nomenclature relating
to this congenital endocrinopathy.
The broad aims in formulating a consensus were 4-fold: (1) to improve the
management of individuals and their families who have an intersex-related
problem; (2) to make recommendations that were evidence-based as far as pos-
sible; (3) to identify areas of diagnosis and management that lacked sufficient
knowledge, and (4) to recommend topics that warranted further research.
Structure of the Consensus
The European Society for Paediatric Endocrinology (ESPE) and the

Lawson Wilkins Pediatric Endocrine Society (LWPES) joined forces to select a
faculty of experts and formulate a syllabus of study. This required the participa-
tion of more than 50 professionals drawn from genetics, endocrinology, surgery
and psychology to enable a sufficiently robust and comprehensive evaluation to
be achieved. Furthermore, representatives of patient advocacy groups also par-
Hughes 48
WT1 Lim1
Urogenital SF1 Emx2
ridge Lhx9
M33
Igf1/Irr/Ir
Bipotential WT1KTS Gata4/Fog2

gonad SF1 Fgf9/Fgfr2
SRY Pod1
SOX9 Pdgfr␣
Anti-testis DMRT1 Vanin-1
46,XX 46,XY
RSPO1 DHH Tescalcin
WNT4 ATRX Dax1
DAX1 TSPYL1 Sox3
Fst Testis SF1 hCG (in utero)
Bmp2 DHH LH (perinatal)
Ovary ARX
CXorf6
Foxl2 ⫹
GDF9 INSL3
Sertoli cells Leydig cells GREAT
Connexin37
BMP15 AMH/MISRII Testosterone
FRAXA SOX9, SF1 DHT
Müllerian Male sex Testicular

regression differentiation descent
Fig. 1. Summary of genes involved in fetal sex development. Genes denoted in lower
case refer to sex reversal observed in the mouse only, when disrupted.
ticipated. The following 6 topic areas were addressed in the formulation of the
consensus statement: (1) genetics and nomenclature; (2) brain programming by
hormones and genes; (3) investigation and medical management; (4) surgical
management; (5) psychosexual management, and (6) outcome in adolescence
and adulthood. A few of these topics are discussed further.
Genetics and Nomenclature
The dimorphic pathway of sex determination from an initial bipotential

gonad and the subsequent sex differentiation of the male/female phenotype is
now remarkably well established with a plethora of genes and hormones identi-
fied in the process (fig. 1). Nevertheless, many genes required for testis or
ovary determination remain to be identified. It is anticipated that application
of techniques such as microarray, comparative genomic hybridization and
tissue-selective disruption of candidate genes will enable progress to be made.
Disorders of Sexual Differentiation 49

Many of the genes depicted in figure 1 have only been demonstrated to lead to
a disorder of sex development in the mouse when disrupted, whereas no syn-
dromes in humans are yet described as a result of mutations in their homo-
logues. For example, disruption of Lim1 and Emx2 in the mouse results in
failure of urogenital ridge development but no equivalent syndrome has been
described in humans. In contrast, WT1 and SF1 disruption leads to profound
sex reversal syndromes in both species.
It has traditionally been considered that ovarian determination occurs only
in the absence of testis-determining genes. Reference to figure 1 indicates that
a number of genes can act as anti-testis genes in a dosage-sensitive manner;
examples include DAX1 and WNT4. Furthermore, genes such as FOXL2 and
RSPO1 appear to be involved directly in ovarian determination. RSPO1 was
identified on chromosome region 1p34 by studying a large consanguineous
Italian family that included four 46,XX individuals who were SRY negative and
had complete male sex reversal [6]. Associated components of this autosomal
recessive sex reversal syndrome included palmoplantar hyperkeratosis and a
predisposition to squamous cell skin carcinoma. Affected members of the fam-
ily had a homozygous nonsense mutation in the RSPO1 gene. This gene
encodes for one of the family of R-spondins that function as growth factors that
interact with ␤-catenin and, via ␤-catenin stabilization, may also synergize with
WNT proteins. The interaction is thus critical for early genital development and
ovarian determination. The identification of the RSPO1 mutation uniquely
demonstrates a cause for complete sex reversal in a 46,XX individual who is
SRY negative. The gonads functioned as testes in somatic aspects as müllerian
structures were inhibited by Sertoli cell production of anti-müllerian hormone,
while interstitial cells synthesized androgens to masculinize the internal and
external genitalia. The absence of Y-specific spermatogenesis genes and the
presence of 2 X chromosomes explains the infertility in affected individuals.
RSPO1 is not required for testis determination based on the observation of nor-
mal male development and fertility in a 46,XY member of the family who car-
ried the homozygous nonsense RSPO1 mutation.
The need to address the nomenclature used by health professionals
involved in the management of individuals with disorders of sex differentia-
tion was 2-fold. Concerns were expressed by patient advocacy groups that
terms such as ‘intersex’ and ‘pseudohermaphroditism’ are confusing and even
perceived as potentially pejorative by patients. Furthermore, the Handbook for
Parents [3], which had been produced on management, included a suggestion
for alternative terminology. Consequently, it was a logical step for the con-
sensus faculty to embrace the ideas from this preparatory work and for-
mulate an alternative term for ‘intersex’. The umbrella term, ‘disorder of sex
development’ (DSD), was proposed and defined as ‘a congenital condition in
Hughes 50
Table 1. A new nomenclature
Previous Proposed
Intersex DSD
Male pseudohermaphrodite
Undervirilization of an XY male 46,XY DSD
Undermasculinization of an XY male
Female pseudohermaphrodite
Overvirilization of an XX female 46,XX DSD
Masculinization of an XX female
True hermaphrodite ovotesticular DSD
XX male or XX sex reversal 46,XX testicular DSD
XY sex reversal 46,XY complete gonadal dysgenesis
which development of chromosomal, gonadal, or anatomical sex is atypical’.

Based on this broad definition, it is possible to include a range of genital
abnormalities from ambiguous genitalia to micropenis, cryptorchidism and
congenital malformation syndromes such as cloacal exstrophy. The inclusion
of congenital in the definition of DSD excludes DSD such as precocious or
delayed puberty.
A karyotype determination is a prerequisite start for the investigation of
any DSD, so this knowledge underpins a new overall nomenclature outlined in
table 1. Thus, female pseudohermaphroditism is replaced by 46,XX DSD and
the male pseudohermaphrodite becomes 46,XY DSD. Also replaced is true her-
maphroditism by the term ‘ovotesticular DSD’, recognizing that this condition
is defined histologically by confirming the presence of testicular and follicle-
containing ovarian tissue in the same individual. No karyotype prefix is listed
as the chromosomes can be 46,XX (the most frequent), 46,XY or chimeric
46,XX/46,XY. ‘Sex reversal’ is also a term which is out of favour with patients,
even though this nomenclature is in standard usage by basic geneticists. Table 2
contains a proposed classification system for causes of DSD which includes the
conventional sex chromosomal anomaly category as 1 of the 3 sub-heads. It is
not an exhaustive list of causes but it is simple, logical and workable in clinical
practice. Under both XX, DSD and XY,DSD sub-groups, there are categories
labelled as other (miscellaneous). Authors are now beginning to include this
new nomenclature and classification system in their revised chapters on DSD in
standard endocrine texts.

Table 2. A proposed classification for DSD
Sex chromosome DSD 46,XY DSD 46,XX DSD
A: 47,XXY (Klinefelter A: Disorders of gonadal (testicular) A: Disorders of gonadal (ovarian)

syndrome and variants) development development
B: 45,X (Turner 1. complete or partial gonadal 1. Gonadal dysgenesis
syndrome and variants) dysgenesis (e.g. SRY, SOX9, SF1, 2. Ovotesticular DSD
C: 45,X/46,XY (mixed WT1, DHH) 3. Testicular DSD (e.g. SRY⫹, dup
gonadal dysgenesis) 2. Ovotesticular DSD SOX9, RSP01)
D: 46,XX/46,XY (chimerism) 3. Testis regression
B: Disorders in androgen B: Androgen excess
synthesis or action 1. Fetal
1. Disorders of androgen synthesis 3␤-hydroxysteroid dehydrogenase
LH receptor mutations 2 HSD3B2
Smith-Lemli-Opitz syndrome 21-hydroxylase (CYP21A2)
Steroidogenic acute regulatory P450 oxidoreductase (POR)
protein mutations 11␤-hydroxylase (CYP11B1)
Cholesterol side chain cleavage Glucocorticoid receptor mutations
(CYP11A1) 3␤-hydroxysteroid 2. Fetoplacental
dehydrogenase 2 (HSD3B2) Aromatase (CYP19) deficiency
17␣ hydroxylase/17,20-lyase Oxidoreductase (POR) deficiency
(CYP17) 3. Maternal
P450 oxidoreductase (POR) Maternal virilizing tumours
17␤-hydroxysteroid (e.g. luteomas)
dehydrogenase (HSD17B3) Androgenic drugs
5␣-reductase 2 (SRD5A2)
2. Disorders of androgen action
Androgen insensitivity syndrome
Drugs and environmental
modulators
C: Other C: Other
1. Syndromic associations of male 1. Syndromic associations (e.g.
genital development (e.g. cloacal cloacal anomalies)
anomalies, Robinow, Aarskog, 2. Müllerian agenesis/hypoplasia
hand-foot-genital, popliteal (e.g. MURCS)
pterygium) 3. Uterine abnormalities
2. Persistent müllerian duct (e.g. MODY5)
syndrome 4. Vaginal atresia (e.g. McKusick-
3. Vanishing testis syndrome Kaufman)
4. Isolated hypospadias (CXorf6) 5. Labial adhesions
5. Congenital hypogonadotrophic
hypogonadism
6. Cryptorchidism (INSL3,
GREAT)
7. Environmental influences
Hughes 52
Early Management
It is axiomatic that initial management of DSD is dependent on establish-

ing a diagnosis, although recognizing this may be difficult in many cases of
XY,DSD. Indeed in this category of DSD, a firm diagnosis may not be possible
in more than half the cases [7]. Nevertheless, it is recommended that all new-
borns with ambiguous genitalia should be assigned a gender but only after
expert evaluation has been performed at a centre with an experienced multi-
disciplinary team. Discussion with the parents at this early critical stage must
be open and their participation in decision-making encouraged.
The literature is replete with numerous protocols of investigation of
ambiguous genitalia of the newborn [8, 9]. Consequently, no single investiga-
tive protocol can be recommended to cover all circumstances. In newborns,
first-line testing will include FISH analysis with X- and Y-specific DNA probes
and a full karyotype, abdominal-pelvic ultrasound and serum measurement of
170H-progesterone, testosterone, gonadotrophins, anti-müllerian hormone,
electrolytes and urinanalysis. This will quickly delineate the category of DSD.
Fluorescent in situ hybridization technology has been a major boost to the early
evaluation of newborn DSD, with a preliminary indication of the chromosomal
sex available within hours of birth.
Second-line investigations are generally confined to XY and XO/XY DSD,
where the hCG stimulation test is pivotal to determine the presence of testes
and whether they are capable of producing age-related normal concentrations of
androgens. A concomitant ACTH stimulation test may also be required if a biosyn-
thetic defect shared by both the adrenals and gonads (e.g., 3␤-hydroxysteroid
dehydrogenase deficiency or a StAR protein defect) is suspected. A number of
different protocols are used for the hCG stimulation test and no consensus has
emerged either for the dose and frequency of injections or at what age the test
should be undertaken in infants. The author uses 1,500 units daily for 3 days
with post-hCG serum samples collected 24 h after the last injection. A pro-
longed stimulation test using twice-weekly injections for 3 weeks may also be
employed. There is certainly no unanimity on when to perform the test while
investigating a newborn with ambiguous genitalia. While it is tempting to pro-
ceed with the test within days of birth, this may give spurious results due to the
effects of placental and fetal adrenal clearance of steroids and lack of assay
specificity if standard unextracted assays are used. Ideally, it makes sense to
delay the tests until about 4 weeks of age and also take advantage of the increased
Leydig cell activity at this age.
More specialized investigations include urinary steroid analysis by spe-
cific gas chromatography-mass spectrometry techniques and DNA analysis of
key genes in the pathway of testis determination and function (see fig. 1).

Table 3. Clinical modes of presentation
for DSD Early Ambiguous genitalia of newborn
Apparent ‘male’ with absent testes
Inguinal swellings in ‘female’ infant
Isolated severe hypospadias
Isolated clitoromegaly
Late Virilized female at puberty
Delayed female puberty
Primary amenorrhea
Gynaecomastia
Male infertility
Urinary steroid analysis is particularly valuable in 46,XY disorders such as 5␣-

reductase deficiency and P450-oxidoreductase deficiency but is curiously not
informative in the neonatal period for the androgen biosynthetic disorder, 17␤-
hydroxysteroid dehydrogenase deficiency. Later, it may be necessary to obtain
gonadal biopsy material for histological examination to be definitive about
diagnoses such as gonadal dysgenesis and ovotesticular DSD. Table 3 summa-
rizes the modes of presentation of DSD, not only in the newborn period but also
later at puberty. Some are not obvious; the apparent male with bilateral cryp-
torchidism may be a fully masculinized female with congenital adrenal hyper-
plasia. The female infant with bilateral inguinal swellings may have herniated
testes and a diagnosis of complete androgen insensitivity syndrome or 17␤-
hydroxysteroid dehydrogenase deficiency. The latter disorder classically pre-
sents at puberty with virilization of a child raised female, also to be seen in
5␣-reductase deficiency. A girl with delayed onset of puberty may have XY
gonadal dysgenesis or 17␣-hydroxylase deficiency. If breast development is
normal but menses are delayed, this may be due to complete androgen insensi-
tivity syndrome.
Psychological Management
The medical and surgical aspects of early management are clearly impor-
tant but the provision of psychological counselling is also paramount at this
stage. What is stated at the outset becomes largely imprinted in the minds of
affected families. Gender assignment is made after a diagnosis has been estab-
lished (if possible), issues of medical and surgical management discussed and
information shared about what is known about outcome in DSDs such as
Hughes 54
5␣-reductase deficiency and partial androgen insensitivity syndrome, if raised
female or male. Health professionals must be clear about the distinction
between gender role and gender identity and the difference in the effects of pre-
natal androgen exposure on these parameters [10, 11]. Definitive statements
can be made that more than 90% of 46,XX infants with congenital adrenal
hyperplasia and 100% of 46,XY individuals with complete androgen insensitiv-
ity syndrome who are female gender assigned continue to identify as females in
later childhood and adulthood. The parity is less clear-cut when the DSD is a
biosynthetic defect and virilization of a child raised female occurs at puberty.
Nevertheless, about 60% of individuals with 5␣-reductase deficiency who are
female gender assigned in infancy and virilize at puberty identify and live
thereafter as male [12]. It must also be cautioned that 25% of individuals with
XY DSD, be it due to partial gonadal dysgenesis, androgen biosynthetic defects
or partial androgen insensitivity syndrome, are later dissatisfied with their gen-
der assignment. This applies whether they are raised female or male [13].
The consensus firmly recommends that a clinical psychologist with exper-
tise in DSD should be an integral part of the management team. It is recognized
that many centres do not yet have such experts employed but the recommenda-
tion may carry benefit when making the case for additional resources. The first
encounter with a family faced with a newborn of indeterminate sex is poten-
tially a recipe for negating the maxim, primum non nocere, recognizing that an
act of good intention may have unwanted consequences. Thus, the use of inap-
propriate terms, information overload or contradictory information delivered by
different members of the team may all conspire to cause harm and misunder-
standing long into the future. Here, the clinical psychologist has a key role in
formulating what information is to be provided, how it should be conveyed and
assess how families react to the process. Various psychological instruments may
be used during detailed counselling sessions to determine how families are best
able to cope with the emotional trauma of having a child with DSD.
Counselling will also need to take cognizance of cultural and religious factors.
The ease of accessing information via the Internet adds another dimension to
the importance of ensuring a sound base of knowledge and understanding at
this early stage which will pay beneficial dividends as the child develops into
adulthood.
Surgical Management
The surgical approach to early DSD management has traditionally

involved performing early reconstructive surgery of the external genitalia to
make their appearance consonant with the chosen gender assignment. It is now

accepted that such haste is not always necessary, although the proponents of a
complete moratorium on any genital surgery during infancy and childhood are
not generally supported by the consensus [14]. There has been a change in prac-
tice regarding the need for clitoral reduction in females with congenital adrenal
hyperplasia, for example. Only severe virilization (as quantified by Prader
stages III-V) should be considered for surgery, which may also be performed at
this stage with concomitant repair of the common urogenital sinus. Studies in
adult females who had surgery in infancy indicate that impaired clitoral sensa-
tion is a common outcome [15]. Such information should form part of the dis-
cussion when planning for any surgery in childhood, with an emphasis placed
on functional outcome, in contrast to only the cosmetic appearance of the exter-
nal genitalia.
Only surgeons who are expert in the complex procedures of genital recon-
struction surgery and aware of the longer-term outcome should be involved in
early management of DSD. It is the surgeon who must be responsible for pro-
viding an outline of surgical sequences and the various consequences which
may arise in later childhood and early adulthood. The discussion will need to
encompass the dimorphic options of gender assignment and more explicit
information subsequently provided consonant with the chosen sex of rearing.
Others issues which also need addressing later include further surgical proce-
dures around the time of puberty, the risk of gonadal tumours and options for
gonadectomy, sexual function and the potential for fertility.
Conclusions
Concentrating the minds of numerous experts worldwide on the optimal

management of individuals with DSD has galvanized collective action across a
number of specialist boundaries. The importance of teamwork, specialist cen-
tres, early gender assignment, more caution with surgical intervention and a
dependence on expert psychosexual counselling have emerged as positive steps
forward. An alternative nomenclature and DSD classification has been pro-
posed and already there is wide acceptance of the proposals based on terms
used in original publications and chapters in textbooks. Considerable efforts are
needed to delineate further the genetic control of sex determination and factors
that influence the development of gender characteristics before and after birth.
Above all, longer-term outcome data are needed, especially in relation to chang-
ing surgical practice and in XY DSD. These goals can best be achieved by spe-
cialist centres pooling their experience on both national and international
fronts. The support of patient advocacy groups is recognized to be essential to
achieve these aims.
Hughes 56
References
1 Achermann JC, Hughes IA: Disorders of sex differentiation; in Larsen PR, Kronenbery HM,
Melmed S, Polonsky KS (eds): Williams Textbook of Endocrinology. Philadelphia, Saunders,
2007, in press.
2 Clayton PE, Miller WL, Oberfield SE, Ritzen EM, Speiser PW; ESPE/LWPES CAH Working
Group: Consensus statement on 21-hydroxylase deficiency from the Lawson Wilkins Pediatric
Endocrine Society and the European Society for Paediatric Endocrinology. Horm Res 2002;58:
188–195.
3 Consortium on the Management of Disorders of Sex Development: Clinical Guidelines and
Handbook for Parents. 2006. www.dsdguidelines.org.
4 Hughes IA, Houk C, Ahmed SF, Lee PA; LWPES Consensus Group; ESPE Consensus Group:
Consensus statement on management of intersex disorders. Arch Dis Child 2006;91:554–563.
5 Houk CP, Hughes IA, Ahmed SF, Lee PA; Writing Committee for the International Intersex
Consensus Conference Participants: Summary of consensus statement on intersex disorders and
their management. International Intersex Consensus Conference. Pediatrics 2006;118:753–757.
6 Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S, Guerra L, Schedl A,
Camerino G: R-spondin 1 is essential in sex determination, skin differentiation and malignancy.
Nat Genet 2006;38:1304–1309.
7 Thyen U, Lanz K, Holterhus PM, Hiort O: Epidemiology and initial management of ambiguous
genitalia at birth in Germany. Horm Res 2006;66:195–203.
8 Ogilvy-Stuart AL, Brain CE: Early assessment of ambiguous genitalia. Arch Dis Child 2004;89:
401–407.
9 Ogilvy-Stuart AL, Brain CE: Practical Neonatal Endocrinology. Cambridge, Cambridge University
Press, 2006, pp 238.
10 Cohen-Bendahan CC, van de Beek C, Berenbaum SA: Prenatal sex hormone effects on child and
adult sex-typed behaviour: methods and findings. Neurosci Biobehav Rev 2005;9:353–384.
11 Hines M: Prenatal testosterone and gender-related behaviour. Eur J Endocrinol 2006;155(suppl 1):
S115–S121.
12 Cohen-Kettenis PT: Gender change in 46,XY persons with 5-alpha-reductase-2 deficiency and
17-beta-hydroxysteroid dehydrogenase-3 deficiency. Arch Sex Behav 2005;34:399–410.
13 Migeon CJ, Wisniewski AB, Gearhart JP, Meyer-Bahlburg HF, Rock JA, Brown TR, Casella SJ,
Maret A, Ngai KM, Money J, Berkovitz GD: Ambiguous genitalia with perineoscrotal hypospa-
dias in 46,XY individuals: long-term medical, surgical, and psychosexual outcome. Pediatrics
2002;110:e31.
14 Greenberg JA: International legal developments protecting the autonomy rights of sexual minori-
ties: who should determine the appropriate treatment for an intersex infant? in Sytsma SE (ed):
Ethics and Intersex. Dordrecht, Springer, 2006, vol 29, pp 87–101.
15 Crouch NS, Creighton SM: Long-term functional outcomes of female genital reconstruction in
childhood. BJU Int 2007;Apr 8 (Epub).
Prof. I.A. Hughes

Department of Paediatrics, University of Cambridge
Box 116, Level 8, Addenbrooke’s Hospital
Hills Road
Cambridge CB2 2QQ (UK)
Tel. ⫹44 1223 336885, Fax ⫹44 1223 336996, E-Mail iahl000@cam.ac.uk

Prenatal and Early Postnatal Treatment

of Congenital Adrenal Hyperplasia
Lucia Ghizzoni, Silvia Cesari, Giulia Cremonini, Lisa Melandri
Dipartimento dell’Età Evolutiva, University of Parma, Parma, Italy
Abstract
Congenital adrenal hyperplasia is a group of monogenic autosomal recessive disorders
due to an enzyme deficiency in steroid biosynthesis. The most frequent form of congenital
adrenal hyperplasia is 21-hydroxylase (21-OH) deficiency, which in its severe form can
cause ambiguous genitalia in the female patient. Recent advances in molecular genetic analy-
sis allow for prenatal diagnosis and treatment of at-risk fetuses. The objective of prenatal
diagnosis and treatment of 21-OH deficiency is the prevention of prenatal virilization in
affected female infants, reducing the risks of sex misassignment and gender confusion, and
the need for corrective genital surgery. Prenatal treatment of 21-OH deficiency is effective in
reducing genital ambiguity, and short-term outcome studies of children exposed to dexam-
ethasone in utero indicate no significant adverse effects. However, more long-term studies of
treated versus untreated pregnancies are warranted to monitor the safety of treatment and
enhance our understanding of the effects of prenatal steroid exposure to the human brain. In
the first year of life, optimization of medical treatment in salt-wasting patients is achieved by
combining the lowest dose of glucocorticoid able to suppress androgen secretion with the
normalization of sodium balance by giving appropriate sodium chloride supplementation.
Congenital adrenal hyperplasia (CAH) is a group of inherited disorders in

steroid biosynthesis, resulting from the deficiency of 1 of the 5 enzymes that
are involved in the conversion of cholesterol to cortisol. We discuss here 21-
hydroxylase (21-OH) deficiency, accounting for more than 90% of cases. CAH
caused by 21-OH deficiency is characterized by cortisol deficiency, with or
without aldosterone deficiency, and androgen excess. The clinical phenotype is
classified as classic, which is the severe form, and nonclassic, which is the mild
or late-onset form. Classic CAH is subclassified as salt-wasting or non-salt-
wasting (simple-virilizing), reflecting the degree of aldosterone deficiency [1].
The incidence of classic 21-OH deficiency, evaluated from almost 6.5 mil-
lion newborns screened in 13 world countries (USA, Canada, Brazil, France, Italy,
UK, Switzerland, Sweden, Germany, Portugal, Spain, Japan and New Zealand) is
estimated between 1 in 13,000 and 1 in 15,000 live births [2]. Therefore, the
carrier frequency of classic 21-OH deficiency is about 1 in 60 individuals. Salt-
wasting 21-OH deficiency accounts for 67% of the cases and non-salt-wasting for
33% of the cases reported [3]. Neonatal screening does not accurately detect
nonclassic CAH, therefore data on the incidence of the milder form of the disor-
der are lacking. However, nonclassic CAH is estimated to be more common than
classic CAH, with a prevalence of 1 in 1,000 in the white population [4].
Clinical consequences of 21-OH deficiency arise primarily from overpro-
duction and accumulation of precursors proximal to the blocked enzymatic
step. These precursors are shunted into the androgen biosynthetic pathway, pro-
ducing virilization in the female fetus or infant, and rapid postnatal growth with
accelerated skeletal maturation, precocious puberty, and short adult stature in
both males and females.
Recent advances in molecular genetic analysis allow for prenatal diagnosis
and treatment of at-risk fetuses. While there is extensive and compelling data
pointing to the benefit of prenatal treatment, controversy remains regarding the
safety of prenatal intervention. Other controversial issues include the optimal
regimen for postnatal treatment, particularly in the critical early postnatal years
of rapid growth. We discuss here prenatal diagnosis and treatment and early
postnatal treatment.
Prenatal Diagnosis
The aim of prenatal diagnosis is to allow prenatal treatment in order to

prevent virilization of external genitalia in affected female infants, thus reduc-
ing the need for corrective genital surgery, and the risk of sex misassignment
and gender confusion. When considering prenatal diagnosis and treatment, the
first step is to offer genetic counseling to the families with an index case for
classic 21-OH deficiency. The role of genetic counseling is to provide to the
parents all available information on the clinical forms of the disease, their con-
sequences and the risk of transmitting the disease to a potential fetus. An accu-
rate prenatal prediction of the clinical form of the disease in a couple at risk
requires the correct molecular genetic analysis of the index case and molecular
genetic analysis and complete hormonal profiling of the parents to be done in
advance of the occurrence of an at-risk pregnancy. This is necessary to deter-
mine whether the family is informative for a diagnosis of 21-OH deficiency in
the fetus.
Prenatal and Early Postnatal Treatment of CAH 59

Several approaches to prenatal identification of affected fetuses have been
used. Initially prenatal diagnosis of 21-OH deficiency was based on second-
trimester amniotic fluid 17-hydroxyprogesterone measurements and HLA hap-
lotyping [5–8]. Despite the accuracy of this technique, the amniocentesis is
performed too late in gestation and does not allow the prevention of the viril-
ization of a female fetus. Therefore, such an approach is currently used only
when molecular diagnosis in unavailable. Recent advances in genotyping of the
CYP21 gene have made molecular genetic studies of fetal DNA the ideal diag-
nostic method [9–12]. DNA is extracted from chorionic villus cells obtained
from chorionic villus sampling performed at around 10–11 weeks of gestation.
Pitfalls occur in a small percentage of the patients undergoing prenatal diagno-
sis, mostly due to undetectable mutations [13], allele dropout [14] or maternal
DNA contamination.
A promising technique that can be applied to the prenatal diagnosis of 21-
OH deficiency is the analysis of free fetal DNA in the maternal circulation. This
technique has the advantage of avoiding invasive procedures such as chorionic
villus sampling that carries a known risk of procedure-related miscarriage. Lo
et al. [15] were the first to show the presence of high concentrations of cell-free
fetal DNA in maternal plasma, estimating that in early pregnancy a mean of
3.4% of the total DNA in the maternal plasma was of fetal origin.
In maternal plasma, fetal DNA molecules circulate among a background
of maternal DNA sequences. Since the fetal DNA allele that the fetus inherits
from its mother is genetically identical to the background maternal sequences,
the allele that the fetus inherits from its father is the most readily distinguish-
able fetal-specific sequence in maternal plasma. Noninvasive prenatal assess-
ment of the fetal genotype can be made on the basis of the detection of a
molecular feature that is unique to the paternally inherited fetal allele.
Examples of these unique molecular features include the Y chromosome or the
SRY gene, and paternally inherited mutations or polymorphisms. Alternatively,
noninvasive prenatal assessment may be made on the basis of excluding the
inheritance of a molecular feature that is unique to the paternally inherited
allele. The prenatal assessment of an autosomal recessive disease, such as
CAH, may be obtained by excluding the fetal inheritance of the paternal muta-
tion if the parents carry a different mutation. If the mother and father carry the
same mutation, the detection of a polymorphism that is uniquely linked to the
paternal wild-type allele can exclude the disease [16].
Rijnders et al. [17] were the first to report successful fetal sex determina-
tion from maternal blood in a pregnancy at risk for 21-OH deficiency. The iden-
tification of the male fetus was based on the presence of the SRY gene in the
maternal circulation starting from 13 weeks of gestation. More recently, Bartha
et al. [18] were able to correctly identify the male sex of a fetus at risk of CAH
Ghizzoni/Cesari/Cremonini/Melandri 60
SRY:PCR
negative
SRY:PCR positive Start
(first sample) dexamethasone
(20 ␮g/kg/day)at 5 Repeat weekly
weeks until 10 weeks
SRY:PCR positive Weekly blood
(second sample) sampling from 5 SRY:PCR
weeks on negative
Diagnostic CVS at 11–12 weeks
Stop
dexamethasone
Not affected Affected
Continue
dexamethasone
Fig. 1. Protocol in pregnancies at risk for 21-OH deficiency in the fetus. Modified
from Rijinders et al. [19]. CVS ⫽ Chorionic villus sampling.
as early as 6 weeks of gestation. A new protocol for prenatal diagnosis has

therefore been proposed that recommends beginning SRY testing at 5 weeks of
gestation (fig. 1). Serial testing is performed up until 11 weeks of gestation or
until male DNA is detected in 2 different samples. If the fetus is established to
be a male, chorionic villus sampling is no longer necessary [19]. This approach
has the advantage of reducing by 50% the need of invasive prenatal diagnosis
and also the time of unnecessary exposure to dexamethasone of the unaffected
fetuses. Although reports of sex determination from fetal DNA show great
promise, more confirmation of the successful results is necessary before such a
protocol can be recommended.
Preimplantation Genetic Diagnosis
This is a procedure that identifies genetic abnormalities in preimplantation

embryos prior to embryo transfer so that only unaffected embryos established
from in vitro fertilization are transferred. So far there are only 2 reports
on preimplantation genetic diagnosis of 21-OH deficiency. In the first report,

2 embryos were transferred and resulted in 2 healthy newborns that were
heterozygous carriers for the screened mutation [20]. In the second study, no
pregnancy was achieved but the reanalysis of untransferred embryos confirmed
the results of the initial diagnosis [21]. Although more than 1,000 apparently
healthy unaffected children have been born after preimplantation genetic diag-
nosis and no increase in birth defects has been described so far, further studies
are necessary to establish the safety of this procedure.
Prenatal Treatment: Outcomes and Risks
The rationale of prenatal treatment is to suppress the activity of the fetal

hypothalamic-pituitary-adrenal (HPA) axis to prevent virilization of the exter-
nal genitalia in the affected female fetus. This is accomplished by the adminis-
tration of dexamethasone to the mother throughout pregnancy. Dexamethasone
is used because it binds only minimally to cortisol-binding globulin in the
maternal blood and, unlike hydrocortisone, escapes inactivation by placental
11-hydroxysteroid dehydrogenase enzyme. Thus, dexamethasone can cross the
placenta from the mother to the fetus and suppress ACTH secretion. To prevent
virilization of the genitalia, treatment needs to be started as early as the 8th
week of gestation and ideally as soon as pregnancy is confirmed. In fact, the
critical period for differentiation of the genitalia is 8–12 weeks after concep-
tion when male differentiation occurs if high levels of testosterone (as secreted
by fetal testes) induce 5␣-reductase type 2 and are then converted to dihy-
drotestosterone in genital skin [22]. Later exposure to testosterone increases
growth of the penis or clitoris but does not induce fusion of the labia majora
and minora into scrotum and penile urethra, respectively. Since ACTH was
thought to start regulating adrenal steroidogenesis at 12 weeks of gestation, it
was hypothesized that the early action of dexamethasone on adrenal steroid
secretion was either due to a direct dexamethasone-suppressive effect on the
fetal adrenal or to an ACTH-independent glucocorticoid feedback pathway
responsible for fetal adrenal steroid production [22]. Recently however, it was
demonstrated that the human fetal adrenal cortex synthesizes cortisol much
earlier than previously documented, an effect associated with transient expres-
sion of the orphan nuclear receptor nerve growth factor IB-like gene and its
regulatory target, the steroidogenic enzyme type 2 3␤-hydroxysteroid dehy-
drogenase. This cortisol biosynthesis is maximal at 8 weeks after conception
under the regulation of ACTH [23]. By 10 weeks of gestation, cortisol secre-
tion begins to decrease, together with a decline in 3␤-hydroxysteroid dehydro-
genase expression. By demonstrating that there is indeed a functioning
fetal HPA axis when the external genitalia are differentiating, a rationale for
prenatal treatment of CAH was provided. Furthermore, it was proposed that
cortisol secretion is relatively high during the period when secretion of dehy-
droepiandrosterone (DHEA) sulphate, the main steroid secreted by the fetal
adrenal cortex, would interfere with genital development in female fetuses.
The high level of cortisol production would suppress the fetal HPA axis, keep-
ing DHEA sulfate production at relatively low levels to produce a transient
mechanism that safeguards the major period of female sex development. This
implies that high doses of dexamethasone are most necessary in the relatively
narrow time window when cortisol levels would normally be high and the
genitalia are differentiating [24]. Elevated androgens later in pregnancy
should have no effect due to placental aromatase and the completed differenti-
ation of the genitalia. Indeed, a reduced dexamethasone dose of 5 ␮g/kg/day
after 23 weeks of gestation effectively prevented prenatal virilization in a
single case [25].
When dexamethasone administration begins as early as the 8th week of
gestation, the treatment is blind to the disease status and sex of the fetus. If the
fetus is later determined upon karyotyping to be a male, or an unaffected female
upon DNA analysis, treatment is discontinued. Otherwise, treatment is contin-
ued to term. Because its half-life is 3.5 h, dexamethasone needs to be adminis-
tered 3 times daily at a dosage of 20 ␮g/kg/day based on the mother’s
prepregnancy weight. The mother’s blood pressure, weight, glycosuria, HbA1c,
symptoms of edema, striae and other possible adverse effects of dexamethasone
treatment should be carefully monitored throughout pregnancy [26]. The largest
series reported to date, by Maria New’s group [12], showed that 49 pregnancies
with an affected female fetus treated by 9 weeks of gestation resulted in normal
or only marginally virilized genitalia compared with the previously untreated
female proband. Treated newborns whose genitalia were rated Prader III-IV had
delayed treatment initiation, were undertreated by the referring physician or
were incorrectly dosed due to maternal noncompliance [12]. The latter might be
difficult to control throughout pregnancy. In dexamethasone-treated mothers,
maternal adrenal suppression is confirmed by suppressed maternal plasma lev-
els of cortisol, 17-hydroxypregnenolone and DHEA or DHEA sulfate; maternal
levels of 17-hydroxyprogesterone are of no value, being scarcely influenced by
the treatment. In the second half of pregnancy, the lack of a rise in maternal
estriol levels is the best indicator of both maternal compliance and suppressed
fetal adrenal secretion. Overall, the accumulated European [27] and American
[12] experiences show that the benefits of dexamethasone treatment clearly out-
weigh the risks and can help to allay anxiety and encourage future pregnancies
in families with 21-OH patients.
Several concerns have been voiced about real and potential harmful effects
of prenatal dexamethasone treatment, to both the mother and the fetus, but these

have been challenged. Mothers treated during pregnancies do gain excess
weight and present more striae and edema, but all these symptoms disappear
upon cessation of therapy [12]. Hypertension or gestational diabetes does not
appear to be more common than in untreated pregnancies [12]. Infants exposed
to dexamethasone prenatally did not differ in weight, length or head circumfer-
ence from untreated, unaffected siblings, and they appeared to develop nor-
mally during childhood [28]. Rare and isolated adverse events have been
reported in treated children, but no harmful effects that can be clearly attributed
to the treatment have been documented. Intrauterine growth retardation and
unexplained fetal death have been observed in 2% or less of treated pregnan-
cies, which is not significantly different from the percentage found in the popu-
lation at large [29]. The risk of overt human fetal defects appears to be low
compared with complications observed in a rodent model of in utero exposure
to high-dose glucocorticoids, which features frequent cleft palate in addition to
fetal growth retardation and/or demise [30]. Pregnant rats treated with the same
dexamethasone weight-based dose used in human prenatal treatment for CAH,
i.e. 20 ␮g/kg/day, produced litters with average birth weights 14% below those
of controls; the offspring were also hypertensive at 6 months, i.e. young adult-
hood [31]. The relevance of these animal studies for human physiology is not
known and the concerns brought forward by data from epidemiological studies,
especially those correlating being small for gestational age with an increased
risk of hypertension, insulin resistance and ischemic heart disease in adult life,
have not been substantiated by follow-up studies so far.
The issue of potential risks is all the more significant for the unaffected
fetuses that are treated with dexamethasone for a few weeks without any bene-
fit of the treatment. In fact, since CAH is an autosomal recessive disease, only
1 of 8 fetuses is likely to be an affected female, and therefore 7 of 8 pregnancies
are unnecessarily exposed to treatment. A long-term follow-up study in
Scandinavia of prenatally treated unaffected boys showed that they had normal
pre- and postnatal growth compared to matched controls [28].
Animal research has shown adverse effects of GC on brain structures such
as the hippocampus [32], raising concerns about possible functional side effects
of dexamethasone on human development. For this reason long-term follow-up
studies have focused on the neuropsychological outcomes in children prenatally
exposed to dexamethasone. A preliminary report of a pilot study on the behav-
ior and development of prenatally treated children, aged 6 months to 5 years,
compared with control untreated children, found no negative effects of dexam-
ethasone on developmental milestones or cognitive development. However, it
did find increased internalizing behavioral traits, such as shyness, less sociability,
greater avoidance, and a marginal increase in emotionality in the children that
were prenatally treated [33]. In a second study phase in which the developmental
outcomes of 174 prenatally treated children, aged 1 month to 12 years, were
compared to 313 unexposed children, none of developmental areas including
cognitive, motor, language and social were different between the 2 groups.
A significant limitation of this study was the fact that it did not examine the
children with psychological or neuropsychological tests but through mother-
administered screening instruments, albeit well standardized [34]. A recent
study, based on direct examination of 26 prenatally treated children, indicates
that dexamethasone treatment is associated with long-term effects on verbal
working memory and on certain aspects of self-perception that could be related
to poorer verbal working memory [35]. Learning and long-term memory are
cognitive functions mediated by neural networks that include hippocampal
structures. The effects of glucocorticoids on hippocampal functions have been
observed in rats, but it is known that the profile of brain development is
species-specific and that sensitivity and expression of corticosteroid receptors
varies between different time points in different species. The observed effects
on verbal working memory/short-term memory would suggest that prenatal
dexamethasone treatment may affect the frontostriatal loop but the present
study does not elucidate whether the effects are functional and/or structural.
Overall, the results of this study should be interpreted cautiously due to the
small sample size and be challenged or confirmed by additional retrospective
studies of larger cohorts. Further follow-up studies in children and adults who
were prenatally treated with dexamethasone should be encouraged and include
direct neuropsychological testing.
Early Postnatal Treatment
In classic 21-OH deficiency, glucocorticoids are given in doses sufficient

to partially suppress adrenal androgen secretion without total suppression of
the HPA axis; mineralcorticoids are given to return electrolyte concentrations
and plasma renin activity to normal. Hydrocortisone is the drug of choice in
childhood because of the relatively low growth-suppressive effect compared
with longer-acting glucocorticoid preparations. Considering that physiological
cortisol secretion rates are about 6–8 mg/m2/day [36], most patients have satis-
factory control of androgen production with daily hydrocortisone doses of
15–20 mg/m2 daily divided in 2 or 3 doses. Replacement doses of glucocorti-
coids for infants have probably been excessive until recently in the mistaken
belief that this was needed to reduce the elevated 17-hydroxyprogesterone concen-
trations and because of misinformation about the cortisol secretion rate. This
was originally thought to be around 12–13 mg/m2/day [37], but more recent
analyses indicate that the true cortisol secretion rate is nearly 50% lower [36]. The

negative effect of high glucocorticoid dosage is elegantly outlined in a recent
study in which the effect of a 10 mg/m2 difference in glucocorticoid dose on
height of patients with salt-wasting 21-OH deficiency was analyzed. In the first
year of life and between the ages of 8 and 14 years, a dose-dependent negative
effect of glucocorticoids on linear growth was shown [38]. Therefore, the daily
glucocorticoid dose in these periods should be sufficient to avoid androgen
excess but as low as possible to allow optimal linear growth and adult height.
This is even more relevant in the first year of life when the risk that androgen
excess will compromise growth is thought to be small. In fact, height velocity
and bone maturation were shown not to be increased in untreated children with
mild forms of simple virilizing 21-OH deficiency in the first year of life [39],
suggesting a relative resistance of bone to androgen activity, that can hypothet-
ically be explained by temporary downregulation or limited expression of the
androgen receptors. After this period there was a progressive increase in height
velocity and bone maturation in strong relation to the duration of androgen
exposition. Therefore, suppression of androgens as required in later years to
prevent growth acceleration is not necessary, and much lower doses of gluco-
corticoids are sufficient for adequate treatment in the first year of life.
Another important factor that seems to affect linear growth of patients with
salt-wasting 21-OH deficiency is sodium depletion. These infants require sup-
plemental mineralcorticoids (usually 0.1–0.2 mg of fludrocortisone) and sodium
chloride (1–2 g or 17–34 mmol of sodium chloride daily) in addition to gluco-
corticoid treatment. The sodium content of either breast milk or infant formulas
is insufficient to compensate for sodium losses in these infants. The need for
sodium chloride supplement is clearly demonstrated by the height at 2 years of
age and final height of a group of patients with salt-wasting 21-OH deficiency
treated with sodium chloride supplements in addition to the regular glucocorti-
coid and mineralcorticoid therapy. The height of these patients was higher than
that of a group of patients treated with a lower dose of glucocorticoid but who
did not receive sodium chloride supplements [40]. A spectrum of salt loss in the
various forms of 21-OH deficiency was demonstrated by the evident decrease
in the aldosterone to renin ratio with increasing phenotypic severity [41]. This
explains why patients with the simple virilizing form of 21-OH deficiency also
benefit from mineralcorticoid therapy. It was shown, in fact, that the addition of
mineralcorticoids to the glucocorticoid therapy in these patients has a positive
effect on linear growth [42].
In conclusion, the accumulated European and American experiences show
that prenatal treatment of 21-OH deficiency, when properly introduced, spares
the affected female from the consequences of genital ambiguity including the
risk of sex misassignment and unnecessary genitoplasty. Studies of treated ver-
sus untreated pregnancies are warranted to monitor the safety of treatment and
enhance our understanding of the effects of prenatal steroid exposure to the
human brain. In the first year of life, optimization of medical treatment is
achieved by combining the lowest dose of glucocorticoid, able to suppress
androgen secretion, with the normalization of sodium balance by giving appro-
priate sodium chloride supplementation in salt-wasting patients.
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Lucia Ghizzoni, MD, PhD

Dipartimento dell’Età Evolutiva, Università degli Studi di Parma
Via Gramsci, 14
IT–43100 Parma (Italy)
Tel. ⫹39 0 521 702 722, Fax ⫹39 0 521 702 209, E-Mail lucia.ghizzoni@unipr.it

Neonatal Diabetes
The Role of KCNJ11 (Kir6.2)
Paolo Tammaro
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
Abstract
ATP-sensitive potassium (KATP) channels are inhibited by intracellular ATP and acti-
vated by MgADP. As a consequence, they couple the metabolic state of the cell to its electri-
cal activity. In pancreatic ␤-cells KATP channels regulate glucose-dependent insulin secretion
and are the target for sulphonylurea drugs clinically employed in the treatment of type 2 dia-
betes. This review discusses recent advances in our understanding of the role of KATP chan-
nels in permanent neonatal diabetes mellitus.
General Properties of the KATP Channel
ATP-sensitive potassium (KATP) channels link cellular metabolism to mem-

brane electrical activity by regulating K⫹ fluxes across the plasma membrane
[1]. Under resting metabolic conditions KATP channels are predominantly open,
allowing K⫹ efflux, membrane hyperpolarisation, and thus preventing action
potential firing. The metabolic generation of ATP that follows glucose uptake
has the opposite effect: closure of KATP channels and stimulation of cell electri-
cal activity. This usually gives rise to downstream events such as release of hor-
mones (e.g. insulin or glucagon) and neurotransmitters or muscle contraction.
KATP channels are found in a number of organs, where they play a multi-
tude of physiological roles. In pancreatic ␤-cells, KATP channels are crucial for
glucose-dependent insulin secretion [1, 2]. In the brain, KATP channels modulate
electrical activity and transmitter release at synapses [3] and protect against
seizures [1, 4]. In the heart, KATP channels are involved in ischaemic precondi-
tioning and the response to ischaemia. Activation of KATP channels during meta-
bolic stress helps preserve myocardial energy stores by promoting membrane
hyperpolarisation, action potential shortening and reduced contraction [5, 6]. In
vascular smooth muscle KATP channels contribute to resting tone, and alter-
ations in channel activity in response to vasoactive agonists cause changes in
arterial diameter that play an important role in blood pressure regulation [7, 8].
KATP channels are octameric complexes of Kir6.x and SUR subunits
[9–11]. Four inwardly rectifying subunits (Kir6.1 or Kir6.2) form the channel
pore, whose opening and closing (gating) is regulated by 4 sulphonylurea
receptor subunits (SUR1, SUR2A or SUR2B). Binding of ATP or ADP to
Kir6.x causes channel closure [12, 13], whereas binding of lipids such as phos-
phatidylinositol bisphosphate [14, 15] or long-chain acyl CoAs [16, 17] to
Kir6.x biases the channel towards the open state and relieves ATP inhibition.
SUR is an ATP-binding cassette transporter. Binding and/or hydrolysis of Mg-
nucleotides by the intracellular nucleotide-binding domains of SUR stimulates
channel opening [18–21]. It is believed that reciprocal changes in the intracel-
lular concentrations of ATP and MgADP are involved, at least in part, in the
metabolic regulation of KATP channels.
Different combinations of Kir6.x and SUR subunits contribute to the diver-
sity of functional properties of native KATP channels. In most tissues, Kir6.2 forms
the pore [11], but in smooth muscle it is Kir6.1. The SUR2B isoform is found in
neurons [22] and both vascular and non-vascular smooth muscles [23]; SUR2A in
heart and skeletal muscle [24], and SUR1 in most other tissues, including ␤-cells
and many neurons [25, 26]. Variation in SUR composition accounts for differ-
ences in metabolic and drug sensitivities of KATP channels in different tissues [27,
28]. In pancreatic ␤-cells, the KATP channel is composed of Kir6.2/SUR1.
Two main classes of therapeutic drug interact with SUR to modulate KATP
channels [29–32]. Sulphonylureas inhibit KATP channels and are used clinically
to stimulate insulin secretion in patients with type 2 diabetes [30, 31]. In con-
trast to sulphonylureas, KATP channel openers, such as diazoxide, open KATP
channels and inhibit insulin secretion [29, 32].
Role of KATP Channels in Pancreatic b-Cells

The central role of KATP channels in insulin secretion was established over
20 years ago [2]. At resting metabolism, KATP channels are open. This permits a
constant outward flow of K⫹ that hyperpolarises the membrane potential to a
level at which voltage-gated Ca2⫹ channels are closed. When plasma glucose
increases, it is taken up by the ␤-cell and metabolised to ATP at the expense of
ADP. This causes closure of KATP channels, producing a membrane depolarisa-
tion that leads to opening of voltage-gated Ca2⫹ channels, a rise in the intra-
cellular Ca2⫹ concentration and exocytosis of insulin granules (fig. 1).
KATP-channel-dependent generation of ␤-cell electrical activity (i.e. Ca2⫹-
dependent action potential) is therefore compulsory for insulin secretion [33].
KATP Channels and Neonatal Diabetes 71

Glucose
Glut 2
Gycolysis/Mitochondria
PIP2
⫹
KATP channel
Kir6.2 ⫺ Increased ATP
SUR1 Decreased MgADP
⫹
Diazoxide ⫺ ⫹
membrane
Sulphonylureas depolarisation
Cav channel ⫹ ⫹ Insulin

Increased [Ca2⫹] exocytosis
Fig. 1. KATP channels and insulin secretion. Blood glucose enters the ␤-cell via
GLUT2 and is metabolised (by glycolytic and mitochondrial metabolism) to ATP at the
expense of MgADP. This leads to KATP channel closure, membrane potential depolarisation,
opening of voltage-gated Ca2⫹ channels and exocytosis of insulin granules. Sulphonylureas
block the KATP channel and produce the same chain of events. Other factors, such as phos-
phatidylinositol bisphosphate (PIP2), may also modulate KATP channel ATP sensitivity.
Permanent Neonatal Diabetes
Neonatal diabetes mellitus is a rare disorder (1 in 400,000 births) charac-

terised by high blood glucose levels that manifest within the first 6 months of
life. It may be either permanent (PNDM) or transient (TNDM): in the latter
case it resolves within the first ⬃3 months of life. TNDM is attributed to pater-
nal imprinting at chromosome 6q24 [34]. However, heterozygous mutations in
Kir6.2 have been reported to cause a remitting relapsing form of neonatal dia-
betes that resembles TNDM [35].
Homozygous or compound heterozygous mutations in glucokinase, the
enzyme that catalyses the rate-limiting reaction of glucose metabolism in pan-
creatic ␤-cells, cause PNDM in a small fraction of cases [36] by reducing gly-
colytic production of ATP. Homozygous mutations in the insulin promoter
factor 1, a transcription factor involved in pancreatic development, or alterations
in forkhead box P3 have been reported to cause neonatal diabetes by reducing
␤-cell mass through impaired pancreatic development [37, 38] or autoimmune
␤-cell reactions [39, 40], respectively. Mutations in the pancreas transcription
Tammaro 72
factor 1␣ [41] and in the translation initiator factor 2-␣ kinase 3 [42] can also
cause PNDM.
The majority of PNDM cases (⬃50%) result from gain-of-function muta-
tions in Kir6.2 or SUR1 that result in reduced KATP channel ATP sensitivity [43,
44]. Until recently, PNDM was typically treated with insulin therapy but the
elucidation of its genetic causes has resulted in better treatments for this condi-
tion [45].
To date, more than 20 single-point mutations in Kir6.2 have been associ-
ated with PNDM. Most are de novo mutations but in some cases familial trans-
mission has been observed [46–49]. In all families examined [50, 51] only
individuals carrying the Kir6.2 mutation had diabetes. A large spectrum of dis-
ease severity has been observed. The majority of Kir6.2 mutations produce
PNDM alone. Patients present with low insulin secretion in response to glucose
without showing type 1 autoantibodies or chromosome 6 abnormalities. Their
diabetes can be successfully treated with sulphonylureas [46, 48, 52]. Patients
with PNDM caused by mutations in Kir6.2 show varying levels of C-peptide
[35, 46, 48, 52, 53] that may partly account for the large spectrum of hypergly-
caemia measured in the patients. Some patients present with additional symp-
toms such as muscle weakness, delayed walking and delayed language
development [46]. A subgroup of mutations that affect KATP channel function
even more markedly produce DEND syndrome, which is characterised by
delayed development of motor, intellectual and social skills, muscle weakness,
epilepsy, facial dysmorphism and neonatal diabetes [46, 54, 55]. As indicated
above, Kir6.2 mutations may also cause remitting relapsing diabetes [35].
There is also variability in the temporal appearance of the disease, which
spans from birth [46] to 5 years of age [35]. It is interesting that a case of Kir6.2
mutation (C42R) caused TNDM, childhood-onset diabetes and an apparently
type 2 diabetes in different carriers of the same pedigree [53].
All PNDM mutations analysed to date result in reduced KATP channel ATP
sensitivity in vitro. This is expected to lead to an increased KATP current ampli-
tude and reduced insulin secretion under resting conditions. The extent of the
reduction of the channel ATP sensitivity mirrors the severity of the clinical phe-
notype. Thus, in heterologous expression systems, mutations associated with
PNDM display a small increase in KATP current in the presence of physiological
concentrations of MgATP (1–5 mM), whereas a larger increase in KATP current
is produced by mutations that cause DEND syndrome.
Direct evidence that Kir6.2 mutations prevent electrical activity and
insulin release has been obtained by transfection of Kir6.2-R201H in the
insulin-secreting cells [56]. Expression of Kir6.2-R201H reduced KATP channel
ATP sensitivity and the metabolic substrate methylsuccinate did not decrease
KATP current and stimulate electrical activity and insulin release.

K⫹
Outside
Membrane
Inside Gating mutations
Binding mutations
Subunit interactions
mutations
Fig. 2. Homology model of Kir6.2 showing location of PNDM-causing mutations.

Structural model of Kir6.2 [59] (side view). For clarity, only 2 transmembrane domains, and
2 separate cytosolic domains, are shown. Residues associated with PNDM are shown in stick
format. ATP is shown docked into its binding site. PNDM mutations that affect KATP channel
gating, ATP binding or Kir6.2 subunit interactions are highlighted.
Molecular Mechanism of Reduced KATP Channel ATP Sensitivity

Mutations in Kir6.2 may affect KATP channel ATP sensitivity in 2 ways: (i)
mutations in the ATP-binding site may affect ATP binding directly [46, 54, 57];
(ii) mutations may also affect ATP sensitivity indirectly by increasing the stabil-
ity of the open state, thereby reducing the time the channel spends in the closed
state to which ATP preferentially binds [54, 55]. The latter lies in regions of the
channel thought to be implicated in channel gating. Some mutations (V59G and
I296L) may affect both ATP binding and channel gating [55]. It is worth men-
tioning that most PNDM mutations also affect the extent by which MgATP acti-
vates the channel via SUR [58]. PNDM mutations that act by affecting the
channel sensitivity to phosphatidylinositol bisphosphate have not yet been
reported but potentially they might occur.
A homology model of the Kir6.2 (fig. 2) [59] suggests that residues R50,
I182, R201, Y330 and F333, which have been shown to cause PNDM when
mutated, form part of the ATP-binding site. The model predicts that R201 and
R50 give rise to electrostatical interactions with the phosphate tail of ATP, I182
directly interacts with the adenine ring of ATP, and F333 and Y330 both lie
Tammaro 74
within 3Å of the phosphate tail of the ATP [59]. The PNDM mutations that
affect ATP sensitivity by increasing the channel open probability (Po) include:
F35, C42R, Q52R, V59G, C166F, I182V, I296L and Y330C. Q52 and V59 are
located within the slide helix region of Kir6.2 that may form part of the physi-
cal link between ATP binding and the channel gate [59]. Mutations that lie in
the helix bundle-crossing region, where the second transmembrane helices in
each Kir6.2 subunit converge to form the gate for K⫹ permeation [59], have
also been reported to cause diabetes (e.g. K170N, K170R [47]).
It is worth mentioning that there is no absolute correlation between the
molecular mechanism of impaired channel ATP sensitivity and severity of
the disease. Rather, it is the magnitude of the unblocked KATP current at phy-
siological levels of MgATP that accounts for the disease severity and presence
of extra-pancreatic symptoms. For example, mutation of arginine 50 into
glutamine caused PNDM but substitution with a proline produced DEND
syndrome [60].
Diabetogenic Mutations in SUR1

Mutations in SUR1 that give rise to neonatal diabetes have recently been
found [43, 44]. SUR1 has 17 transmembrane domains (arranged in 3 groups of
5, 6 and 6 helices termed TMD0, TMD1, TMD2, respectively). The first muta-
tion to be identified was F132L, which caused DEND syndrome [44]. Residue
F132 lies in TMD0, a region that physically interacts with Kir6.2 and is known
to modulate channel Po. The F132L mutation increased the KATP channel Po and
thereby decreased its sensitivity to MgATP. Like Kir6.2 mutations associated
with DEND syndrome, F132L resulted in a large unblocked KATP current at
physiological concentrations of MgATP. Two other mutations causing PNDM
(L213R and I1424V) and 5 causing TNDM (C435R, L582V, H1023Y, R1182
and R1379) have been described. Functional analysis of I1424V and H1023Y
showed an increased channel Po and enhanced SUR1-dependent MgATP activa-
tion on Kir6.2 pore [43]. Unlike the F132L mutation, these mutant channels
were blocked by sulphonylureas and treatment with these drugs resulted in eug-
lycaemia [43].
Effects of Heterozygosity on ATP Sensitivity of KATP Channels Containing

Kir6.2 Mutations
To date, all patients with PNDM caused by mutations in Kir6.2 are het-
erozygotes. Because Kir6.2 assembles as a tetramer [10, 11], their ␤-cells will
contain a mixed population of channels with a variable number of mutant sub-
units (from 0 to 4). The ATP sensitivity of each of these channel subtypes will
depend both on the number of mutant subunits that make up the channel and on
the contribution of each mutant subunit to the channel ATP sensitivity.

It is known that binding of 1 molecule of ATP is sufficient to cause chan-
nel closure [61]. This implies that a mutation that reduces the affinity of ATP
for its binding site will affect the channel ATP sensitivity only when all 4 Kir6.2
subunits are mutated. If wild-type and mutant Kir6.2 subunits assemble ran-
domly, the binomial theory predicts that only 1/16 of the channels in the het-
erozygous population will contain 4 mutant subunits and have reduced ATP
sensitivity; thus, the shift in the ATP sensitivity of the heterozygous population
(in the absence of Mg2⫹) will be small [54]. However, although experiments
carried out in the absence of Mg2⫹ can help elucidate the molecular mechanism
by which ATP interaction with Kir6.2 is altered by the PNDM mutation, it
should not be forgotten that it is the MgATP sensitivity of the channel that is
important for the disease phenotype, and mutation in Kir6.2 may modulate
MgATP stimulation of channel activity [58].
Other Possible Effects of Kir6.2 Mutation on b-Cell Function

It is interesting that transgenic mice overexpressing an overreactive KATP
channel displayed changes in islet architecture, with abnormal distribution of
␣- and ␤-cells throughout the pancreas [62]. It cannot be excluded that patients
with diabetes caused by mutations in Kir6.2 might also be affected by similar
islet dysfunction.
Effects of Permanent Neonatal Diabetes

Mellitus Mutations in Extra-Pancreatic Tissues
Kir6.2 is also expressed in skeletal muscle, cardiac muscle and neurons.

This histological distribution explains the spectrum of symptoms associated
with DEND syndrome. Interestingly, extra-pancreatic symptoms are seen only
for mutations that cause a substantial reduction in ATP sensitivity and the larger
increases in KATP current. Possible reasons for why larger currents are required
to affect extra-pancreatic tissues are: (i) lower expression of KATP channels in
extra-pancreatic tissues and/or less contribution to the cell resting membrane
potential; (ii) association of Kir6.2 with a different SUR subunit (SUR2 vs.
SUR1); or (iii) cell-specific differences in metabolism or in channel regulators,
including phosphatidylinositol bisphosphate.
Too much KATP channel activity in inhibitory neurons may underlie
epilepsy, since a decrease in the inhibitory tone is expected to enhance
excitability of target neurons [63]. Interestingly the electrocardiogram of
PNDM and DEND patients was normal [46, 48]. This is likely to be due to the
association of Kir6.2 with SUR2A in cardiac myocytes, since SUR2A enhances
channel activity in response to MgATP less than SUR1 [64, 65] (fig. 3). Thus
Tammaro 76
SUR1
1.0
homomeric
0.8
0.6
G/GC
heteromeric
0.4
0.2
wild-type
0.0
10⫺6 10⫺5 10⫺4 10⫺3 10⫺2
a [MgATP] (M)
SUR2A
1.0
homomeric
0.8
0.6
G/GC
heteromeric
0.4
0.2 wild-type
0.0
10⫺6 10⫺5 10⫺4 10⫺3 10⫺2
b [MgATP] (M)
Fig. 3. Effect of the PNDM mutation Q52R on KATP channels composed of SUR1 or
SUR2A. Mean relationship between MgATP and KATP conductance (G) expressed relative to
the conductance in the absence of the nucleotide (GC) for Kir6.2-Q52R coexpressed with
either SUR1 (a) or SUR2A (b). The smooth curves are the best fit of the Hill equation to the
data with IC50 of 16, 640 and 4,900 ␮M (SUR1) and 25, 45 and 1,400 ␮M (SUR2A) for
wild-type, heteromeric and homomeric mutant channels, respectively. The grey shaded bars
indicate the range of physiological intracellular MgATP concentration. Data are re-plotted
from Tammaro et al. [64].
cardiac KATP channels are substantially closed under resting conditions [66].
Kir6.2 is expressed in both skeletal muscle [67] and nerve termini [3]. However,
since Kir6.2 associates with SUR2A (and not with SUR1) in skeletal muscle,
muscle weakness in DEND patients seems more likely to be of neuronal origin.

The discovery of a DEND-causing mutation in SUR1 [44] further supports the
idea that muscle weakness and delayed motor development seen in DEND
patients are of neuronal origin (although whether this is of central or peripheral
origin yet remains to be determined).
Glucose uptake by skeletal muscle and adipose tissue is influenced on KATP
channel activity [68] and PNDM mutations may impair this function. In unpub-
lished studies (Tammaro and Ashcroft), we found that coexpressions of Kir6.2-
F333I with SUR2B (the SUR isoform found in smooth muscle) are activated
(not inhibited) by MgATP, raising the possibility that some PNDM mutations
may affect smooth muscle function and suggesting that further clinical investi-
gation in this direction may be warranted.
Implications for Therapy and Conclusions
Before the discovery of forms of PNDM caused by mutations in KATP

channel genes, patients affected by this disorder were treated with daily insulin
injections as they were believed to suffer from type 1 diabetes. Patients with
Kir6.2 mutations that cause PNDM alone can be controlled solely on sulphony-
lureas [46, 48, 52]. Several patients have now transferred from insulin to gliben-
clamide therapy, and their glycaemia has been well managed for ⬎6 months
[48, 52]. In some cases the effective oral dosage of sulphonylurea required was
several-fold greater than that commonly used to treat type 2 diabetes [52, 69].
Further studies will be required to clarify the efficiency of sulphonylureas in
patients affected by KATP channel mutations that markedly influence channel
gating (as found with some DEND syndrome mutations). This is because stud-
ies conducted in vitro show that mutations which cause an increase in channel
Po also exhibit reduced inhibition by sulphonylureas [46, 54, 70]. It is worth
mentioning that while insulin can control the diabetes of patients with DEND
(or intermediate DEND) syndrome, it cannot ameliorate the extra-pancreatic
symptoms caused by mutations in KATP channel genes. The extent to which
sulphonylureas may be able to ameliorate the neurological complications of
these patients remains to be seen.
Acknowledgements
I wish to thank Professor Frances Ashcroft for her critical reading of the manuscript
and for her guidance during my research. P.T. holds a Junior Research Fellowship at the
Wolfson College.
Tammaro 78
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Tammaro 80
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Paolo Tammaro
Department of Physiology, Anatomy and Genetics, University of Oxford
Parks Road
OX1 3PT Oxford (UK)
Tel. ⫹44 1865285817, Fax ⫹44 1865285813, E-Mail paolo.tammaro@dpag.ox.ac.uk
Tammaro 82
Diagnosis of Neonatal and

Infancy-Onset Diabetes
Fabrizio Barbetti
San Raffaele Biomedical Park Foundation and Bambino Gesù Pediatric Hospital and
Department of Internal Medicine, University of Tor Vergata, Rome, Italy
Abstract
Until 1995, the etiology of ‘neonatal’ diabetes was totally unknown. In about a decade,
mutations in 8 different genes (IPF1, EIF2AK3, GK, FOXP3, KCNJ11, ABCC8, PTF1A and
GLIS3) have been discovered in patients with the permanent form of the disease, and 3
genetic abnormalities (defects in the paternally imprinted chromosomal region 6q24 and
‘mild’ activating mutations in KCNJ11 or ABCC8) have been detected in subjects with tran-
sient neonatal diabetes. Together with the advances in the understanding of the pathophysiol-
ogy of this condition, clearly different from type 1 diabetes, also the temporal criterion by
which one clinically defines a patient as being affected by neonatal diabetes has changed. In
1995, neonatal diabetes was defined as hyperglycemia that requires insulin treatment and
occurs during the first month of life. In some patients with defects of KCNJ11, ABCC8 or
EIF2AK3 genes however, diabetes can present at 6 months of age and beyond. It is now time
to adopt a new definition in order to avoid the confusion originating by the misuse of the
term ‘neonatal’. I would suggest monogenic diabetes of infancy, which includes both the per-
manent and the transient types, irrespectively of the mechanism of disease.
Neonatal and Infancy-Onset Diabetes: A Brief History
About a decade ago (1995) the seminal paper of von Muhlendahl and
Herkenhoff laid the basis for the clinical definition of neonatal diabetes melli-
tus (NDM) [1]. At that time neonatal diabetes was defined (for that study) ‘as
hyperglycemia that requires insulin treatment, occurs during the first month of
life and lasts more than two weeks’. The reappraisal of 57 cases of the literature
and the investigation of the long-term course of some of those patients led the
authors to conclude that there are 2 clinical variants of neonatal diabetes: a transient
form (33 cases out of 57; TNDM) that remits in a variable interval of time
(17–1,914 days from onset), but can relapse later in life (13 subjects out of 57),
and a permanent form (24/57; PNDM). Most of the cases included in the paper
(47/57) fulfilled the criteria established in the definition mentioned above. For
the others however (10/57 or 17%), the age at diabetes onset was comprised
between day 31 and day 90 from birth [1]. Interestingly (we will learn later
why), among these 10, five patients with the Wolcott-Rallison syndrome (dia-
betes and short stature) and 3 with transient-relapsing diabetes were included
[1]. What about the etiology of neonatal diabetes? Von Muhlendahl and
Herkenhoff reckoned intrauterine growth retardation, a feature common to most
cases with this condition, as a possible agent (rather than the consequence) of
PNDM and reasoned that autoimmunity was not a likely cause. Seven years
later, the Diabetes Study Group of the Italian Society of Paediatric Endocrinology
and Diabetes published the results of its study on 111 patients who presented
with diabetes in the first year of life. With the exception of a few subjects with
diabetes associated with (autoimmune) enteropathy, most of the patients with
onset of hyperglycemia within 180 days from birth (36 individuals) were nega-
tive for the search of autoantibodies of type 1 diabetes (T1D) (for some patients
data were not available) and had a nonpredisposing HLA. In contrast, T1D
autoantibodies and predisposing HLA haplotypes were frequent in the second
group of patients (diabetes onset between 180 and 365 days of life; 75 individ-
uals) [2]. In addition, small for date births were many in the first group (⬍180
days ⫽ 64.3%) and few in the second group (⬎180 and ⬍365 ⫽ 14.3%) [2].
These findings reinforce the notion that neonatal diabetes is clinically distin-
guishable from T1D of very early onset and set a new temporal cutoff (i.e. onset
⬍180 days from birth) to select patients to the aim of searching for the cause(s)
of this supposedly genetic form of diabetes.
Between 1995 and 2002, several groups of researchers identified a handful
of genetic defects causing neonatal diabetes. The serendipitous discovery in
1995 of paternal uniparental isodisomy of chromosome 6 in a patient with
TNDM (and in a second unrelated subject with transient diabetes) [3] opened
the route for more extensive investigations in patients with this clinical variant.
Subsequent studies reached the conclusion that about 80% of the patients with
TNDM carry a genetic defect which results in the overexpression of paternally
imprinted genes (ZAC and HYMAI) [4–6]. Distinctive features characterize
patients with defects of imprinting at 6q24: very low birth weight (1,930 g on
average, equal or below second percentile), neonatal onset of hyperglycemia
(average age at presentation of diabetes 7 days), apparent remission with a
median of 3 months (average 111 days) and minor dysmorphic findings
(1/3 macroglossia) [5]. Though the creation of transgenic mice overexpressing
ZAC and HYMAI has shed light on the pathophysiology of TNDM, mechanism
Barbetti 84
of ␤-cell dysfunction caused by paternal uniparental isodisomy of chromosome
6 remains not fully understood [6].
In 1997, the first mutation leading to PNDM was identified in a subject
with pancreatic agenesis. The patient carried a single nucleotide homozygous
insertion causing a frameshift and a premature stop codon in the IPF1/PDX1
gene, encoding a transcription factor crucial in pancreatic embryonic develop-
ment [7, 8]. Another patient with 2 loss-of-function mutations of IPF1 was
reported in 2003 [9]. As expected, in both patients lacking the whole pancreas
diabetes presented within few days from birth along with exocrine pancreas
insufficiency. In addition, both had an extremely low birth weight, a sign of
poor/absent fetal insulin secretion. Three years later, a new breakthrough in the
field was accomplished when the cause of the recessive Wolcott-Rallison syn-
drome was identified [10]. Patients with this syndrome show a permanent,
infancy-onset diabetes (range: 1.5–30 months from birth with mean age at onset
of 3 months), epiphyseal dysplasia and other associated features (e.g. liver dys-
function, mental retardation) [10–14]. Homozygous or compound heterozygous
loss-of-function mutations of the EIF2AK3 gene encoding for PKR-like ER
kinase (PERK, a eukaryotic translation initiation factor kinase residing in the
endoplasmic reticulum) are found in virtually all patients with this condition
[10–14]. Animal models (Perk knockout mice, devoid of PERK) have been
instrumental for the understanding of the molecular mechanisms underpinning
the multiple organ defects found in patients with Wolcott-Rallison syndrome
[15–17]. As for the pancreatic ␤-cell failure, it turns out that mice lacking PERK
activity have a normal pancreas at birth, but a progressive loss of ␤-cells is
observed postnatally as a consequence of massive apoptosis [15, 16]. This is in
good agreement with what is found in patients with Wolcott-Rallison syndrome,
i.e. birth weight often normal [C. Julier, pers. commun. to F. B., and 13], indicat-
ing normal insulin secretion during intrauterine life and neonatal, but frequently
infancy-onset, diabetes. Fourteen different mutations of EIF2AK3 (13 homozy-
gous, 1 compound heterozygous) have been published to date [10–14].
The first report (2001) on a gene causing permanent neonatal diabetes in iso-
lation (not syndromic) described 2 patients with homozygous inactivating muta-
tions of glucokinase (GK), the enzyme that serves as ‘glucose sensor’ for
glucose-stimulated insulin secretion [18]. The discovery did not come as a sur-
prise because GK knockout mice show hyperglycemia and ketoacidosis at birth
and die 3–5 days after delivery [19–21]. In addition, heterozygous inactivating
mutations of GK give rise to a relatively frequent form of autosomal dominant
diabetes called maturity-onset diabetes of the young 2. Patients with complete
GK deficiency had a very low birth weight (1,600 g) and diabetes from day 1 of
life; further investigations found that both homozygous or compound heterozy-
gous mutations of GK can cause PNDM (6 patients), always with the same
Diagnosis of Neonatal Diabetes 85

phenotype [22, 23]. These findings confirmed the pivotal role of GK in insulin
secretion and that GK function cannot be substituted by other hexokinases (e.g.
hexokinase II) in the human (and mouse) ␤-cell [18, 22]. Attempts to wean patients
from insulin using sulfonylureas, though theoretically possible [20], were unsuc-
cessful (F. Cerutti and F. B., unpubl. obs., and P. Njolstad, pers. commun. to F.B.).
The genetic origin of a form of neonatal autoimmune diabetes associated
with enteropathy, eczema and thyroid autoimmunity denominated IPEX
(immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome)
was reported in the same year (2001) by 2 independent groups of investigators
[24, 25]. They found that loss-of-function mutations of the FOXP3 (forkhead
box protein 3) gene, located in chromosome Xp11.23, cause the syndrome in
male children. FOXP3 encodes for a transcription factor which is expressed in a
subpopulation of T lymphocytes called regulatory T cells, which exert a crucial
role in maintaining self-tolerance [26, 27]. Abnormalities of FOXP3, also of
unusual kind such as new, upstream AUG translation initiation site [28], cause a
dysfunction of regulatory T cells leading to IPEX [24–26, 28–30] or related
conditions [31]. To date 17 FOXP3 mutations have been reported, but interest-
ingly, variations of FOXP3 gene are not associated with common T1D [32]. The
therapeutical possibilities available for IPEX, which is often fatal, was recently
enriched by successful bone marrow transplantation [33].
The year 2004 was a true turning point in the hunt for new PNDM genes
with the discovery by A. Hattersley’s group of dominant, activating mutations
of KCNJ11 in sporadic as well as familial cases with permanent neonatal and
infancy-onset diabetes mellitus [34]. The KCNJ11 gene encodes for the pore
forming unit of the ATP-sensitive potassium channel (KATP) of the pancreatic
␤-cell, a molecular ‘switch’ for insulin secretion. The mechanism of disease in
patients bearing genetic defects of KCNJ11 is thoroughly explained in another
paper in this issue [see chapter by Tammaro, pp. 70–82]. Suffice it to say here
that KCNJ11 (previously known as Kir6.2) mutations represent a frequent
cause of neonatal and infancy-onset diabetes mellitus, accounting for 40–64%
of all cases in large series [35–38]. In addition, KCNJ11 mutations can cause
TNDM, usually relapsing later in life [39–41]. Age at diabetes presentation in
patients with mutations of Kir6.2 ranges from day 1 to day 220 from birth
[35–38, 42 and F.B., unpubl. obs.] with birth weight ranging from low (i.e.
small for gestational age) to normal according to age at diabetes onset (i.e. the
earlier the presentation, the lower the birth weight) [37]. As associated features,
Kir6.2 mutants can also show a wide spectrum of motor and mental develop-
mental delays ranging from minor muscle weakness to DEND syndrome
(developmental delay, epilepsy and neonatal diabetes) [34–38, 43–45]. In the
Italian series, some degree of neurological impairment is present in ⬎50% of
cases [37, 45–47], with the phenotype determined by the genotype. As already
Barbetti 86
reported by A. Hattersley [34, 38, 42], we also found that patients with the
mutation KCNJ11/V59M (6 out of 19 KCNJ11 mutants with permanent dia-
betes of the Italian collection) or R201C (2 patients) show neurological features
and the so-called incomplete DEND (no epilepsy) [37, 47]. Patients with neu-
rological features did not usually show a severer ␤-cell phenotype [37, 42, 47].
The KATP channel is a hetero-octameric structure formed by 4 Kir6.2 subunits
and, in the ␤-cell, 4 SUR1 (the sulfonylurea receptor) subunits: binding of sul-
fonylureas to SUR1 closes the channel and triggers insulin secretion [34, 38].
Most of the patients with KCNJ11 mutations (but not all) can thus be weaned
from insulin and transferred to sulfonylureas (as reviewed elsewhere in this
issue), quite ‘prodigious’ a result and a nice example of pharmacogenomics
[34, 35, 41, 45–48]. In 2006, also mutations of SUR1 (encoded by the ABCC8
gene) were found in patients with neonatal and infancy-onset diabetes [49, 50].
Mutations of the ABCC8 gene account respectively for 7% of cases with the
permanent and 15% of cases with the transient form of the French series [50].
In patients with defects of SUR1 diabetes presents between day 3 and 125 from
birth [49, 50] with some patients (4/10) with low birth weight and some (5/10)
with motor/mental developmental delay, but no epilepsy [49, 50]. As expected,
SUR1 mutants respond to sulfonylurea therapy [50]. As of February 2007,
patients with neonatal and infancy-onset diabetes due to an activating KCNJ11
or ABCC8 mutation have probably exceeded the number 80 [34–38, 42, 49, 50].
Two other recessive genes causing syndromic neonatal diabetes have been
discovered respectively in 2004 and 2006: PTF1A and GLIS3. Mutations of
PTF1A cause pancreatic and cerebellar agenesis and the 4 patients identified so
far are characterized by very low birth weight, PNDM with onset on day 1 of
life and irregular movements [51]. Investigation of Ptf1a knockout mice con-
firmed the cerebellar agenesis [51] and the requirement of this basic helix-loop-
helix transcription factor for the correct development of GABAergic neurons in
the spinal cord [52] and retinogenesis [53]. The 6 patients carrying GLIS3
mutations presented with neonatal diabetes (onset day 1–2 from birth) and
hypothyroidism (TSH: 100–965 mIU/ml) due to absent thyroid gland [54].
They were all born with low birth weight (1,400–2,200 g). Computerized
tomography or abdominal ultrasonography documented a small, hypoplastic
pancreas in 3 patients. Interestingly, the glucagon basal level (measured in 2
patients) was found normal [54, supplementary online information]. GLIS3 was
not known as a transcription factor required for pancreatic development and/or
␤-cell lineage specification and/or survival, therefore this finding was some-
how unexpected.
In summary, mutations in 8 different genes (IPF1, EIF2AK3, GK, FOXP3,
KCNJ11, ABCC8, PTF1A and GLIS3) can cause permanent neonatal/infancy-
onset diabetes (table 1), while defect of imprinting in the chromosomal region

Table 1. The 8 genes which cause permanent neonatal or infancy-onset diabetes
Barbetti
Gene IPF1 PTF1A GLIS3 GK KCNJ11 ABCC8 EIF2AK3 FOXP3
Protein TF TF TF Hexokinase KATP channel KATP channel Kinase TF

function
Days of life 1 1 1 1–12 1–220 7–125 4–900 1–180
at diabetes
presentation
Birth 1,700– 1,200a– 1,400–2,200 1,650– 1,400a–3,500 2,200–3,065 Normal Lowa to
weight (g) 2,140 1,540 1,900 normal
Associated Exocrine Cerebellar Hypothyroidism None Motor/mental Motor/mental Epiphyseal Autoimmune
features insufficiency agenesis developmental developmental dysplasia; Enteropathy
delay; delay osteopenia
epilepsy
Number of 2 4 6 6 ⬎80 3 22 ⬍20
cases
Trait Recessive Recessive Recessive Recessive Dominant Dominant Recessive X-linked
(recessive)
TF ⫽ Transcription factor.
a
In some cases the gestational age was below 38 weeks.
88
6q24 or ‘mild’ mutations in KCNJ11 or ABCC8 genes give rise to transient/
relapsing neonatal diabetes. PNDM/TNDM genes fall within 4 main categories:
(1) genes encoding for transcription factors important for the embryonic devel-
opment of pancreas (IPF1, PTF1A and GLIS3), (2) genes encoding proteins
which couple glucose metabolism inside the ␤-cell with insulin secretion (GK,
KCNJ11, ABCC8), (3) a gene controlling protein translation, a crucial function
for survival of professional secretory cells (EIF2AK3), and (4) a master gene in
the regulation of the immune system (FOXP3).
Take Home Messages for the Clinicians
Genetic screening has practical consequences for counselling (diabetes

associated with dominant mutations can be passed to the next generation) and
the correct therapeutical approach (see KNCJ11 and ABCC8 mutants) for
patients with neonatal and infancy-onset diabetes. However, the current defini-
tion of PNDM (diabetes diagnosed within the first 3 months of life) is mislead-
ing and can give rise to uncertainty: as a matter of fact it is apparent that
diabetes caused by genetic mutations can present within the first 6 months of
life and beyond. I believe that patients negative to the search of T1D autoanti-
bodies and diabetes onset within 1 year of age have to be regarded as likely
bearers of a single gene defect (not FOXP3) and reasonable candidates for
mutation screening (according to the phenotype). This is my personal opinion,
which partially disagrees with the International Society for Pediatric and
Adolescent Diabetes Clinical Practice Consensus Guidelines [55]. Thus, in
order to avoid the confusion deriving from the adjective ‘neonatal’ applied to
patients with diabetes onset at 6 or 7 months of age, we proposed in 2005 to call
this condition ‘permanent diabetes of infancy’ [37]. As an alternative, I would
suggest to adopt the more comprehensive ‘monogenic diabetes of infancy
(MDI)’, which would also include the transient form of the disease.
Regardless of the semantics, my personal experience is that 50% of the
patients with diabetes onset within 7 months of age, even if the T1D autoanti-
bodies are unknown (‘historical’, adult cases), carry a heterozygous activating
mutation of KCNJ11 [37, 41, 45–47 and F.B. unpubl. obs.] and in another 25%
a mutation in a new MDI gene is detected (F.B., unpubl. obs.). In the Italian
series SUR1 and GK mutations probably account for another 7–8% and 2–3%,
respectively (F.B., unpubl. results). Recessive mutations, which can be sus-
pected on a phenotypic basis, are very rare and are usually found in populations
with a high rate of consanguinity. Therefore, the genetic cause of about 80% of
the Italian patients with MDI has been discovered and 90% of those carrying a

mutation of KCNJ11 have been switched from insulin to sulfonylureas, a result
that no one would ever have imagined only 12 years ago.
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Fabrizio Barbetti
S. Raffaele Biomedical Park Foundation, Laboratory of Molecular Endocrinology and
Metabolism, Room B303
Via di Castelromano 100
IT–00128 Rome (Italy)
Tel. ⫹39 0680 319 073, Fax ⫹39 0680 319 054, E-Mail mody2@libero.it

Management of Neonatal and

Infancy-Onset Diabetes Mellitus
Oddmund Søvika, Mojca Zerjav Tansekd, Jørn V. Sagenb,
Pål Rasmus Njølstad a,c
Departments of aClinical Medicine and bMedicine, University of Bergen, and
c
Department of Pediatrics, Haukeland University Hospital, Bergen, Norway;
d
Medical Center Ljubljana University Children’s Hospital, Ljubljana, Slovenia
Abstract
Diabetes mellitus is a rare disorder during the first 2 years of life, amounting to about
3–5% of all cases diagnosed before the fifteenth birthday. However, in spite of low numerical
values, this is an important diagnosis, since we are dealing with a vulnerable age group with
major and special problems related to diagnosis, treatment and psychosocial follow-up.
Efforts should be made to establish a molecular genetic diagnosis as early as possible
(e.g. homozygous glucokinase deficiency, defects of the ATP-sensitive potassium channel,
chromosome 6 imprinting abnormalities). This is particularly important, since patients with
Kir6.2 and SUR1 defects can now be treated with oral sulfonylureas. Major advancements
have been obtained and continue to be made with respect to diagnosis and classification.
Differentiation between transient and permanent neonatal diabetes can only be done after
long-term follow-up. Patients should be scrutinized for comorbidity (e.g. celiac disease,
Wolcott-Rallison syndrome). Type 1 diabetes is probably the most prevalent subtype, partic-
ularly after the first year of life. Insulin treatment in infancy continues to represent major
technical, medical and psychological challenges. Family support is mandatory and close
attention should be paid to psychosocial issues.
In spite of diagnostic and therapeutic improvements, diabetes mellitus in

neonates and infants continues to be a major challenge in clinical pediatrics.
The presenting symptoms may be unspecific, causing delayed diagnosis.
Insulin treatment and blood glucose monitoring are particularly demanding.
Episodes of hypoglycemia and ketoacidosis are relatively frequent. The devel-
oping brain is vulnerable to low blood glucose, increasing the risk of hypo-
glycemic seizures and epilepsy.
Diabetes mellitus in infants was traditionally subdivided into (1) neonatal
forms, (2) classical type 1 diabetes, and (3) diabetes associated with special
syndromes. Recently, we have witnessed a spectacular advancement in molecu-
lar genetic diagnostics. Thus, a molecular diagnosis is now possible in glucoki-
nase deficiency [1], defects of the pancreatic ATP-sensitive potassium (KATP)
channel [2–4], Wolcott-Rallison syndrome (WRS) [5] and chromosome 6
imprinting abnormalities [6]. Based on a genetic diagnosis, oral treatment with
sulfonylurea has become a real and attractive alternative [7, 8].
We here review recent advancements in diagnosis and treatment of neona-
tal and infancy-onset diabetes. In this article, infancy is defined as the first 2
years of life.
Early-Onset Diabetes: Magnitude of the Problem
The occurrence of diabetes in the youngest age groups has been estimated
in several reports, covering neonates, infants 0–1 year and 0–2 year olds.
Among 3,847 juvenile diabetic patients treated at the Joslin Diabetes Center in
Boston, 1922–1956, 118 had onset before the age of 2 years [9]. Of these, only
13 had onset before the age of 1. Since that time, the relative frequency of dia-
betes in young children may have increased [10], but still, in 4,702 Swedish
patients with type 1 diabetes observed 1983–1998, there were only 51 with
onset in the first year of life. In terms of numerical values the problem of early-
onset diabetes mellitus is therefore small. However, as will be stressed in the
following, diabetes in the youngest age groups represents major and distinct
medical, psychosocial as well as technical problems [11].
A recalculation of the data of Jeffersen et al. [12] gives an age-specific
incidence of 6.2/100,000 per year for the age group 0–2 years. Studying the age
group 0–2 years in a Norwegian cohort, Mjellem et al. [13] found a consider-
ably higher incidence (10.9/100,000). In an Italian study [14], the incidence of
diabetes in the first year of life was 1.7/100,000, corresponding well with an
incidence of 2/100,000 in a German work [15]. It is quite clear that whereas
diabetes is rare in the first year of life, there is a considerable increase already
in the second year [9, 10, 13].
How frequent is autoimmune diabetes in the youngest age groups? Iafusco
et al. [14] concluded that diabetes during the first 6 months of life is most often
not associated with autoimmune markers, indicating that type 1 diabetes is rare
in this age group. Furthermore, in this age group a ‘protective’ HLA genotype
was often present. It is still interesting that there are case reports suggesting
autoimmunity in neonatal diabetes [16, 17].
Diabetes Mellitus in Infancy 95

Neonatal Diabetes
Neonatal diabetes is a clinical concept, defined as hyperglycemia during

the first month of life, requiring insulin therapy and lasting ⬎2 weeks. The con-
cept may become superfluous as more and more biochemical and genetic enti-
ties are established. As shown below, a number of these patients may now be
successfully classified on a molecular genetic basis. Thus, we think in the
future diabetes will most likely be grouped on a molecular basis, for instance
Kir6.2 diabetes and glucokinase diabetes. However, the concept of neonatal
diabetes may in some instances still be useful, and there is a considerable liter-
ature on the subject.
Traditionally, a distinction has been made between transient neonatal dia-
betes (TNDM) and permanent neonatal diabetes. This classification is notori-
ously difficult, since ‘transient’ cases may have relapses, and ‘permanent’ cases
may show remissions; depending upon the duration of follow-up. It is also
doubtful whether this distinction has any biochemical or genetic foundation.
The most common cause of neonatal diabetes seems to be imprinting anomalies
on chromosome 6q24, followed by mutations in Kir6.2.
Fösel [18] reviewed 139 cases of neonatal diabetes reported prior to
1995. With very few exceptions the patients had been treated with insulin in
doses of 0.2–1.0 IU/kg. TNDM had been treated for 2–16 weeks and seldom
beyond the first year of life. Several patients with TNDM showed impaired
glucose tolerance on follow-up, and 13 of 65 cases classified as TNDM later
developed permanent diabetes. Mühlendahl and Herkenhoff [19] reviewed 57
infants with neonatal diabetes. Transient diabetes was found in about 50%.
Forty-one cases were small for gestational age. About 50% of the patients did
not have typical type 1 diabetes. HLA-DR3 and/or DR4 haplotypes were
found in only 7 cases.
Oral treatment of neonatal diabetes with sulfonylurea is mentioned in sev-
eral early reports. Successful chlorpropamide treatment was reported by Oseid
et al. [20] and Parameswanappa and Douglas [21]. Pagliara et al. [22], studying
TNDM, observed insulin response during a tolbutamide tolerance test at 52
days of age but no response in the early neonatal period. In the patient studied
by Sodoyez-Goffaux and Sodoyez [23] tolbutamide did not trigger insulin
secretion in a twin with transient diabetes. Nielsen [24] experienced unsuccess-
ful chlorpropamide treatment.
Fösel [18] described development of mental retardation and/or spasticity,
attributed to severe hypoglycemia in some patients. However, according to
Mühlendahl and Herkenhoff [19], the prognosis for health and mental develop-
ment is generally good.
Søvik/Tansek/Sagen/Njølstad 96
Monogenic Diabetes
Monogenic diabetes in children and young adults was recently comprehen-

sively reviewed by Slingerland [25]. We here discuss monogenic diabetes in
neonates and infants from a clinical management point of view. This is based on
our own experiences and the International Society for Pediatric and Adolescent
Diabetes Clinical Practice Consensus Guidelines 2006–2007 [26] (table 1).
Glucokinase Deficiency
Homozygous glucokinase deficiency presents as congenital diabetes mel-
litus [1, 31, 32] in growth-retarded newborn infants. Hyperglycemia may be
present on the first day of life, and insulin requirement is absolute. The man-
agement is a demanding task, requiring diluted insulin solutions. With insulin
replacement, rapid catch-up growth is obtained. In our experience, the insulin
dose during long-term follow-up is close to 1 IU/ kg body weight. Good meta-
bolic control is difficult to obtain. On theoretical grounds, one would expect
response to sulfonylurea. However, in a trial with glibenclamide we observed
no effect on neither blood glucose or plasma insulin response [unpubl. data].
Furthermore, there was no insulin response during loading tests with glucagon
or arginine.
Mutations of the KATP Channel

Activating mutations in either part of the pancreatic KATP channel, Kir6.2
and SUR1, can cause neonatal diabetes [2–4]. Kir6.2 is more often associated
with the permanent form and SUR1 the transient and relapsing form, but the
opposite situations also exist [4, 33, 34]. Although the molecular mechanisms
of mutations in Kir6.2 and SUR1 are distinct, the cellular mechanism reducing
insulin release is common to both. In line with this, the key clinical features low
birth weight, age at diagnosis ⬍7 months and a severe hyperglycemia or
accompanying ketoacidosis are not significantly different [2, 4]. Due to some
common features with type 1 diabetes, such as presence of ketoacidosis and
undetectable C-peptides, these patients are often treated with insulin. In the
first subject successfully switched from insulin to sulfonylurea, detectable
C-peptides after 1 oral tolbutamide dose revealed endogenous insulin production
could be stimulated by a sulfonylurea. Subsequently, a treatment trial was set up
[7]. As figure 1 shows, we titrated the sulfonylurea dose by minute doses and
increments until the insulin requirement suddenly decreased at a sulfonylurea
dose between 0.1 and 0.2 mg/kg/day. A dose of 0.4 mg/kg/day was necessary to
wean insulin. This girl has now been off insulin for 3 years and 2 months.
Interestingly, she is presently treated with a dose of 0.2 mg/kg/day, about the

Table 1. Monogenic diabetes mellitus in neonates and infants (modified from references [25, 26])
Gene; clinical Number Median birth Median age Other features

syndrome; of cases weight, g at diagnos
inheritance described in weeks
ZAC/HYAMI ⬵150 2,100 0.5 [0–4] macroglossia (23%)

imprinting (–2.94)
defect on 6q24
Kir6.2 (KCNJ11); ⬵100 2,580 6 [0–260] developmental delay (20%), epilepsy
spontaneous (–1.73) (6%), diabetic ketoacidosis (30%)
dominant (60%)
SUR1 (ABCC8) 11 3,040 4 [0–17] developmental delay (18%)
EIF2AK3; WRS; 30 13 [6–65] epiphyseal dysplasia (90%),
recessive osteopenia (50%), acute liver failure
(75%), developmental delay (80%),
hypothyroidism (25%)
FOXP3; IPEX 14 2,860 6 [0–30] chronic diarrhea with villous atrophy
syndrome; X-linked (–1.2) (95%), pancreatic and thyroid
autoantibodies (75%), thyroiditis
(20%), eczema (50%), anemia (30%)
GCK (glucokinase); 6 1,720 ⬍2 parents have fasting hyperglycemia
dominant (–2.75) as heterozygotes
IPF1; dominant 2 2,140 parents may have early-onset
(–2.97) diabetes as heterozygotes
HNF-1␤ spontaneous 2 1,900 ⬍3 renal developmental disorders,
dominant (60%) (–3.21) epilepsy, mental retardation,
elevated liver enzymes
PTF1A; recessive 3 1,390 severe neurological dysfunction and
(–3.8) cerebellar hypoplasia
GLIS3; recessive 6 1,725 0 congenital hypothyroidism,
glaucoma, hepatomegaly, liver
fibrosis, facial dysmorphology
CFTR; cystic- 4 2,800 12 [3–88] failure to thrive, respiratory and
fibrosis-related diabetes gastrointestinal symptoms
Figures in parentheses represent standard deviation scores and values in square brackets are ranges. Data for
cystic fibrosis are from [27–30]. IPEX ⫽ Immunodysregulation, polyendocrinopathy, enteropathy, X-linked.
same dose as the threshold dose (fig. 2). Several other reports support these
initial findings [35–41], and a large international multicenter study has recently
established that these patients cannot only successfully be treated with sulfony-
lurea but also achieve an improved metabolic control without increased frequency
18 10
Start sulfonylurea
16 9
Sulfonylurea dose (mg/day) or insulin dose (U/day)

8
14
Blood glucose (mmol/l) or HbA1c (%)
7
12
6
10
5
8
4
6
3
4
2
2 1
0 0
⫺28 ⫺18 ⫺8 2 12 22 32 42 52 62 72 82 92 102 112 122
Time (days after startof sulfonylurea treatment)
Fig. 1. An infant diagnosed as having permanent neonatal diabetes due to a mutation in

Kir6.2 (F333I) successfully switched from insulin injections to a sulfonylurea (gliben-
clamide). Measurements of capillary glucose ( ), HbA1c ( ), administered dose of sulfony-
lurea (x) and insulin (䉭) are presented. Each point of capillary glucose is an average of 5–6
daily measurements performed by a standard glucose monitoring at home. HbA1c was mea-
sured at the hospital. The arrow indicates the initiation of sulfonylurea treatment. The dose
was increased every 3 days, and the dose of insulin was reduced in parallel, according to glu-
cose monitoring. There was no deterioration of metabolic control during a 6-month period
evaluated by the HbA1c values. With permission from The American Diabetes Association.
of hypoglycemia [42]. Moreover, many patients can reduce the dose sulfony-
lurea without worsening the metabolic control. Compared with adults, the sul-
fonylurea dose is high, most often in the range of 0.2–0.6 mg/kg/day, although
some may temporarily need as much as 1 mg/kg/day. Our experience is that side
effects are few and the children achieve normal growth and development. One
report interestingly suggests the neurological delay associated with some muta-
tions in Kir6.2 improves on sulfonylurea treatment [42].
Chromosome 6 Imprinting Abnormalities

The most common chromosome 6 imprinting abnormalities associated
with neonatal diabetes mellitus encompass paternal uniparental isodisomy and

0.70
0.60
Insulin (U/kg/day) or sulfonylurea
0.50
(mg/kg/day)
0.40
0.30
0.20
0.10
0.00
⫺2 ⫺1.5 ⫺1 ⫺0.8 ⫺0.3 0 1 3 5 8 9 10 12 14 15 22 25 29
a Time off insulin (months)
10.00
9.00
8.00
7.00
6.00
HbA1c (%)
5.00
4.00
3.00
2.00
1.00
0.00
⫺2 ⫺1.5 ⫺1 ⫺0.8 ⫺0.3 0 1 3 5 8 9 10 12 14 15 22 25 29
b Time off insulin (months)
Fig. 2. a Long-time treatment with sulfonylurea of a child with a Kir6.2 mutation

(F333I). Initially, the dose of sulfonylurea ( ) was increased every 3 days, and the dose of
insulin (x) was reduced in parallel and eventually discontinued. After a near normalization of
HbA1c values, the dose of sulfonylurea was reduced. b HbA1c values in the same patient that
was switched from insulin to sulfonylurea. There was an improved metabolic control during
treatment with sulfonylurea evaluated by reduction in and stabilization of the HbA1c values.
This was accomplished even with a simultaneous decrease of the sulfonylurea dose.
parentally inherited duplication of 6q24 [6]. Methylation defects are, however,

more frequently identified as a cause of TNDM [43]. These patients may be
growth retarded at birth but show normal catch-up growth as soon as insulin
is introduced. The diabetes is usually classified as transient, but permanent
diabetes may appear after shorter or prolonged remission periods (unpubl.
obs.). Initially, these patients require insulin, but during follow-up, sulfonylurea
may be tried. If insulin is required in short- or long-term management, small
doses (0.5 IU/kg body weight) may be sufficient. Importantly, too much insulin
may lead to hypoglycemia, variable blood glucose and increasing glycosylated
hemoglobin.
Wolcott-Rallison Syndrome
Typical features of WRS are permanent neonatal or early-onset infancy
diabetes mellitus, multiple epiphyseal dysplasia, postnatal growth retardation,
and hepatic and renal dysfunction [44]. A molecular genetic diagnosis is now
possible [5]. Prenatal diagnosis may thus be offered. If WRS is not recognized
in an infant with early-onset diabetes, postnatal growth retardation and meta-
bolic crises may not be correctly diagnosed and managed. The metabolic crisis
in WRS presents with nonketotic hyperglycemia, a clinical condition that may
be confusing in a patient with diabetes, where diabetic ketoacidosis is the
expected complication. In a patient with WRS, we noticed that her insulin
requirement was similar to that of type 1 diabetes.
Type 1 Diabetes
Although type 1 diabetes is uncommon in small children, this disease still

seems to be the most prevalent type of diabetes during the first 2 years of life.
Once it occurs, the autoimmune attack against ␤-cells in very young children
may be particularly strong. In the study by Komulainen et al. [45] ⬎50% of the
youngest patients tested positive for 4 antibodies. Furthermore, in that study
children with a diagnosis established before the age of 2 years showed a high-
risk HLA genotype more often than older children.
The diagnosis of diabetes mellitus, which is easy in most children, may
cause problems in the youngest ones. Typical symptoms, such as polydipsia,
polyuria and weight loss, may be seen in only 50% [13]. Instead, the disease
may present with infection, fever, diarrhea and failure to thrive [13, 46, 47]. It is
therefore strongly recommended that the physician who first sees these infants
performs tests for sugar in blood and/or urine. At presentation in hospital, there
are often severe hyperglycemia and ketoacidosis [13]. Komulainen et al. [45]
observed pH ⬍7.10 in 10% of their cases. Clinically, the patients may be ill
appearing and dehydrated, with apathy and restlessness [13, 47]. A prolonged
stay in hospital (⬎4 weeks) is not uncommon, to secure stable blood glucose,
adequate nutrition and growth, and provide practical and theoretical know-how
to parents.

During follow-up, several important points should be noted. A typical
remission period may not appear [13] or may come at a lower rate than at an
older age [47]. Episodes of ketoacidosis as well as severe hypoglycemia are rel-
atively common, requiring hospitalization. After the initial stay in hospital, 14
patients were readmitted altogether 38 times [13]. In 13 of these instances there
were hypoglycemic convulsions. Celiac disease as a complicating disorder is
relatively common [47].
Insulin treatment and glucose control are technically demanding in uncoop-
erative subjects [13, 48]. Lteif and Schwenk [49], who reported a higher insulin
requirement and higher glycosylated hemoglobin in younger than in older chil-
dren, concluded that tight control is achievable but at an expense of more hypo-
glycemic reactions. Insulin pump treatment should always be considered [50].
Particularly close attention should be paid to psychosocial issues. It is
imperative that the families receive adequate support. An infant with diabetes
may require supervision 24 h a day, causing an intolerable amount of parental
stress and strain. A broken home may be the consequence. The treatment team
should always include a child psychologist and a social worker. Whenever pos-
sible, a good family network should be established.
Conclusions
It is well established that diabetes mellitus is a heterogeneous condition.

This fact is particularly relevant in the young age groups. In very young chil-
dren with diabetes mellitus, efforts should be made to obtain a molecular
genetic diagnosis. On such a basis, patients may be allocated to replacement of
insulin by oral treatment with sulfonylurea. A molecular genetic diagnosis is
also the basis of genetic counseling. Insulin treatment and blood glucose con-
trol are demanding and require close contact between the family and the treat-
ment team, with physician, nurse, dietician, psychologist and social worker.
Close attention should be paid to psychosocial issues.
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Pål Rasmus Njølstad

Department of Pediatrics, Haukeland University Hospital
NO–5021 Bergen (Norway)
Tel. ⫹47 55 975153, Fax ⫹47 55 975159, E-Mail pal.njolstad@uib.no

Insights in Congenital Hyperinsulinism

Khalid Hussain
London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street
Hospital for Children NHS Trust and Developmental Endocrinology Research Group
Molecular Genetics Unit Institute of Child Health, University College London,
London, UK
Abstract
Congenital hyperinsulinism is characterized by the unregulated secretion of insulin
from pancreatic ␤-cells. The inappropriate insulin secretion causes severe and persistent
hypoglycaemia, which is a potent cause of brain damage if inappropriately managed. So far
mutations in 5 different genes have been described which lead to inappropriate insulin
secretion. The most common cause of congenital hyperinsulinism is autosomal recessive
mutations in the genes ABCC8 and KCNJ11 encoding the 2 subunits (SUR 1 and Kir6.2,
respectively) of the pancreatic ␤-cell ATP-sensitive potassium channel. Autosomal domi-
nant mutations in the genes encoding glucokinase (GCK) and glutamate dehydrogenase
(GLUD1) lead to inappropriate insulin secretion by increasing the ATP/ADP ratio in
the ␤-cells. Autosomal recessive mutations in the HADHSC gene (encoding the enzyme
short-chain L-3-hydroxyacyl-CoA dehydrogenase) have been linked to defects in fatty acid
oxidation and hyperinsulinism. Finally some patients have been described with exercise-
induced hyperinsulinaemic hypoglycaemia but the genetic basis of this is unclear at
present. Recent advances in 18fluoro-L-Dopa positron emission tomography scanning sug-
gest that this is a highly sensitive method for differentiating diffuse from focal disease as
well as accurately locating the focal lesion. Despite huge advances in the last 10 years the
mechanisms leading to hyperinsulinaemic hypoglycaemia are still unknown in ⬎50% of
patients.
Congenital hyperinsulinism (CHI) is the most common cause of persistent

and recurrent hypoglycaemia in neonates and infants during their first year of
life [1]. CHI can be a major cause of severe mental retardation and epilepsy if
not treated properly [2]. Both sporadic and familial variants of CHI are recog-
nized, with sporadic forms being relatively uncommon (incidence 1 per 35,000
live births), and familial forms being common in communities with high rates
of consanguinity; in these communities, the incidence may be as high as 1 in
2,500 live births [3].
In patients with CHI there is marked heterogeneity with respect to clinical
presentation, histology and molecular biology [4, 5].
The diagnosis of CHI is suggested by the persistent and recurrent episodes
of hypoglycaemia associated with inappropriately raised serum insulin levels
as well as suppressed serum fatty acids and ketone bodies [6]. Pathologically
CHI can be classified into 2 major subgroups: ‘channelopathies’ and
‘metabolopathies’ [7]. Channelopathies refer to defects in the pancreatic ␤-cell
ATP-sensitive potassium (KATP) channels that lead to unregulated insulin secre-
tion. Metabolopathies cause CHI by either altering the concentration of intra-
cellular signalling molecules (such as ATP/ADP) or by the accumulation of
intermediary metabolites.
The histological differentiation of CHI into focal and diffuse disease has
radically changed the surgical management of patients with CHI [8]. Correct
localization and limited excision of the focal lesion will result in complete
cure of the patient. Recent advances in 18fluoro-L-Dopa positron emission
tomography (PET) scanning are beginning to provide greater accuracy in
preoperative differentiation of focal and diffuse disease and correct localiza-
tion of focal lesions [9]. In contrast some forms of medically unresponsive dif-
fuse disease will still require a near total pancreatectomy, greatly increasing
the risk of postpancreatectomy diabetes mellitus and pancreatic exocrine
insufficiency.
The clinical severity of CHI varies mainly with age at onset of hypogly-
caemia and has major consequences in terms of therapeutic outcome and
genetic counselling. Therapy is aimed at preventing brain damage from hypo-
glycaemia, allowing normal psychomotor development, establishing normal
feeding pattern (content and frequency for the age of the child), to ensure nor-
mal tolerance to fasting for age without developing hypoglycaemia and to
maintain family integrity. This chapter provides an overview on CHI, firstly
focussing on the role of ␤-cell KATP channels in regulating insulin secretion,
then describing the molecular mechanisms that lead to unregulated insulin
secretion and finally reviewing the recent advances in 18fluoro-L-Dopa PET
scanning for differentiating focal from diffuse CHI. The clinical aspects of the
disease are covered in other reviews [1, 6] and will not be the focus of this
chapter.
Congenital Hyperinsulinism 107

Congenital Hyperinsulinism Due to Channelopathies
(Defects in Pancreatic ␤-Cell KATP Channels)
Role of Pancreatic b-Cell KATP Channels in

Glucose-Induced Insulin Secretion
KATP channels are a combination of transport ATPases complexed with
potassium ion channel subunits. KATP channels are heteromultimers of 2 types
of subunit, inward rectifiers, KIR6.x, and sulphonylurea receptors, SURs,
members of the ATP-binding cassette (ABC) superfamily. Differing combina-
tions of Kir6.1 or Kir6.2 and SUR1, SUR2A or SUR2B (where SUR2A and
SUR2B are splicing variants) constitute KATP channels with distinct nucleotide
and pharmacological sensitivities [10, 11]. The ABC proteins are characterized
by well-conserved nucleotide-binding folds and multispanning transmembrane
domains. ABC proteins are found in most cells of all species from prokaryotes
to humans and hence make up one of the largest protein superfamilies [12].
KATP channels have a key role in the physiology of many cells, and defects
either in the channel itself or in its regulation can lead to diseases in humans
[13, 14]. Functionally KATP channels provide a means of linking the electrical
activity of a cell to its metabolic state by sensing changes in the concentration
of intracellular nucleotides and in some cases they mediate the actions of hor-
mones and transmitters. Opening and closing of the KATP channels is influenced
by the intracellular concentrations of nucleotides, particularly ATP and ADP
[15], lipids such as phosphatidylinositol-4,5 phosphate [16], and long-chain
acyl-CoA esters [17]. In addition to being regulated by various nucleotides,
KATP channels are also modulated by hormones and neurotransmitters, and there
is some evidence that intracellular signals such as G proteins may also modu-
late KATP channel function [18].
In the pancreatic ␤-cell Kir6.2 and SUR1 components (encoded by the
genes ABCC8 and KCNJ11, respectively) constitute the KATP channel [19]. The
function of the KATP channel is best understood in the pancreatic ␤-cell, where
it couples changes in plasma glucose concentration to electrical excitability and
insulin release [20]. Under euglycaemic conditions, KATP channels are main-
tained in an open state, resulting in K⫹ efflux and thus clamping the resting
membrane potential at approximately ⫺70 mV [21]. In ␤-cells, the KATP chan-
nel is proposed as a critical link in the pathway of glucose-induced insulin
release. Insulin secretion from ␤-cells occurs in 2 phases, an early transient
phase and a secondary sustained phase. Glucose stimulates insulin secretion by
generating triggering (the mechanism of regulated insulin secretion involving
the KATP channels is referred to as the KATP-channel-dependent pathway or first
phase of insulin secretion) and amplifying signals (KATP-channel-independent
pathway or second phase of insulin secretion) in ␤-cells [22]. Following the
Hussain 108
ingestion of a meal, the plasma glucose concentration increases. Glucose is
taken up by the pancreatic ␤-cells and is metabolized by glucokinase and mito-
chondrial events raising the intracellular glucose concentration. This results in
an increase in the intracellular concentration ratio of ATP/ADP [23, 24].
Oxidative glucose metabolism leads to the generation of NADH and FADH2,
which in turn donate electrons to the mitochondrial inner membrane electron
transport chain. As electrons move down this chain, protons are pumped out of
the mitochondrial matrix by complexes I (NADH-ubiquinone oxidoreductase),
III (ubiquinone-cytochrome-c oxidoreductase) and IV (cytochrome oxidase),
creating a proton electrochemical gradient. This proton motive force is then
used by ATP synthase, an inner membrane protein complex, to generate ATP
from ADP, hence increasing the ATP/ADP ratio.
The free concentrations of ADP and ATP in the ␤-cell cytoplasm are esti-
mated to be about 40 ␮M of ADP and ATP to be approximately 100-fold higher
[24]. Glucose metabolism causes a reduction in the concentration of ADP, lead-
ing to an increase in the ATP/ADP ratio. This is presumed to depolarize the
plasma cell membrane, leading to Ca2⫹ entry via voltage-gated calcium chan-
nels. The rise in intracellular Ca2⫹ concentration then triggers the exocytosis of
a small pool of secretary granules that is responsible for the first phase of glu-
cose-stimulated insulin release. KATP channels are open in resting, unstimulated
␤-cells and, along with the Na⫹-K⫹-ATPase, establish a resting membrane
potential of approximately ⫺65 mV [25]. The mechanism responsible for spon-
taneous channel openings has been proposed to involve the low intracellular
ATP/ADP ratio that exists in resting ␤-cells. ADP has been shown to be both a
potent agonist of KATP channels and to reverse the inhibitory effects of ATP
even when there is a ⬎20-fold excess in the concentration of ATP relative to
ADP at the cell membrane [25].
Circulating insulin stimulates glucose uptake in insulin-sensitive tissues
(mostly liver, adipose tissue and muscle), lowering the blood glucose concen-
tration. Conversely, a fall in the intracellular ATP/ADP ratio during the inter-
prandial state is presumed to open KATP channels, causing membrane
hyperpolarization and cessation of insulin release.
KATP channels also determine second-phase insulin secretion from ␤-cells,
which is brought about by the gradual augmentation and potentiation of Ca2⫹-
triggered insulin release, a process that entails the preparation of previously
nonreleasable granules for exocytosis. This pathway, which is termed the
‘amplification’ or ‘augmentation’ pathway, is also referred to as ‘KATP-channel-
independent’. The amplifying pathway also depends on glucose metabolism but
does not involve a further increase in intracellular Ca2⫹ concentration – it
serves to amplify the efficacy of Ca2⫹ on exocytosis of insulin granules through
biochemical mechanisms that remain incompletely identified [26, 27]. The

molecular mechanism by which glucose metabolism augments distal signalling
events is not completely resolved. Proposed coupling factors include increased
ATP/ADP and GTP/GDP ratio, cytosolic levels of long-chain acyl-CoA, the
pyruvate-malate shuttle and glutamate export from mitochondria [28].
Molecular Mechanisms of Congenital Hyperinsulinism

Due to Channelopathies
The commonest genetic causes of CHI are autosomal recessive mutations in

ABCC8 and KCNJ11 genes encoding the 2 subunits of the pancreatic ␤-cell KATP
channels [29–32]. Autosomal dominant mutations have also been described
[33, 34]. These mutations result in differing abnormalities of recombinant KATP
channels including protein folding, protein synthesis defects, assembly and
trafficking defects, and alterations in both nucleotide regulation and open-state
frequency. In ⬎50% of patients, screening has failed to define the genetic basis
of this disease [35]. In some populations mutations in the ABCC8 and KCNJ11
genes account for only about 20% of cases of CHI, hence suggesting that there
may well be other genes involved [36]. Potential molecular mechanisms that can
lead to CHI include defects in KATP channel turnover, channel trafficking, chan-
nel assembly and alterations of channel sensitivity to nucleotides.
Rate of Turnover of Channel

The mechanisms that control the maturation and assembly of KATP chan-
nels are not well understood. Pulse-labelling studies have shown that when
Kir6.2 is expressed individually, its turnover is biphasic with about 60% being
lost with a half-life of 36 min. The remainder converts to a long-lived species
(half-life 26 h) with an estimated half-time of 1.2 h. SUR1 has a long half-life of
25.5 h when expressed alone. When Kir6.2 and SUR1 are co-expressed, they
associate rapidly and the fast degradation of Kir6.2 is eliminated [37]. Two
mutations, Kir6.2 (W91R) and SUR1 (⌬F1388), identified in patients with the
severe form of familial hyperinsulinism, profoundly alter the rate of Kir6.2 and
SUR1 turnover, respectively. Both mutant subunits associate with their respec-
tive partners but dissociate freely and degrade rapidly. The 2 subunits are able to
form complexes with their partners but the complexes are short-lived, implying
the subunits dissociate and degrade.
Trafficking Defects
Trafficking of KATP channels requires that the endoplasmic reticulum-
retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during
channel assembly. Some mutations in the ABCC8 gene such as R1437Q(23)X,
Hussain 110
⌬F1388 and R1394H cause a trafficking defect by affecting the exit of channel
subunits from the endoplasmic recticulum compartment [31, 38, 39]. The
R1437Q(23)X mutation in exon 35 of ABCC8 truncates 200 amino acids from
the COOH-terminal region of the protein, an area that contains the anterograde
signalling sequence (L1566, L1567.F1574) and residue L1544, which is part of
the cloaking region for the RKR sequence. This defect will affect the exit of
channel subunits from the endoplasmic recticulum.
The pivotal role of the RKR signal in allowing the channels to express cor-
rectly on the ␤-cell membrane is illustrated by the fact that inactivation of the
RKR signal in ⌬F1388 SUR1 by mutation to AAA (⌬F1388 SUR1AAA) and co-
expression of ⌬F1388 SUR1AAA with Kir6.2 leads to partial surface expression
of the mutant channel [38]. Moreover, mutant channels were active. Compared
with wild-type channels, the mutant channels have reduced ATP sensitivity and
do not respond to stimulation by MgADP or diazoxide. The RKR → AAA muta-
tion alone has no effect on channel properties. These studies showed that that
F1388 in SUR1 is critical for normal trafficking and function of KATP channels
[38]. Other mutations such as the R1394H-SUR1 cause a trafficking disorder by
effecting retention of mutant proteins in the trans-Golgi network [39]. Moreover,
some trafficking defects caused by the DF1388-SUR1 mutation can be partially
overcome by inactivation of the RKR ER-retention signal in SUR1 in vitro [38].
Mutations in the KCNJ11 gene can also cause defective trafficking and
truncated nonfunctional proteins. For example the Kir6.2 mutation (Y12X)
causes the synthesis of a truncated nonfunctional protein [32], whereas another
mutation (W91R) showed defective channel assembly with a rapid degradation
in the endoplasmic reticulum [37] Recently a new homozygous mutation
(H259R) in Kir6.2 has been shown to lead to nonfunctional KATP channels with
impaired trafficking to the cell membrane [40].
Channel Regulation
The SUR1 subunit plays a key role in determining the pharmacological
regulation of KATP channels, with SUR1 acting as a conductance regulator of
Kir6.2. The sensitivity of KATP channels to changes in ATP, ADP and guanosine
(GTP, GDP) nucleotides involves both subunits. The functional regulation of
KATP channels is induced by changes in the ATP/ADP ratio. This involves coop-
erative interactions of nucleotides at both subunits, with the actions of ATP-
induced inhibition of Kir6.2 being countered by the activating of ADP at SUR1.
Hence mutations that affect the regulation of the KATP channels by altering its
sensitivity to changes in ADP/ATP will lead to unregulated insulin secretion.
Several mutations have now been described that result in the loss of ADP-
dependent gating properties of the channel [33, 41–43]. Loss of ADP-depen-
dent gating results in the constitutive inhibition of KATP channels by ATP.

Recently a dominant missense CHI causing mutation F55L in Kir6.2 has
been shown to greatly reduce the open probability of KATP channels in intact
cells without affecting channel expression [44]. It was shown that the low chan-
nel activity was likely due to reduced channel response to membrane phospho-
inositides and/or long-chain acyl CoAs, as application of exogenous
phosphatidylinositol-4,5 phosphate or oleoyl CoA restored channel activity
similar to that seen in-wild type channels. This may provide a link between KATP
channels and their regulation by membrane phosphoinositides and/or long-
chain acyl CoAs.
Congenital Hyperinsulinism Due to Metabolopathies
Metabolopathies cause CHI either by altering the concentration of intracel-

lular signalling molecules (such as ATP/ADP) or by the accumulation of inter-
mediary metabolites. Autosomal dominant mutations in the genes encoding
glutamate dehydrogenase (GDH) (GLUD1) and glucokinase (GCK) lead to
inappropriate insulin secretion by increasing the amount of ATP in the ␤-cells.
More recently autosomal recessive mutations in short-chain L-3-hydroxyacyl-
CoA dehydrogenase (HADHSC) have been linked to defects in fatty acid oxida-
tion and hyperinsulinism.
Congenital Hyperinsulinism Due to Gain of Function Mutations in

Glutamate Dehydrogenase
GDH is encoded by a single nuclear gene (GLUD1) located on chromo-

some 10q23.3. It is a mitochondrial matrix enzyme that is expressed in high
levels in some tissue such as the brain, liver and ␤-cells. GDH catalyzes the
reversible oxidative deamination of glutamate to ␣-ketoglutarate and ammonia.
The increased ␣-ketoglutarate enters the citric acid cycle to generate ATP,
which then has the effect of closing the ␤-cell KATP channels. The enzyme activ-
ity of GDH is regulated by the complex interplay of inhibitors and activators
acting at specific inhibitory and activating allosteric sites [45]. GTP is a potent
inhibitor of GDH, whereas leucine is an activator of GDH activity.
Activating mutations in GDH are the second commonest cause of CHI.
These mutations in GDH underlie the molecular basis of the hyperinsulinism/
hyperammonaemia syndrome (HI/HA) and may explain the ‘leucine-sensitive’
hypoglycaemia described in previous years [46]. The HI/HA syndrome is
caused by missense mutations of GDH that reduce the sensitivity of the enzyme
to allosteric inhibition by the high-energy phosphates, GTP and ATP. Mutations
Hussain 112
which lead to loss of inhibition by GTP cause leucine to increase the oxidation
of glutamate, thereby raising the ratio of ATP/ADP in the pancreatic ␤-cell. The
increased ratio of ATP/ADP then triggers closure of the KATP channel, opening
the voltage-gated calcium channel, raising cytosolic calcium and triggering
release of insulin. Mutations that cause HI/HA syndrome are single amino acid
substitutions which occur either in the GTP inhibitory allosteric binding site or
in an antenna region of the enzyme which plays a role in communicating with
adjacent enzyme subunits. Mutations have also been reported in the presumed
catalytic site and outside the GTP allosteric domain of the enzyme [47]. The
majority of cases (80%) are de novo, but about 20% of cases are familial and
transmitted in an autosomal dominant fashion [45].
Patients with the HI/HA syndrome can present with hypoglycaemia either
in the neonatal period or later on in childhood. The hyperinsulinaemic hypo-
glycaemia may be detected during fasting but more importantly the postpran-
dial blood glucose response to a protein meal is more sensitive than prolonged
fasting for detecting hypoglycaemia in the HI/HA syndrome [48]. These
patients also have a mildly elevated plasma ammonia concentration which
appears to be asymptomatic. Patients show no signs of lethargy or headaches,
typical of other forms of hyperammonaemia. The mechanism of hyperammon-
aemia is still unclear at present. Children with HI/HA syndrome have an
unusual frequency of absence-type seizures [49]. These children have an EEG
pattern of generalized epilepsy that resembles the seizures associated with
mutations of plasma membrane ion channels. It is unlikely that this seizure
pattern is a manifestation of ammonia toxicity. Patients with HI/HA syndrome
respond to diazoxide.
Recently GDH transgenic mice have been generated to express the human
GDH-HI H454Y mutation [50]. GDH enzyme activity is increased in islets
expressing the H454Y transgene with decreased sensitivity to GTP inhibition.
The H454Y GDH transgenic mice display hyperinsulinaemic hypoglycaemia.
Further studies confirmed that the H454Y GDH transgenic islets were more
sensitive to leucine- and glutamine-stimulated insulin secretion but had
decreased response to glucose stimulation. Further studies in these mice will
provide detailed insights into the regulation of GDH activity.
Congenital Hyperinsulinism Due to Gain of Function

Mutations in Glucokinase
Glucokinase
The low-affinity glucose-phosphorylating enzyme glucokinase (GCK) is
the flux-limiting glucose sensor in the liver and ␤-cells of the pancreas.

Activation of GCK lowers the threshold for glucose-stimulated insulin secre-
tion (‘resetting’ of the glucose-stimulated insulin release threshold), thus caus-
ing hypoglycaemia. The first activating mutation in GCK was the Val455Met,
which was a single base change, resulting in the substitution of methionine for
valine at codon 455 [51]. When expressed in vitro, the Val455Met mutation
increased the affinity of glucokinase for glucose. Several other patients with
activating mutations in GCK have now been reported [52, 53], all responsive to
medical therapy with diazoxide. However a case of severe CHI due to a ‘de
novo’ mutation in the GCK (Y214C) gene was reported which failed to respond
to medical therapy [54]. Functional studies of this mutant showed a 6-fold
increase in its affinity for glucose and the histology of the resected pancreas in
this patient revealed abnormally large and hyperfunctional islets. It is unclear
why this patient failed to respond to diazoxide, one possibility being that the
dose of diazoxide was insufficient.
Short-Chain Acyl-CoA Dehydrogenase Deficiency

Short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), encoded by
the HADHSC gene, is an intramitochondrial enzyme that catalyzes the penulti-
mate reaction in the ␤-oxidation of fatty acids, the NAD⫹-dependent dehydro-
genation of 3-hydroxyacyl-CoA to the corresponding 3-ketoacyl-CoA. The
gene is localized to chromosome 4q22-26. SCHAD expression is high in pan-
creatic ␤-cells, reflecting the use of fat as oxidative fuel. So far 3 patients with
mutations in the HADHSC gene and CHI have been reported [55–57]. The
clinical presentation can be heterogeneous either with mild late-onset intermit-
tent hypoglycaemia or severe neonatal hypoglycaemia. The first patient identi-
fied had a homozygous 773C-T transition in exon 7 of the HADHSC gene,
resulting in a Pro258-to-Leu substitution in 1 of the ␣-helices of the C-terminal
domain [55]. The mutation was predicted to prevent normal protein folding.
In vitro functional expression studies showed that the mutant enzyme had no
catalytic activity. The parents were heterozygous for the mutation. Molvern at
al. [57] demonstrated a 6-bp deletion in their patient that removed the acceptor
splice site adjacent to exon 5 of the HADHSC gene. They demonstrated that
exon 5 was skipped during the mRNA splicing process so that exon 4 was cou-
pled directly onto exon 6. This led to an in-frame deletion of 90 nucleotides in
the mature mRNA, resulting in a protein product predicted to lack 30 amino
acids. Both parents were heterozygous. The acylcarnitine profile in all
reported patients has demonstrated raised hydroxybutyrylcarnitine and urine
organic acids showed raised 3-hydroxyglutarate with decreased expression
and function of the SCHAD enzyme. The significance of the raised hydroxy-
butyrylcarnitine and 3-hydroxyglutarate to insulin secretion is unclear at
present.
Hussain 114
Table 1. Outline of the location, symbol, title of genes causing CHI and different treatment options
Gene location Symbol Title/disease inheritance and response to treatment OMIM number
11p15.1 ABCC8 ABC, subfamily C, 600509

(member 8 sulphonylurea receptor); autosomal
recessive disease usually unresponsive to diazoxide; focal:
limited pancreatectomy, diffuse: 95% pancreatectomy
11p15.1 KCNJ11 potassium inwardly-rectifying channel; autosomal recessive 600937
disease usually unresponsive to diazoxide; focal: limited
pancreatectomy, diffuse: 95% pancreatectomy
10q23.3 GLUD1 GDH; 138130
autosomal dominant disease responsive to diazoxide
7p15-p13 GCK glucokinase (hexokinase-4); 138079
autosomal dominant disease responsive to diazoxide
4q22-q26 HADHSC SCHAD; 601609
autosomal recessive disease responsive to diazoxide
Focal CHI due to mutations in the ABCC8 and KCNJ11 genes will require only a limited resection of the focal
lesion. Diffuse disease due to mutations in ABCC8 and KCNJ11 may require a 95% pancreatectomy. CHI due to
mutations in GCK, GLUD1 and HADHSC is responsive to diazoxide.
Fatty acids increase insulin secretion by affecting the concentrations of

long-chain fatty acyl derivatives as a result of the inhibitory effect of citrate and
malonyl-CoA on the rate-controlling enzyme carnitine palmitoyltransferase-1.
The mechanism of how a defect in the HADHSC gene leads to dysregulated
insulin secretion is unclear at present. However 2 recent studies [58, 59] are
beginning to provide some insights into how HADHSC may be involved in reg-
ulating insulin secretion. The first study has shown that Foxa2 (HNF3␤) is
involved in regulating the expression of HADHSC gene with studies in Foxa2-
deficient ␤-cells showing a 3-fold downregulation of HADHSC transcripts
along with the ability of Foxa2 to bind to and activate this gene [58]. More
recently an in vitro model of reduced SCHAD expression has demonstrated for
the first time that SCHAD is required directly in ␤-cells for the regulation of
basal insulin release [59]. RNAi-mediated gene suppression of HADHSC in
insulinoma cells and primary rodent islets revealed enhanced basal but normal
glucose-stimulated insulin secretion. This increase in basal insulin secretion
was not attenuated by opening of the KATP channel with diazoxide, suggesting
that SCHAD regulates insulin secretion through a KATP-channel-independent
mechanism [59]. Table 1 summarizes the location, symbol, title of genes caus-
ing CHI as well as response to medical and surgical therapy. Figure 1 outlines
the different genetic mechanisms that lead to CHI.

Defective Insulin
KATP channels exocytosis
Pancreatic ␤-cell
Loss of HADHSC
ATP/ADP
function? Mechanism
Increased of insulin secretion
GCK activity
Glucose Glucose-6-phosphate
␣-Ketoglutarate ⫹ Ammonia
GDH
Glutamate
Fig. 1. Outline of the known mechanisms that lead to CHI. Defects in the KATP chan-
nels due to mutations in the ABCC8 and KCNJ11 genes are the commonest cause of CHI.
Gain of function mutations in GLUD 1 and GCK cause an increased ATP/ADP ratio. The
increased ratio of ATP/ADP then triggers closure of the KATP channel, opening the voltage-
gated calcium channel, raising cytosolic calcium and triggering release of insulin. As
HADHSC is a negative regulator of insulin secretion, loss of function mutations in this gene
cause inappropriate insulin release.
Exercise-Induced Hyperinsulinaemic Hypoglycaemia

In exercise-induced hyperinsulinaemic hypoglycaemia strenuous physical
exercise leads to inappropriate insulin release from ␤-cells causing postexercise
hypoglycaemia [60]. These patients show increased insulin secretion in
response to intravenous pyruvate in comparison to control patients [60]. The
molecular mechanism/s of how exogenous pyruvate triggers inappropriate
insulin secretion in these patients is still unclear.
Recent Advances in Differentiating Diffuse and

Focal Congenital Hyperinsulinism
Two major histological forms of CHI have been described, diffuse and
focal [61]. Both the diffuse and focal forms share a similar clinical presentation
but result from different path-physiological and molecular mechanisms. In
addition diffuse CHI usually presents as an autosomal recessive disorder,
whereas focal CHI is sporadic. The typical diffuse form affects all the ␤-cells
and is most commonly due to autosomal recessive mutations in the genes
encoding the 2 subunits of the KATP channel.
Hussain 116
The ‘focal’ form (focal adenomatous pancreatic hyperplasia) of CHI is found
in about 50% of children and appears to be localized to one region of the pancreas.
The genetic defect in the focal form consists of germline mutations in the paternal
allele of ABCC8 and KCNJ11 encoding SUR1 and Kir6.2 on chromosome 11p15.
In addition the lesion exhibits a somatic loss of a part of the maternally inherited
chromosome 11p, which includes imprinted maternally expressed tumour sup-
pressor genes (H19 and P57KIP2), paternally expressed insulin-like growth factor-2
as well as (nonimprinted) ABCC8/KCNJ11 genes [62, 63]. This results in a corre-
sponding reduction to homozygosity of the paternal mutation and the outcome is
unregulated insulin secretion. ␤-Cells within the focal lesion do not express
p57KIP2 but insulin-like growth factor-2 is mildly increased. The somatic loss of
heterozygosity is associated with increased proliferation [62, 63].
Identification of the children who have the focal form of the disease pre-
operatively is a critical part of the management of patients with CHI. The pre-
operative localization allows radically different treatment options and medical
outcomes. Focal disease is curable with limited (partial) pancreatectomy with
few long-term complications. Until recently highly invasive methods such as
intrahepatic pancreatic portal venous sampling, the arterial calcium stimula-
tion/venous sampling and the acute insulin response testing to intravenous glu-
cose, calcium and tolbutamide were used for identifying the children with focal
and diffuse forms of the disease. Now 18fluoro-L-Dopa PET has been success-
fully used to localize the focal domain [64–66]. The principle of this test is
based on the fact that islets take up L-3,4-dihydroxyphenylalanine (L-dopa) and
convert it to dopamine by dopa decarboxylase, present in the islet cells [67].
However the precise role of dopamine in the pancreatic ␤-cells is currently
unclear. 18fluoro-L-Dopa PET can also accurately locate ectopic focal lesions
[68, 69]. 18fluoro-L-Dopa PET is highly sensitive in detecting focal lesions
compared with the previous highly invasive techniques [65].
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Dr. K. Hussain
Developmental Endocrinology Research Group
Molecular Genetics Unit, Institute of Child Health, University College London
30 Guilford Street
London WC1N 1EH (UK)
Tel. ⫹44 20 7 905 2128, Fax ⫹44 20 7 404 6191, E-Mail k.hussain@ich.ucl.ac.uk

A Clinical Approach to Severe

Insulin Resistance
David B. Savagea, Robert K. Semplea, V. Krishna K. Chatterjeea,
Jeremy K.H. Walesb, Richard J.M. Rossc, Stephen O’Rahillya
a
Departments of Clinical Biochemistry and Medicine, University of Cambridge,
Cambridge, and bDepartment of Paediatric Endocrinology, Sheffield Children’s
Hospital, and cDepartment of Diabetes and Endocrinology, Northern General
Hospital, Sheffield, UK
Abstract
Extreme forms of insulin resistance are a rare cause of type 2 diabetes. However, indi-
viduals with severe insulin resistance pose unique diagnostic and therapeutic challenges,
and have often acted as ‘experiments of nature’ providing important novel information
regarding endocrine physiology and mechanistic insights relevant to the study of more com-
mon disorders. Progress in understanding the molecular pathogenesis of such syndromes is
also beginning to yield novel therapeutic options. Severe insulin resistance typically pre-
sents in 1 of 3 ways: (1) disordered glucose metabolism including both diabetes and/or
paradoxical hypoglycaemia; (2) acanthosis nigricans, a velvety hyperpigmentation of axil-
liary and flexural skin often associated with skin tags; or (3) hyperandrogenism in girls (hir-
sutism, oligo-/amenorrhoea and polycystic ovaries). Lipodystrophy is a major cause of
severe insulin resistance and needs to be looked for very carefully, particularly in the
patients with significant dyslipidaemia and fatty liver. Specific treatments are now available
for some forms of severe insulin resistance; for example, leptin replacement in patients with
generalized lipodystrophy. In the absence of a specific diagnosis and therapy, metformin is
a useful insulin sensitizer and should be used in conjunction with aggressive diet and exer-
cise interventions.
Case History
A 13-year-old girl was referred to an endocrine centre with hirsutism, sec-

ondary amenorrhoea and lower abdominal masses. On initial clinical assessment,
she was also noted to have axilliary acanthosis nigricans. Further investigation
revealed the masses to be large fallopian tube cysts and biopsy of her ovaries
showed polycystic changes. At that time she was neither obese nor overtly
lipodystrophic. A 75-gramm oral glucose tolerance test revealed impaired glu-
cose tolerance (fasting glucose 4.4 mmol/l, 120-min glucose 8.5 mmol/l). The
corresponding insulin levels were massively increased [baseline 276 pmol/l and
120 min 9,730 pmol/l (normal fasting insulin ⬍60 pmol/l)], confirming the
presence of severe insulin resistance. The amenorrhoea and hirsutism were
treated with Dianette (an oral contraceptive with antiandrogenic properties) and
DNA was sent for candidate gene screening.
At the age of 21 years her fasting glucose was 3.9 mmol/l with a corre-
sponding insulin of 346 pmol/l. At this time she was also noted to be hyperten-
sive (BP 150/110 mm Hg) and hypertriglyceridaemic (fasting triglycerides
10.1 mmol/l). Liver enzymes (alanine aminotransferase and ␥-glutamyl trans-
ferase) were mildly increased in keeping with hepatic steatosis, leptin levels
were within the expected range for a woman with a BMI of 29 (12.4 ug/l; refer-
ence range 8.6–38.9 ␮g/l) and adiponectin levels were unremarkable (6.6 mg/l;
BMI/gender-matched 95% CIs 3.5–15.5 mg/l).
Screening of the PPARG gene identified a novel heterozygous frameshift
premature stop mutation [(A553⌬AAAiT)fs.185(stop 186)] [1]. Family screen-
ing (fig. 1) identified 6 additional carriers of this PPARG variant, 4 of whom
were also severely insulin resistant. The other 2 carriers were the proband’s
grandfather, a 70-year-old man, who was found to be diabetic without any fea-
tures to suggest severe insulin resistance, and her uncle, a lean 32-year-old man,
with normal fasting glucose (4.6 mmol/l) and insulin levels (46 pmol/l). In vitro
characterization of this mutation suggested that it was a null allele without
dominant negative activity, effectively rendering carriers haploinsufficient for
PPAR␥ [1]. The apparent absence of severe insulin resistance in 2 carriers of
this variant, together with the absence of insulin resistance in PPAR␥ heterozy-
gous knockout mice, prompted ongoing candidate gene studies in this kindred.
A second heterozygous frameshift premature stop mutation [(C1984⌬AG)
fs.662(stop 668)] was identified in an unlinked gene, phosphoprotein phos-
phatase 1 regulatory subunit 3A (PPP1R3A). PPP1R3A is a muscle-specific
isoform of this family of phosphoprotein phosphatase 1 regulatory proteins.
Phosphoprotein phosphatase 1 dephosphorylates glycogen synthase and
glycogen phosphorylase, promoting glycogen synthesis and increasing muscle
glycogen content. This second variant was present in the proband, all 4 insulin-
resistant relatives and 2 unaffected relatives, strongly suggesting that co-
inheritance of both variants (i.e. digenic) was required to induce severe insulin
resistance.
Severe Insulin Resistance 123

i ii
I
71 years 71 years
⫹/P ⫹/⫹
⫹/⫹ ⫹/R
i ii iii iv v vi
II
50 years 49 years 47 years 41 years 37 years 32 years

⫹/⫹ ⫹/P ⫹/P ⫹/P ⫹/⫹ ⫹/P
⫹/R ⫹/R ⫹/R ⫹/⫹ ⫹/⫹
i ii iii iv
III
21 years 20 years 25 years 21 years

⫹/⫹ ⫹/⫹ ⫹/P ⫹/P
⫹/⫹ ⫹/R ⫹/R ⫹/R
Fig. 1. Family pedigree. The age and genotype of members is indicated. ⫹ ⫽ Wild
type; P ⫽ PPAR␥ frameshift mutation; R ⫽ PPP1R3A frameshift mutation.
Severe Insulin Resistance
Insulin resistance is a core pathophysiological feature in most people with

type 2 diabetes. It is also the principal link between obesity and type 2 diabetes,
and is probably the primary metabolic defect in the metabolic syndrome.
Clinical Features of Severe Insulin Resistance
Although syndromes of severe insulin resistance are commonly diagnosed

after ␤-cell decompensation and the onset of diabetes, they have a range of clin-
ical features which may predate this by many years and may lead to primary
presentation to non-endocrinological specialists such as dermatologists,
gynaecologists or clinical geneticists. Typical manifestations include the
following. (1) Insulin-resistant diabetes mellitus – arbitrarily, a requirement for
⬎200 U/day or ⬎3 U/kg/day of exogenous insulin has been suggested to define
severe insulin resistance in the context of established diabetes. However, prior
to complete insulinopaenia clinicians should note the intermediate state
denoted by the presence of acanthosis nigricans in diabetes treated with more
modest insulin doses. In this situation determination of fasting C peptide or
Savage/Semple/Chatterjee/Wales/Ross/O’Rahilly 124
proinsulin may give some additional clue as to the degree of the underlying
insulin resistance. (2) Hypoglycaemia – paradoxically, hypoglycaemic episodes
can be a major early feature of severe insulin resistance. This can be either fast-
ing hypoglycaemia (frequently seen in patients with genetic defects in the
insulin receptor) or postprandial (3–4 h) hypoglycaemia. The latter may occur
because the normal accuracy of the physiological systems that control circulat-
ing insulin and glucose levels cannot be maintained at such extreme levels of
insulin resistance, leading to a period of ‘overcompensation’. The markedly
delayed insulin clearance that occurs in some of the disorders may also con-
tribute to reactive hypoglycaemic episodes. (3) Other manifestations – as illus-
trated by the case above, there are a group of conditions that most frequently are
present, not with diabetes, but with other clinical manifestations of the underly-
ing disorder such as the skin lesion acanthosis nigricans, ovarian hyperandro-
genism, altered growth or acral enlargement. Acanthosis nigricans is a dark
velvety hyperpigmented skin lesion, often accompanied by multiple skin tags,
occurring most strikingly in flexural locations such as the axillae, the back of
the neck and in the groin. It is also frequently seen over pressure points. In the
most extreme cases it can be generalized but the palms and soles are usually
spared. Histologically, it is a hyperkeratotic epidermal papillomatosis with
some evidence for increased melanocyte number. Some patients with long-
standing acanthosis nigricans recall vigorous maternal efforts to clean their
‘dirty necks’.
Amenorrhoea/oligomenorrhoea, hirsutism, acne and ultrasonographically
demonstrable polycystic ovaries are perhaps the most common presenting man-
ifestations of severe insulin resistance, and their presence in lean adolescent
girls ought to prompt evaluation of this possibility. Plasma testosterone levels
may be as high as 10 mmol/l, often leading to a fruitless search for an adrenal
or ovarian tumour if the association with severe insulin resistance is not
recognized.
Abnormal growth is yet another manifestation – this can either be growth
retardation as seen in Donohue’s syndrome and other complex syndromes, e.g.
severe insulin resistance associated with some types of primordial dwarfism;
or somatic overgrowth as reported in pseudoacromegaly [2]. The presence of
acanthosis nigricans should alert clinicians to severe insulin resistance in these
settings.
Classification of Severe Insulin Resistance Syndromes
We usually place lipodystrophic disorders and the genetic disorders with

complex phenotypic anomalies into separate categories (table 1); leaving a third

Table 1. Differential diagnosis of inherited severe insulin resistance syndromes
Syndrome Clinical features
Primary disorders of insulin action

Donohue’s syndrome Dysmorphic facies, acanthosis nigricans, hirsutism, abdominal
distension, lipoatrophy, fasting hypoglycaemia, postprandial
hyperglycaemia; death in childhood
Rabson-Mendenhall syndrome Acanthosis nigricans, thick and rapidly growing hair, abnormal
dentition and fingernails, diabetes in childhood; death in
teenage years
Type A insulin resistance Acanthosis nigricans, features of hyperandrogenism in girls,
variable onset of diabetes
Hyperandrogenism, insulin resistance Hyperandrogenism, insulin resistance and acanthosis nigricans
and acanthosis nigricans in obese girls – similar to type A plus obesity
Lipodystrophies
Congenital generalized lipodystrophy Lipodystrophy from birth, acanthosis nigricans frequent,
prominent dyslipidaemia, fatty liver, hyperphagia
Partial lipodystrophy – LMNA mutations Lipodystrophy apparent from puberty with excess facial
– commonly known as Dunnigan- and neck fat, dyslipidaemia, fatty liver, polycystic ovary
Kobberling syndrome or FPLD1 syndrome in women; may be associated with muscular
(Werner’s syndrome and mandibulo- dystrophy, cardiomyopathy, progeroid features
acral dysplasia are also caused by (overlap syndromes)
LMNA mutations.)
Partial lipodystrophy – PPARG variants Predominantly limb lipodystrophy, dyslipidaemia, fatty liver,
– also known as FPLD3(?) polycystic ovary syndrome in women, hypertension
Insulin resistance plus other
syndromic features
Alstrom’s syndrome Obesity, retinal dystrophy, neurosensory deafness, acanthosis
nigricans
Myotonic dystrophy Myopathic facies, myotonia
category, which we refer to as ‘primary disorders of insulin action’. Ultimately

genetic insight into the primary molecular abnormality/ies will refine this
somewhat arbitrary classification. Lipodystrophic syndromes1 and primary
disorders of insulin action can be further defined as either acquired or congeni-
tal, and in the case of lipodystrophy as partial or generalized. Patients can usu-
ally be assigned to 1 of these 3 categories on clinical grounds, although partial
1
Substantial progress has been made in understanding the genetic basis of inherited
lipodystrophies (see Garg [3] for review).
lipodystrophy may be difficult to distinguish from type A insulin resistance in
lean children and adolescents. The subject described herein illustrates this prob-
lem in so far as she does not have obvious lipodystrophy, but she does have less
femoro-gluteal fat than is commonly seen in women and one of her aunts with
both mutations clearly has partial lipodystrophy.
Recent work suggests that the biochemistry of insulin receptoropathies is
distinct from other severe insulin resistance syndromes – for example,
adiponectin levels are typically very high in patients with insulin receptor muta-
tions [4] and in type B insulin resistance. Thus screening the large insulin
receptor gene can be reserved for the patients with the unusual combination of
hyperadiponectinaemia and severe insulin resistance. Severe dyslipidaemia
suggests the possibility of a lipodystrophic syndrome. Triglycerides are typi-
cally normal in patients with mutations in the insulin receptor [5].
Measuring Insulin Sensitivity
Although the actions of insulin include effects on carbohydrate, lipid and

protein metabolism, insulin resistance is typically defined as a reduced ability
of a given concentration of insulin to lower blood glucose levels. The reference
method for measuring insulin resistance remains the hyperinsulinaemic eugly-
caemic clamp, which when used together with stable isotopes, measures both
peripheral insulin-induced glucose turnover and endogenous (primarily
hepatic) glucose production. Frequently sampled intravenous glucose tolerance
tests are marginally less cumbersome and provide comparable data (again sta-
ble isotopes can be incorporated to facilitate measurements of endogenous glu-
cose production). The labour-intensive nature, complexity and cost of these
investigations mean that for practical purposes they remain research tools. The
oral glucose tolerance test is a much simpler test and when combined with mea-
surements of insulin (and free fatty acids) can provide useful indices of insulin
action (see Pacini and Mari [6] for review of insulin sensitivity parameters
derived from OGTT data). The simplest way to assess insulin sensitivity is to
measure fasting insulin and glucose concentrations. In many cases, this is suffi-
cient to identify clinically significant insulin resistance, but significant limita-
tions include: (1) the fact that fasting parameters primarily reflect hepatic
insulin action and in rare cases may fail to detect isolated peripheral insulin
resistance (isolated tissue-specific insulin resistance is a very rare phenomenon
in humans as opposed to mice, where several genetically modified models man-
ifest tissue-specific insulin resistance); (2) difficulties understanding data after
the development of overt type 2 diabetes due to the use of exogenous insulin in
the face of varying degrees of ␤-cell dysfunction.

In a patient who is suspected of having severe insulin resistance but does
not have insulin-treated diabetes a clinically useful and simple way of estab-
lishing the condition is to undertake an oral glucose tolerance test with mea-
surements of plasma insulin. While no formal criteria for severe insulin
resistance are widely accepted, a fasting insulin level ⬎150 pmol/l and/or a
postglucose load insulin level of ⬎1,500 pmol/l indicate a marked degree of
insulin resistance. The two-hour insulin levels in the patient described above
were amongst the highest we have seen outside of those with insulin receptor
mutations.
Management
Improving insulin sensitivity will alleviate most associated problems and

ought to be the primary treatment goal (see fig. 2 for management algorithm).
Treating Insulin Resistance

In the case of type B insulin resistance, where insulin resistance is a direct
result of autoantibody production, immunosuppression may be very effective
[7], though in practice it is often reserved for cases associated with other
autoimmune features for which this therapy is indicated. Acquired generalized
lipodystrophy is also commonly accompanied by additional autoimmune condi-
tions which tend to respond to immunosuppression, although in this case the
lipodystrophy and its associated metabolic derangements are not likely to be
reversed. Insulin resistance and the associated metabolic abnormalities noted in
generalized lipodystrophy, whether acquired or congenital in origin, can how-
ever be very effectively treated by leptin replacement [8]. Current evidence
suggests that leptin replacement is most likely to be effective in patients with
leptin levels ⬍4 ␮g/l (healthy lean men frequently have leptin levels within this
range). Leptin replacement will inevitably be required for life – to date studies
have reported ongoing metabolic benefits for at least 2 years [9]. We are aware
of 1 patient with acquired generalized lipodystrophy and C3-nephritic-factor-
associated mesangiocapillary glomerulonephritis (type 2) in whom renal dis-
ease appeared to deteriorate following leptin replacement. As leptin deficiency
is known to be associated with impaired lymphocyte reactivity [10], we surmise
that leptin replacement may re-activate a quiescent immune system and suggest
caution when replacing leptin in people with other autoimmune disorders. In
the absence of leptin, restricting energy intake and dietary fat, and increasing
energy expenditure (exercise) are key components of treatment. In fact, we
believe that the primary mechanism of leptin action in lipodystrophy is to
Clinical assessment: 1. Diabetes requiring high doses of insulin
2. Acanthosis nigricans
3. Hyperandrogenism in adolescent girls
OGTT with insulin measurement (fasting insulin ⬎150pM

and/or postload insulin ⬎1,500pM) Also screen for
dyslipidaemia and hypertension
? Lipodystrophy, especially if
Check adiponectin
associated with dyslipidaemia
Normal or low High
Screen insulin receptor

See classification for ? Short stature, skeletal
and/or check for insulin
appropriate candidate dysplasia or other
receptor antibodies if suspect
gene screening syndromic features
acquired insulin resistance
Check leptin
If ⬍4 ␮g/L, consider Consider IGF-1/IGFBP3
leptin therapy therapy
Refer for candidate gene studies if no genetic cause identified
Fig. 2. Severe insulin resistance management algorithm. Additional points: (1) check
complement levels and if C3 low, C3 nephritic factor in acquired lipodystrophy (especially
partial), as it may be associated with glomerular disease and should heighten awareness of
the need for renal assessment; (2) human immunodeficiency virus infection and antiretrovi-
ral therapy are a common cause of partial lipodystrophy, insulin resistance and dyslipi-
daemia, but we would not consider the insulin resistance to be severe. IGF-1 ⫽ Insulin-like
growth factor-1; IGFBP3 ⫽ insulin-like-growth-factor-binding protein-3.
reduce hyperphagia in a setting in which the capacity to store excess energy as

fat is severely curtailed [11].
Other options include the use of insulin sensitizers such as metformin or
thiazolidinediones. Whilst the latter appear a logical choice in lipodystrophy,
we have not found them to be useful in generalized lipodystrophy and mouse

data suggest that they may even worsen hepatic steatosis in the setting of gener-
alized lipodystrophy [12]. Reports suggesting that thiazolidinediones are useful
in partial lipodystrophy do exist [13], and whilst we would agree that metabolic
abnormalities do improve in some patients with partial lipodystrophy, we are
also aware of fat accumulation in cosmetically unfavourable areas such as the
head and neck in LMNA-associated partial lipodystrophy. We described 1
patient with a PPARG mutation who benefited significantly from thiazolidine-
diones [14], but this option remains largely unexplored. Thus we favour the use
of metformin as first-line therapy, although it is frequently not very effective.
Just as in generalized lipodystrophy, leptin replacement may be effective in
patients with partial lipodystrophy and low leptin levels. Restricting energy and
fat intake is also very important. In many cases where high-dose insulin therapy
is still necessary, it is important to deliver sufficient insulin to control blood
sugars. U500 insulin, which has been successfully delivered using pumps, is a
useful option [15].
Insulin receptoropathies tend to cause severe, early-onset diabetes and in
most cases severe morbidity and early mortality. Attempts to circumvent the
insulin receptor defect by stimulating the insulin-like growth factor-1 receptor
appear to be beneficial, but have to date been limited by adverse side-effects
[16]. Ongoing studies are exploring the use of insulin-like growth factor-1
together with insulin-like-growth-factor-binding protein-3 (somatokine); a com-
bination which appears to reduce side effects [17]. Leptin replacement also
appeared to be of benefit in 2 children with Rabson-Mendenhall syndrome [18].
Treating the Consequences of Severe Insulin Resistance

The principles behind the treatment of ovarian hyperandrogenism in
insulin resistance syndromes are similar to those that pertain in typical polycys-
tic ovary syndrome; however, women with severe insulin resistance are more
likely to be resistant to treatment. They frequently need potent antiandrogens to
ameliorate severe hirsutism, acne and androgenic alopecia together with oestrogen/
progestagen combinations to ensure regular menses, and assistance with fertil-
ity when appropriate. Acanthosis nigricans is a disfiguring skin lesion which
reduces quality of life for many patients. Other than treating the insulin resis-
tance per se and using cosmetic measures to mask the skin lesion, there is little
reliable specific therapy for acanthosis. There have been case reports of its
improvement with etretinate and with calcipotriol. Dyslipidaemia is not infre-
quent, particularly in the lipodystrophic disorders, where it may be severe
enough to result in eruptive xanthomata and pancreatitis. Leptin replacement
and/ or strict dietary restriction can be very effective in people with lipodystro-
phy. Fibrates are also useful and statins almost certainly indicated for cardio-
protective effects.
Acknowledgements
D.B.S., R.K.S., V.K.K.C. and S.O. are supported by the Wellcome Trust. We are grate-
ful to the patients for their helpful co-operation.
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prospective. Medicine (Baltimore) 2004;83:209–222.
6 Pacini G, Mari A: Methods for clinical assessment of insulin sensitivity and beta-cell function.
Best Pract Res Clin Endocrinol Metab 2003;17:305–322.
7 Coll AP, Morganstein D, Jayne D, Soos MA, O’Rahilly S, Burke J: Successful treatment of type B
insulin resistance in a patient with otherwise quiescent systemic lupus erythematosus. Diabet Med
2005;22:814–815.
8 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, Wagner AJ, DePaoli AM, Reitman
ML, Taylor SI, Gorden P, Garg A: Leptin-replacement therapy for lipodystrophy. N Engl J Med
2002;346:570–578.
9 Javor ED, Cochran EK, Musso C, Young JR, Depaoli AM, Gorden P: Long-term efficacy of leptin
replacement in patients with generalized lipodystrophy. Diabetes 2005;54:1994–2002.
10 Farooqi IS, Matarese G, Lord GM, Keogh JM, Lawrence E, Agwu C, Sanna V, Jebb SA, Perna F,
Fontana S, Lechler RI, DePaoli AM, O’Rahilly S: Beneficial effects of leptin on obesity, T cell
hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin defi-
ciency. J Clin Invest 2002;110:1093–1103.
11 Savage DB, O’Rahilly S: Leptin: a novel therapeutic role in lipodystrophy. J Clin Invest
2002;109:1285–1286.
12 Kim JK, Fillmore JJ, Gavrilova O, Chao L, Higashimori T, Choi H, Kim HJ, Yu C, Chen Y, Qu X,
Haluzik M, Reitman ML, Shulman GI: Differential effects of rosiglitazone on skeletal muscle and
liver insulin resistance in A-ZIP/F-1 fatless mice. Diabetes 2003;52:1311–1318.
13 Arioglu E, Duncan-Morin J, Sebring N, Rother KI, Gottlieb N, Lieberman J, Herion D, Kleiner DE,
Reynolds J, Premkumar A, Sumner AE, Hoofnagle J, Reitman ML, Taylor SI: Efficacy and safety
of troglitazone in the treatment of lipodystrophy syndromes. Ann Intern Med 2000;133: 263–274.
14 Savage DB, Tan GD, Acerini CL, Jebb SA, Agostini M, Gurnell M, Williams RL, Umpleby AM,
Thomas EL, Bell JD, Dixon AK, Dunne F, Boiani R, Cinti S, Vidal-Puig A, Karpe F, Chatterjee
VK, O’Rahilly S: Human metabolic syndrome resulting from dominant-negative mutations in the
nuclear receptor peroxisome proliferator-activated receptor-gamma. Diabetes 2003;52:910–917.
15 Cochran E, Musso C, Gorden P: The use of U-500 in patients with extreme insulin resistance.
Diabetes Care 2005;28:1240–1244.
16 Jabri N, Schalch DS, Schwartz SL, Fischer JS, Kipnes MS, Radnik BJ, Turman NJ, Marcsisin VS,
Guler HP: Adverse effects of recombinant human insulin-like growth factor I in obese insulin-
resistant type II diabetic patients. Diabetes 1994;43:369–374.

17 Mecasermin rinfabate: insulin-like growth factor-I/insulin-like growth factor binding protein-3,
mecaserimin rinfibate, rhIGF-I/rhIGFBP-3. Drugs R D 2005;6:120–127.
18 Cochran E, Young JR, Sebring N, DePaoli A, Oral EA, Gorden P: Efficacy of recombinant
methionyl human leptin therapy for the extreme insulin resistance of the Rabson-Mendenhall syn-
drome. J Clin Endocrinol Metab 2004;89:1548–1554.
David B. Savage
Department of Clinical Biochemistry, University of Cambridge
Box 232, Level 4, Addenbrooke’s Hospital
Hills Road
Cambridge, CB2 2QQ (UK)
Tel. ⫹44 1223 767 923, Fax ⫹44 1223 330 598, E-Mail dbs23@cam.ac.uk
Inherited Endocrine Diseases Involving

G Proteins and G Protein-Coupled Receptors
Allen M. Spiegel
Albert Einstein College of Medicine, Bronx, N.Y., USA
Abstract
Naturally occurring mutations in the G protein Gs-␣ subunit and in a number of
G protein-coupled receptors (GPCRs) have been identified in human diseases. Loss-of-
function mutations in GPCRs for various hormones lead to hormone resistance manifest as
hypofunction of the gland expressing the affected GPCR. Conversely, GPCR gain-of-
function mutations lead to hormone-independent activation and hyperfunction of the
involved gland. Our laboratory has focused on the extracellular calcium-sensing GPCR
(CaR) expressed primarily, but not exclusively, in parathyroid glands and kidney. Loss-of-
function CaR mutations lead to a form of hyperparathyroidism, an apparent exception to the
general pattern described above, but in fact reflecting resistance to the normal inhibition of
parathyroid hormone secretion by the ‘hormone’ agonist, extracellular Ca2⫹. CaR gain-of
function-mutations cause autosomal dominant hypocalcemia due to activation of the receptor
at subphysiologic concentrations of serum Ca2⫹, leading to ‘inappropriate’ inhibition of
parathyroid hormone secretion. I will describe our recent work that helps inform design of
novel therapeutics targeting this important GPCR.
Just as mutations in genes encoding a variety of enzymes have been identi-

fied in the diseases termed ‘inborn errors of metabolism’ by Garrod, mutations
in genes encoding G proteins and G protein-coupled receptors (GPCRs) have
been identified in a number of endocrine diseases that may be termed ‘inborn
errors of signal transduction’. Inborn errors of metabolism are caused by loss-
of-function mutations leading to deficient enzymatic activity with a corre-
sponding excess of substrate and deficiency of metabolic product. In contrast,
inborn errors of signal transduction comprise both loss- and gain-of-function
mutations of G proteins and GPCRs. The former manifest as hormone resis-
tance syndromes in which there exists a deficiency of hormone action despite
an excess of hormone (resulting from the usual feedback regulation mecha-
nisms). The latter manifest as hormone-independent endocrine hyperfunction.
Mutations in G proteins and GPCRs may occur, not only as germline mutations
leading to inborn errors of signal transduction, but also as somatic mutations
that may cause more focal phenotypes in adults. For germline mutations, the
particular phenotype caused by a given mutation will be a function of the range
of expression of the involved gene, with genes more widely expressed leading
to a more pleiotropic phenotype. For somatic mutations, focal manifestations
may result even from mutation in a ubiquitously expressed gene.
Mutations in G proteins and GPCRs may impair function at any of several
steps in the GTPase cycle (fig. 1). Naturally occurring, germline loss-of-function
mutations in the gene encoding the ␣-subunit of the ubiquitously expressed
G protein, Gs, coupling many GPCRs to stimulation of cAMP formation cause
the pleiotropic manifestations of the archetypical hormone resistance disorder,
pseudohypoparathyroidism. Somatic gain-of-function mutations of the same
gene occurring early in development cause McCune-Albright syndrome with
endocrine, skin and bone manifestations, whereas somatic mutations of the
gene occurring later in life cause more focal manifestations such as soma-
totroph pituitary tumors. A more detailed description of the complex regulation
of the imprinted Gs-␣ gene and the disorders resulting from mutations in the
gene can be found in a recent review [1].
Endocrine Diseases Caused by GPCR Gene

Loss-of-Function Mutations
Clinically significant impairment of signal transduction generally requires

loss of function of both alleles of a GPCR gene; thus, most such diseases are
autosomal recessive, but there are several exceptions (table 1). Loss-of-function
mutations may be missense as well as nonsense or frameshift mutations that
truncate the normal receptor protein. They may involve any portion of the recep-
tor, although the membrane-spanning helices are a particularly frequent site.
Loss-of-function mutations of receptors for ACTH, TSH, FSH and the hypothal-
amic hormones – gonadotropin-releasing hormone (GnRH), thyrotropin-releasing
hormone (TRH), and growth-hormone-releasing hormone (GHRH) – mimic
deficiency of the respective hormones. Subjects with heterozygous loss-of-
function mutations of the TSH receptor gene are generally euthyroid with com-
pensatory elevated serum TSH, but homozygous mutations result in congenital
hypothyroidism associated with a hypoplastic or even absent thyroid gland.
Loss-of-function mutations in LH and parathyroid hormone (PTH)/PTH-related
protein (PTHrP) receptors cause developmental anomalies, reflecting the critical
Spiegel 134
1. Synthesis and targeting
of components
Adenylyl
␣s ␥ cyclase
Gs-Coupled receptor ␤
GDP
5. GTPase 2. Receptor
activation
Cholera toxin by agonist
␣s activating
mutations
Agonist Agonist
Adenylyl Adenylyl
␥ cyclase ␣s ␣s ␥ cyclase
Gs-Coupled receptor ␤ Gs-Coupled receptor ␤
GDP GTP GDP

3. Receptor activation
cAMP of G protein
PKA 4. G protein-effector interaction
PKA substrate
phosphorylation
Physiologic effects
Fig. 1. The G protein GTPase cycle. Potential sites for disease-causing abnormalities
are numbered. In each panel, the stippled region denotes the plasma membrane with extra-
cellular above and intracellular below. Under physiologic conditions, effector regulation by G
protein subunits is transient and is terminated by the GTPase activity of the ␣-subunit. The
latter converts bound GTP to GDP, thus returning the ␣-subunit to its inactivated state with
high affinity for the ␤␥-dimer, which reassociates to form the heterotrimer. The figure shows
the G protein Gs with its effector, adenylyl cyclase. Activation of adenylyl cyclase generates
the intracellular second messenger, cAMP, which activates protein kinase A (PKA). The lat-
ter enzyme phosphorylates a variety of proteins that mediate the physiologic effects of ago-
nists for Gs-coupled receptors. Cholera toxin covalently modifies the Gs-␣ subunit blocking
its GTPase activity. Somatic mutations of the Gs-␣ subunit likewise block GTPase activity.
In both cases, constitutive activation and agonist-independent cAMP formation result.
role of the respective hormones in normal development. Loss-of-function muta-

tions of both copies of the LH receptor gene cause a rare form of 46,XY male
pseudohermaphroditism known as Leydig cell hypoplasia. Absence of func-
tional PTH/PTHrP receptors causes a rare, lethal form of dwarfism known as
G Protein and G Protein-Coupled Receptor Diseases 135

Table 1. Endocrine diseases caused by GPCR loss-of-function mutations
Receptor Disease Inheritance
V2 vasopressin nephrogenic diabetes insipidus X-linked

ACTH familial ACTH resistance aut. rec
LH male pseudo-hermaphroditism aut. rec
TSH familial hypothyroidism aut. rec
CaR familial hypocalciuric hypercalcemia/ aut. dom
neonatal severe primary
hyperparathyroidism aut. rec
FSH hypergonadotropic aut. rec
ovarian failure
TRH central hypothyroidism aut.rec
GHRH GH deficiency aut. rec
GNRH hypogonadotropic hypogonadism aut. Rec
PTH Blomstrand chondrodysplasia aut. rec
Blomstrand chondrodysplasia. X-linked nephrogenic diabetes insipidus (renal

vasopressin resistance) is caused by loss-of-function mutations in the V2 vaso-
pressin receptor gene located on the X chromosome. Males inheriting a mutant
gene develop the disease, whereas most females do not show overt disease
because random X inactivation results, on average, in 50% normal receptor
genes. Identification of the mutation in carrier females facilitates early treatment
of affected male neonates to avoid hypernatremia and brain damage. Loss-of-
function mutations in the gene encoding the melanocortin 4 receptor, which reg-
ulates hypothalamic pathways controlling appetite and energy metabolism,
result in a distinct obesity syndrome characterized by hyperphagia and increased
linear growth. Inheritance is codominant, with homozygotes showing a severer
phenotype than heterozygotes.
Endocrine Diseases Caused by GPCR Gene

Gain-of-Function Mutations
Given the dominant nature of activating mutations, most diseases caused

by GPCR gain-of-function mutations are inherited in an autosomal dominant
manner (table 2). Unlike loss-of-function mutations, GPCR gain-of-function
mutations are almost always missense mutations. Activating missense muta-
tions are thought to disrupt normal inhibitory constraints that maintain the
receptor in its inactive conformation. Mutations disrupting these constraints
Spiegel 136
Table 2. Endocrine diseases caused by GPCR gain-of-function mutations
Receptor Disease Inheritance
LH familial male precocious puberty aut. dom.

LH sporadic Leydig cell tumors somatic
TSH sporadic hyperfunctional thyroid nodules somatic
TSH familial nonautoimmune aut. dom.
hyperthyroidism
CaR familial hypoparathyroidism aut. dom.
PTH/PTHrP Jansen metaphyseal aut. dom.
chondrodysplasia
mimic the effects of agonist binding and shift the equilibrium toward the acti-
vated state of the receptor.
Germline gain-of-function mutations in the LH and TSH receptor genes
may mimic states of hormone excess, familial male precocious puberty and
familial nonautoimmune hyperthyroidism, respectively. Women inheriting
gain-of-function mutations in the LH receptor gene do not show precocious
puberty because, unlike in males, the combined action of LH and FSH is
required for female pubertal development. As with activating Gs-␣ mutations,
increased cAMP in many endocrine cells leads to increased proliferation and
hormone hypersecretion. Thus, somatic gain-of-function mutations of the LH
and TSH receptor genes cause sporadic tumors of Leydig cells and the thyroid
cells, respectively. Activating mutations of the PTH/PTHrP receptor gene cause
Jansen’s metaphyseal chondrodysplasia. The phenotype includes hypercalcemia
and hypophosphatemia mimicking the effects of PTH hypersecretion but also
abnormal bone development (short-limb dwarfism), reflecting the critical role
of PTHrP in endochondral bone formation. Activating mutations of the V2
vasopressin receptor were identified in neonates manifesting a syndrome of
inappropriate antidiuresis but lacking the elevated serum vasopressin typically
associated with this syndrome.
Overview of the Extracellular Calcium-Sensing Receptor
The cloning of the extracellular Ca2⫹-sensing receptor (CaR) provided a

new paradigm in signal transduction in which an extracellular ion, Ca2⫹, serves
as an agonist for a cell surface receptor [2]. The CaR is expressed abundantly
in parathyroid and kidney, where its activation inhibits PTH secretion and

Fig. 2. Schematic diagram showing amino acid sequence of the hCaR with boundaries
of transmembrane helices based on alignment with rhodopsin. The location of signal peptide,
Spiegel 138
promotes urinary Ca2⫹ excretion, respectively [3]. The CaR is expressed in
other tissues, where it might have roles beyond extracellular Ca2⫹ homeostasis
[see 4 for a review].
Notwithstanding its unique agonist, the CaR is a member of the GPCR
family 3 or C [5]. All GPCRs share the signature 7-transmembrane-spanning
(7TM) domain. The assumption is that GPCR activation involves a conforma-
tional change of the membrane-spanning ␣-helices, altering the disposition of
intracellular loops, and thereby promoting activation of G proteins. For
rhodopsin, a member of GPCR family 1, the 3-dimensional structure of the
receptor with its covalently bound ligand, retinal, has been solved, providing
direct evidence for the interaction of ligand with specific residues of the mem-
brane-spanning helices [6]. For members of GPCR family 3, which include, in
addition to the CaR, multiple subtypes of metabotropic glutamate receptor
(mGluR), the GABA-B receptor and certain taste and pheromone receptors,
evidence indicates that agonists bind to a dimeric, Venus-flytrap-like (VFT)
domain within the large N-terminal extracellular domain (ECD) of the receptor.
The VFT domain is linked to the 7TM domain by a cysteine-rich domain.
Understanding how agonist binding to the VFT domain leads to receptor activa-
tion has important implications for designing drugs targeting family 3 GPCRs.
The human CaR (hCaR) is a 1,078-amino-acid polypeptide comprising an
N-terminal ECD, the 7TM domain and intracellular C terminus (fig. 2); see Hu
and Spiegel [7] for review. The ECD contains 11 potential N-linked glycosyla-
tion sites, of which at least 3 must be glycosylated for cell surface expression.
Ca2⫹ activates the CaR at millimolar concentrations, implying a much lower
affinity Ca2⫹-binding site than for intracellular Ca2⫹-binding proteins such as
calmodulin.
Solution of the three-dimensional structure of the VFT domain of the rat
mGluR1 [8] offers important insights into agonist-promoted conformational
changes, which are probably relevant for the CaR and other members of family
3. The crystal structure of the glutamate-bound form of the mGluR1 VFT
revealed the key residues in lobe 1 and lobe 2 involved in agonist binding.
N-linked glycosylation sites and the sequence of synthetic polypeptide used to raise mono-
clonal antibody ADD is indicated. All cysteines are shown in black background. The begin-
ning and end of the VFT domain and the 4 loops in lobe 1 of the VFT are indicated. Naturally
occurring activating mutations identified in the hCaR, as well as the inactivating V817I
mutation (boxed) are indicated. Glu837, shown to be involved in binding of the allosteric
modulators NPS R-568 and NPS 2143, and Pro823, reported to be critical for the function of
the receptor, are shown in bold print. The 2 regions with clustering ADH mutations, residues
116–131 and residues 819–837, are shaded.

Studies of chimeric receptors show that the predominant agonist-binding site
for the CaR, and probably most other family 3 GPCRs, resides within the VFT
domain. The specific amino acids responsible for Ca2⫹ binding to the CaR have
not been definitively identified, but 3 residues, Ser147, Ser170 and Asp190,
corresponding to amino acids in the mGluR1 glutamate binding site, when arti-
ficially mutated to alanine, impair CaR activation. L-Amino acids allosterically
enhance CaR sensitivity to Ca2⫹, and studies of the Ser170Ala mutant suggest
that the amino-acid-binding site is related to that for Ca2⫹ itself.
The CaR is a homodimer linked by intermolecular disulfides at cysteines
129 and 131, as well as by noncovalent interactions along a dimer interface
involving both lobes 1 and 2 of the VFT domain. Comparison of the glutamate-
bound, ‘active’ versus antagonist-bound, ‘inactive’ structures of the mGluR1
VFT revealed several important differences [8]: (1) the VFT is closed in the
glutamate-bound and open in the antagonist-bound structures; (2) residues
equivalent to hCaR 117–123 in loop 2 form an ordered extension of an ␣-helix
of lobe 1 in the inactive form but are disordered, along with the remainder of
loop 2, in the active form; (3) agonist-promoted VFT closure leads to a 70⬚ rota-
tion of 1 monomer relative to the other about an axis perpendicular to the dimer
interface; and (4) VFT closure-promoted rotation of the monomers permits lobe
2 domains to move 26Å closer than in the open VFT conformation, where elec-
trostatic repulsion keeps them further apart. Apposition of the lobe 2 domains
in the agonist-bound state might cause concomitant movement of the cysteine-
rich domains linked to lobe 2.
The VFT and 7TM domains are linked by an 84-residue region contain-
ing 9 closely spaced cysteines (fig. 2), termed the cysteine-rich domain. With
the exception of the GABA-B receptor, which lacks this domain, other family
3 GPCRs contain the same 9 cysteines with conserved spacing. Mutation of
any of these cysteines to serine severely impairs the expression and function
of the CaR. Although chimeric hCaRs, in which the mGluR1 cysteine-rich
domain is substituted for that of the hCaR, preserve some degree of function,
deletion of the cysteine-rich domain abolishes CaR activation, in spite of the
preservation of some cell surface expression. This suggests that the cysteine-
rich domain plays a key role in signal transmission between the VFT and 7TM
domains.
A truncation mutant with N-terminal residues 1–20 of bovine rhodopsin
fused to hCaR Ala600 (Rho-C-hCaR) shows excellent cell surface expression
and is activated by Ca2⫹ when added with an allosteric modulator, NPS R-568.
These results suggest that the 7TM domain, in addition to the VFT, might con-
tain sites for polycation binding and CaR activation. Mutagenesis of the acidic
residues in extracellular loops 1–3, however, does not abolish Ca2⫹ activation of
the receptor. Much of the 216 residue C terminus of the receptor (residues
Spiegel 140
889–1078) can be truncated without impairing cell surface expression and acti-
vation. Nonetheless, the C terminus might be responsible for other properties of
the CaR, such as binding to a scaffold protein, filamin-A.
Diseases Caused by Loss- and Gain-of-Function Mutations of the

Calcium-Sensing Receptor
The importance of the CaR in extracellular Ca2⫹ homeostasis is under-

scored by the identification of inactivating mutations in the CaR gene as the
cause of familial hypocalciuric hypercalcemia (FHH) and neonatal severe pri-
mary hyperparathyroidisim (NSPHT) and the identification of activating muta-
tions as the cause of autosomal dominant hypocalcemia/hypoparathyroidism
(ADH).
Inactivating mutations of the CaR cause a right shift in set point for Ca2⫹
inhibition of PTH secretion and for stimulation of urinary Ca2⫹ excretion, lead-
ing to relative hypercalcemia and hypocalciuria in subjects with FHH and
NSPHT. The severity of alteration in the biochemical phenotype correlates with
the type of mutation. Null mutations that prevent CaR expression cause mild
FHH when heterozygous, but cause NSPHT when homozygous or compound
heterozygous. Heterozygous mutations that permit CaR expression but impair
function might cause severer FHH or NSPHT by acting as dominant negatives
of the wild-type CaR, presumably through heterodimerization. Truncation of
the hCaR proximal to residue 888 disrupts receptor function; thus, frameshift
and nonsense mutations causing such truncation are inactivating mutations.
Missense mutations causing FHH/NSPHT might inactivate the CaR by impair-
ing normal folding and cell surface expression or by preventing Ca2⫹ activation
of the properly expressed receptor. Over 30 inactivating missense mutations in
FHH/NSPHT have been identified to date, and their distribution is nonrandom.
More than half cluster between residues 13 and 297 of the ECD, whereas only 1
has been reported between residues 298 and 548.
Heterozygous, activating mutations in subjects with ADH generally cause
a left shift in the Ca2⫹ set point, leading to relative hypocalcemia and hypercal-
ciuria. With the exception of an in-frame deletion, Ser895-Val1075, activating
mutations in ADH are missense mutations. Such mutations presumably act by
relieving inhibitory constraints that maintain the CaR in its inactive conforma-
tion. Most ADH mutations increase Ca2⫹ sensitivity rather than causing consti-
tutive activation. As with naturally occurring inactivating mutations, ADH
mutations are clustered in particular regions of the CaR (fig. 2). Most occur at
the presumptive dimer interfaces of lobe 1 (particularly those within loop 2
shaded in fig. 2) and of lobe 2 (Pro221Leu, Glu228Gln and Gln245Arg). We

have suggested that these mutations enhance Ca2⫹ sensitivity by facilitating
agonist-induced dimer rotation.
Within the 7TM domain, a cluster of mutations at the junction of TM
helices 6 and 7 suggests that movement of these helices relative to each other
could be a crucial event in CaR activation [9]. Artificial mutation of proline 823
in TM6 (a residue highly conserved in family 3 GPCRs) to alanine drastically
impairs Ca2⫹ activation of the receptor, despite intact expression of the mutant
receptor at the cell surface. In contrast, a unique ADH mutation, Ala843Glu in
TM7, leads to constitutive activation of the CaR, even when expressed in the
ECD-deleted Rho-C-hCaR. These mutations further underscore the key role of
TM6 and 7 in CaR activation.
Allosteric Modulators of the Calcium-Sensing Receptor
The central role of the CaR in regulating PTH secretion has made it an
attractive target for positive and negative allosteric modulators, so-called cal-
cimimetic and calcilytic drugs, respectively. Positive allosteric modulators of
the CaR inhibit PTH secretion and could be useful in the treatment of sec-
ondary hyperparathyroidism (e.g. in end-stage renal disease), in parathyroid
cancer and other forms of primary hyperparathyroidism not amenable to surgi-
cal treatment [10]. Negative allosteric modulators would increase PTH secre-
tion and with appropriate pharmacokinetics could be useful as anabolic agents
for the treatment of osteoporosis [11].
Phenylalkylamines such as NPS R-568 act as positive allosteric modula-
tors of the CaR, enhancing its sensitivity to Ca2⫹ without activating it by them-
selves. They are selective for the CaR, failing to modulate closely related family
3 GPCRs such as mGluR1. Presumably selectivity reflects sequence differences
at the drug-binding site, which has been shown to be within the 7TM domain.
In particular, glutamate 837 has been identified as critical for binding of both
positive and negative allosteric modulators such as NPS 568 and NPS 2143 [9].
Since both of these compounds share a positively charged central amine, direct
interaction with the negatively charged side chain of glutamate 837 may be crit-
ical for drug binding. Similarities between the action of the negative allosteric
modulator, NPS 2143, and the Pro823Ala mutation in TM6 suggest that nega-
tive modulators may constrain the 7TM domain in a conformation that ‘resists’
activation by signals transmitted from the agonist-bound VFT [9].
In in vitro studies, NPS 2143 inhibited Ca2⫹ activation of mutant forms of
the CaR corresponding to those identified in subjects with ADH. Since patients
with ADH are often hypercalciuric and at risk for development of kidney stones
when treated with vitamin D and calcium to correct hypocalcemia, negative
Spiegel 142
allosteric modulators might be particularly useful in the treatment of ADH.
Further studies are needed to test the possibility that the treatment of such
patients with negative allosteric modulators would increase serum PTH and
Ca2⫹ without the hypercalciuria seen with conventional treatment.
Conclusions and Future Studies
Studies of naturally occurring mutations of the hCaR have provided sub-

stantial insight into the structure and function of this unique GPCR. Its pivotal
role in the maintenance of extracellular Ca2⫹ homeostasis has spurred the
development of positive and negative allosteric modulators, some of which
have already proved useful clinically. Further study of hCaR mutations and of
novel allosteric modulators combined with efforts to model the structure of the
CaR 7TM domain and modulator-binding site(s) should prove fruitful in help-
ing us understand the mechanism of CaR activation, and in developing more
potent and selective drugs to modulate CaR activity.
Acknowledgment
I am grateful to many fellows in my former laboratory at NIH and collaborators from

other laboratories who have contributed to our studies of the CaR. I would especially like to
thank Dr. Jianxin Hu, staff scientist in my laboratory, Dr. Ken Jacobson and his colleagues in
the Laboratory of Bioorganic Chemistry, NIDDK, who synthesized many of the compounds
we studied, and Dr. Stefano Mora of Milan, who identified many of the activating mutations
of the CaR we studied.
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Allen M. Spiegel
Albert Einstein College of Medicine, Belfer 312
1300 Morris Park Avenue
Bronx, NY 10461 (USA)
Tel. ⫹1 718 430 2801, Fax ⫹1 718 430 8822, E-Mail spiegel@aecom.yu.edu
Spiegel 144
Stem Cells
From Animal Research to Clinical Applications
Carlo Alberto Redia, Manuela Montib, Valeria Mericob, Tui Nerib,

Mario Zanonib, Maurizio Zuccottic, Silvia Garagnab
a
Direzione Scientifica Fondazione IRCCS Policlinico San Matteo and bLaboratorio di
Biologia dello Sviluppo, University of Pavia, Pavia, and cDipartimento di Medicina
Sperimentale, University of Parma, Parma, Italy
Abstract
The application of stem cells to regenerative medicine is one of the actual hot topics in
biomedicine. This research could help the cure of a number of diseases that are affecting a
large share of the population. Some good results in cell replacement have already been
obtained (infarcted heart, diabetes, Parkinson disease), apart from those of more traditional
applications like severe burns and blood tumors. We are now facing crucial questions in stem
cell biology. One of the key questions is how a cell begins to proliferate or differentiate.
Genome reprogramming, both following nuclear transfer and cytoplast action, will likely
highlight some of the molecular mechanisms of cell differentiation and dedifferentiation. In
turn, these clues should be useful to the production of populations of reprogrammed cells
that could develop into tissues or, in the future, into proper organs. We will overview what
stem cells are, what roles they play in normal developmental processes and how stem cells
could have the potential to treat diseases.
The Biology of Stem Cells
Stem cells (SC) are unspecialized cells that have 2 defining properties: the
ability to differentiate into other cells and the ability to self-renewal. The ability to
differentiate is the potential to develop into other cell types. A totipotent SC (e.g.
the fertilized egg, the zygote) can develop into any cell type. A pluripotent SC can
develop into cells from all 3 germinal layers (e.g. cells from the inner cell mass).
Other cells can be pluripotent, multipotent, oligopotent, bipotent or unipotent
depending on their ability to develop into several, few, 2 or 1 other cell type(s).
Self-renewal is the ability of SC to divide and to produce again SC: during early
development the cell division is symmetrical (i.e., each pluripotent cell divides to
give rise to daughter cells each with the same potential), while later on the cell
divides asymmetrically, producing an SC and a more differentiated cell [1].
SC play a crucial role in mammalian development: the zygote is the totipo-
tent SC with the ability to produce all the cell types of the new individual
including the trophoblast. The zygote undergoes several cell divisions and at the
32- to 64-cell stage, each cell (blastomere) sticks together to form a tight ball of
cells (morula). Each blastomere is pluripotent. The next developmental stage is
the blastocyst, which consists of a hollow ball of cells, while later on the gas-
trula is composed of 3 germ layers, the ectoderm, the mesoderm and the endo-
derm, each of which gives rise to the future different type of tissue. As
development proceeds, there is a loss of potential and a gain of specialization, a
process called determination. The germ layer SC are multipotent, giving rise to
all of the terminally differentiated cells of the individual. The number of SC
present in an adult is far lower than that seen in early development because most
of the SC have differentiated and multiplied. This makes it extremely difficult
to isolate SC from an adult organism, which is why there is a need to use
embryonic stem cells (ES) for research and therapy: because ES are much eas-
ier to obtain and possess a very high proliferative rate. Increasing evidences
support the view that cultured ES have a high potential for therapeutic applica-
tions [2]. Thus, it is clear that there is a need for methodological advances with
the aim of getting rapid and noninvasive technical tools to monitor SC differen-
tiation in culture. At this regard, we are studying the changes in the expression
of proteins and nucleic acids during the first 14 days of spontaneous ES differ-
entiation by Fourier transform infrared microspectroscopy. We are now able to
detect variations in intensity and peak position for specific infrared bands, such
as the protein ␣-helix component in the amide 1 absorption region
(1,700–1,600 cm⫺1) and several bands in the nucleic acid region from 1,050 to
850 cm⫺1. The protein ␣-helix band at 1,657 cm⫺1 increases from the beginning
(1–4 days) up to day 10 of differentiation, while in the same span of time, 2
RNA bands (at 994 and 914 cm⫺1) decrease. These data suggest that mRNA
translation takes place in ES, with the production of the specific proteins
required for the development of the new phenotype. Interestingly, the second
derivative analysis of the amide 1 band provides information about the sec-
ondary structure of these proteins. Furthermore, between day 4 and 7 of differ-
entiation, it is possible to observe the response of the DNA/RNA hybrid (954
and 899 cm⫺1): likely, the transcriptional switch of the genome starts at this
stage of differentiation. As supported by cytochemical assays, these spectral
changes can be taken as ‘fingerprints’ for the identification of specific molecu-
lar events occurring in ES cytodifferentiation.
Redi/Monti/Merico/Neri/Zanoni/Zuccotti/Garagna 146
The role of somatic SC (also called adult SC) is believed to be the replace-
ment of damaged and injured tissue. Observed in continually replenished cells
such as blood and skin cells, SC have recently been found in other tissue, such
as neural tissue.
Organ regeneration has long been believed to occur through organ- and tis-
sue-specific SC: hematopoietic SC were thought to replenish blood cells, SC of
the gut to replace cells of the gut and so on. Recently, using cell lineage track-
ing, SC from one organ that divide to form cells of another organ have been dis-
covered. Hematopoietic SC can give rise to liver, brain and kidney cells. This
plasticity of adult SC has been observed under several experimental conditions.
Tissue regeneration is achieved by 2 mechanisms: (1) circulating SC
divide and differentiate under appropriate signaling by cytokines and growth
factors, e.g. blood cells, and (2) differentiated cells which are capable of divi-
sion can also self-repair, e.g. hepatocytes, endothelial cells, smooth muscle
cells, keratinocytes and fibroblasts. These fully differentiated cells are limited
to local repair. For more extensive repair, SC in the quiescent state can then be
activated and mobilized to the required site. For example, for wound healing in
the skin, epidermal SC and bone marrow progenitor cells both contribute. Thus,
it is likely that organ-specific progenitors and hematopoietic SC are involved in
repair, even for other organ repair.
Great hopes have been raised that human ES will one day be used to
replace damaged cells and to provide therapies beyond the reach of conven-
tional drugs. On the other hand, human ES research has been highly controver-
sial due to the ethical issues concerned with the culture and use of SC derived
from human embryos. However, a serious obstacle to their use is our lack of
insight into the mechanisms that regulate the SC biology; more specifically,
whether an SC undergoes self-renewal or differentiates to become a more spe-
cialized type of cell.
Understanding how self-renewal and pluripotency are controlled may
allow generation of SC lines from somatic tissues, thus avoiding the ethically
contentious need to derive them from embryos [3]. Differentiated cells can be
reprogrammed to an embryonic-like state by transfer of nuclear contents into
oocytes or by fusion with ES. A step forward in this understanding was recently
taken by 2 teams. Takahashi and Yamanaka [4], making use of an ingenious
strategy, successfully searched for factors that are able to reprogram somatic
cells. They showed that the introduction of just 4 selected factors proved to be
enough to give fibroblasts from mouse skin some of the characteristics of
pluripotent cells. This astonishing effect [5] revealed a new opportunity for
studying the mechanisms that regulate pluripotency, with the longer-term aim
of producing pluripotent cells from people either for research or therapy. Among
these factors there are some shown previously to be essential for proliferation of
Stem Cells 147

ES, factors known to be expressed in tumors and others that are expressed in
ES. These genes were Oct4, Sox2, cMyc, and Klf4. A great deal of debate has
begun as to the unexpected contribution of some of these genes and the appar-
ent lack of a need for other factors, such as Nanog. No doubt this debate will
continue until the role of candidates has been defined. A key step in this process
will be the identification of genes downstream of these factors. These data
demonstrate that pluripotent SC can be directly generated from fibroblast cul-
tures by the addition of only a few defined factors.
Ivanova et al. [6] chose an RNA interference strategy to identify known and
novel transcriptional regulators from 2 distinct pathways that control self-
renewal in mouse ES. They used short hairpin RNA loss-of-function techniques
to downregulate a set of gene products whose expression patterns suggest self-
renewal regulatory function. By doing this, they found out that Oct4 is required
to prevent trophectodermal differentiation, Nanog and Sox2 appear to be global
regulators that repress multiple differentiation programs, whereas Esrrb, Tbx3
and Tcl1 are necessary to block the differentiation into epiblast-derived lineages.
All together, these studies confirm the involvement of Oct4 and Sox2 in the
maintenance of ES identity and further underscore their ability to induce
nuclear reprogramming providing a number of insights into the mechanisms
that are able to genetically reprogram cells. Noteworthily, these studies reveal
how self-renewal-regulating genes appear to be connected in a transcriptional
network that governs self-renewal and differentiation programs [1, 3].
Particularly, 2 separate pathways seem to regulate self-renewal: one including
Nanog, Oct4 and Sox2, and the other Esrrb, Tbx3, Tcl1 and Dppa4.
The efficiency of reprogramming can be estimated at between approxi-
mately 1 per 2,500 to approximately 1 per 30,000. To explain why the proportion
of cells that are reprogrammed is so low, being of the order of 0.07–0.002%, one
can speculate that this reflects heterogeneity in the cell cultures with only that
proportion of cell being in a state that is amenable to reprogramming. Because of
the low frequency of the observed phenomenon, definitive proof of mature cell
nuclear reprogramming will emerge when similar studies are performed with dif-
ferentiated cells (genetically marked) and in various laboratories. In fact, the use
of cell extracts for inducing cell dedifferentiation could be a powerful system to
obtain large quantities of pluripotent cells. It is thus of crucial importance that the
robustness of this method of cell transdifferentiation is tested by other laborato-
ries before it is advanced to a more ambitious use in cell therapy programs. A
recent remarkable study has shown that when mouse NIH-3T3 fibroblasts are
exposed to an ES extract (ESC), the majority of them express the Oct4 gene and
form ESC-like colonies and embryoid bodies that differentiate into cells of the 3
germ layers [7]. We used the same reprogramming protocol on STO and NIH-3T3
mouse fibroblasts. Three are the main results we got: first, we confirmed an
enduring reprogramming activity of the ESC extract, although on a much smaller
number of fibroblasts (⬃0.04%) and with an effect limited to the induction of
Oct4 gene expression and alkaline phosphatase activity; second, transcripts failed
to be translated as the expression of OCT-4, SSEA-1 and Forssman antigen pro-
teins was never detected; third, our work has clearly demonstrated that ESCs may
survive the procedure of extract preparation, may be source of contamination that
is expanded in culture and may give false-positive results. Several explanations
can be put forward to account for the low reprogramming frequency, as said
before, including (a) potential variableness in laboratory practice that still exists
with this protocol; (b) the possibility that the extract treatment acts only on the
tiny population of SC (⬃0.067%) that has been described to be present in mam-
malian skin cells and that may still be present in fibroblast cultures. The consis-
tent presence of a number, though small, of fibroblasts that are expressing
pluripotency markers only following extract treatment is nevertheless encourag-
ing and motivates to try to isolate these cells and expand them in culture.
Clinical Applications of Stem Cells
Even though SC are used to investigate questions to further our basic

knowledge on: (a) biological processes such as development of the organism and
progress of cancer; (b) drug discovery and (c) functional genomic studies, the
most prominent use of SC is cell therapy for treating pathological conditions.
The identification of hematopoietic SC in mice by Till and McCulloch [8]
in 1961 heralded the use of SC therapy. Adult SC (and ES) now offer hope for
reversing the symptoms of many diseases and conditions including cancer, neu-
rodegenerative diseases, spinal cord injuries and heart disease. Nowadays, in
fact, many diseases are treated by bone marrow and SC therapy with success,
just to remember a few of them, leukemia, diabetes, infarcted heart, breast can-
cer, osteogenesis imperfecta and Parkinson’s disease.
Adult SC offer hope for cell therapy to treat diseases in the future because
ethical issues do not impede their use. In addition, if the patient’s own cells are
used, immunological compatibility is not an issue. SC cord blood, from the
umbilical cord, was believed to be an alternate source of hematopoietic SC; how-
ever, it is impossible to obtain sufficient numbers of SC from most cord blood
collections to engraft an adult of average weight. Development continues on
techniques to increase the number of these cells ex vivo. Cord blood contains
both hematopoietic and nonhematopoietic SC. However, ES have been found to
be superior for both differentiation potential and ability to divide in culture. ES
can be induced to differentiate in vitro by culturing in suspension to form
3-dimensional cell aggregates called embryoid bodies. The cells spontaneously
Stem Cells 149

differentiate into various cell types, e.g. neurons, cardiomyocytes and pancreatic
␤-cells. The addition of growth factors to the culture directs differentiation to
specific cell types. Human ES lines are derived from blastocyst-stage embryos,
which occurs at about 5 days after fertilization in humans, that are excess after in
vitro fertilization procedures. Human ES have been investigated by multiple
techniques, including gene expression profiling, mitochondrial sequencing,
immunocytochemistry, genotyping and functional assays. Human ES are unique
in their abilities to maintain pluripotence and a normal diploid karyotype over
long periods in culture. These properties make human ES leading candidates for
use in cell therapy and for studies of early human development. However, it is
still challenging to isolate pure differentiated cell types. Following injection of
ES into immunodeficient mice, teratomas develop with derivatives of all 3 germ
layers. This is a major disadvantage of using ES for cell therapy, since any cont-
aminating undifferentiated cells could give rise to cancer.
SC therapies involve more than simply transplanting cells into the body
and waiting for them to go to work. A successful SC therapy requires an under-
standing of how SC work, combined with a reliable approach to ensuring that
the SC perform the desired action in the body. In other words, at least 3 steps
must be entailed.
Step 1: finding the right type of SC.
Step 2: match the SC with the transplant recipient.
Step 3: put the SC in the right place.
A good example of how therapies are developed comes from real life: an
SC therapy to treat Parkinson’s disease in humans. This therapy made its debut
in the late 1980s and was based on a successful treatment in a rat model of
Parkinson’s disease. Since the therapy was introduced, several research groups
have been evaluating its long-term success in separate trials. Parkinson’s dis-
ease is the second most common neurodegenerative disease following
Alzheimer’s. Millions of people suffer from Parkinson’s disease worldwide,
which is caused when 80% or more of dopamine-producing neurons in the sub-
stantia nigra of the brain die; thus, the movement of the body is no longer
smooth and coordinated. It has been recognized that dopamine-producing cells
are required to reverse Parkinson’s disease. Many types of dopamine-producing
cell have been used for transplantation and successes with animal models led to
clinical trials. Fetal tissue transplantation has been performed in more than 400
patients. The success of these therapies to reverse Parkinson’s disease using
fetal tissue has been quite good and the majority of the patients have been able
to lead an independent life without L-dopa treatment, even though some of
them developed uncontrolled flailing movements (dyskinesias).
Other good examples of SC therapies entering the clinical practice are those
for cardiovascular disease and diabetes. The high rate of mortality associated with
heart diseases is the inability to repair damaged tissue. Interruption of blood sup-
ply to the tissue causes infarction of the myocardium and death of myocardiocytes.
Somatic SC have been used in cell therapy for the heart. Skeletal muscle
myoblast transfers showed contraction but did not differentiate into cardiomy-
ocytes and did not integrate with the host myocardium. Ideally, both contraction
and integration with host myocardium should have occurred in order for the
therapy to be effective. Endothelial progenitor cell transplants halted the degen-
erative process but did not initiate regeneration. Human ES-derived cardiomy-
ocytes transplanted into the pig’s heart work well as a pacemaker: the ES
survived, functioned and integrated with the host cells, which is promising for
future myocardial regeneration using human ES.
As for diabetes, something like 150 million people worldwide (just 6% of
the population in the USA), pancreas transplantation has been performed in dia-
betics as more recently has pancreatic islet cell transplantation. Thanks to the
Edmonton protocol, which transplants a large amount of islet cells, early clinical
testing showed the possibility to reverse diabetes in all of the patients tested.
As illustrated, remarkable progress has been achieved in studying SC and
in the future, ideally, somatic SC from the patient will be extracted and manip-
ulated and then reintroduced into the same patient. However, it must be stressed,
more basic research to highlight the biology of SC has to be done before SC-
based therapy is widely used.
References
1 Boiani M, Scholer HR: Regulatory networks in embryo-derived pluripotent stem cells. Nature Rev
Mol Cell Biol 2005;6:872–884.
2 Avery S, Inniss K, Moore H: The regulation of self-renewal in human embryonic stem cells. Stem
Cells Dev 2006;15:729–740.
3 Bilodeau M, Sauvageau G: Uncovering stemness. Nat Cell Biol 2006;8:1048–1049.
4 Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult
fibroblast cultures by defined factors. Cell 2006;126:663–676.
5 Wilmut I: An astonishing experiment. Cloning Stem Cells 2006;8:235–236.
6 Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y, Lemischka I-R:
Dissecting self-renewal in stem cells with RNA interference. Nature 2006;442:533–538.
7 Taranger CK, Noer A, Sorensen AL, Hakelien AM, Boquest AC, Collas P: Induction of dediffer-
entiation, genome-wide transcriptional programming, and epigenetic reprogramming by extracts
of carcinoma and embryonic stem cells. Mol Biol Cell 2005;16:5719–5735.
8 Till JE, McCulloch EA: A direct measurement of the radiation sensitivity of normal mouse bone
marrow cells. Radiat Res 1961;14:213–222.
Carlo Alberto Redi

Fondazione IRCCS Policlinico San Matteo
Viale Camillo Golgi, 19
IT–27100 Pavia (Italia)
Tel. ⫹39 0382 503 451, Fax ⫹39 0382 986 270, E-Mail c.redi@smatteo.pv.it
Stem Cells 151

Author Index
Achermann, J.C. 36 Lanfranchi, F. 28 Ravazzolo, R. 1

Lin, L. 36 Redi, C.A. 145
Barbetti, F. 83 Lorini, R. VIII Ross, R.J.M. 122
Buzi, F. 28
Sagen, J.V. 94
Maghnie, M. VIII
Carapella, T. 28 Savage, D.B. 122
Maiorana, A. 16
Cesari, S. 58 Savage, M.O. VII
Melandri, L. 58
Chatterjee, V.K.K. 122 Semple, R.K. 122
Mella, P. 28
Cianfarani, S. 16 Søvik, O. 94
Merico, V. 145
Cremonini, G. 58 Spiegel, A.M. 133
Monti, M. 145
Ferraz-de-Souza, B. 36 Tammaro, P. 70
Neri, T. 145 Tansek, M.Z. 94
Njølstad, P.R. 94
Garagna, S. 145
Wales, J.K.H. 122
Geremia, C. 16
O’Rahilly, S. 122 Woods, K. 6
Germani, D. 16
Ghizzoni, L. 58 Zanoni, M. 145
Pilotta, A. 28 Zuccotti, M. 145
Hughes, I.A. 47 Prandi, E. 28
Hussain, K. 106 Puglianiello, A. 16
152
Subject Index
Acanthosis nigricans, management in trafficking defects 110, 111

severe insulin resistance 130 turnover of channels 110
Acid labile subunit (ALS) permanent neonatal diabetes defects
deficiency phenotypes 13 extra-pancreatic effects 76–78
function 12, 13 Kir6.2 gene mutations and
knockout mouse 13 mechanisms 72–76, 86, 87, 97–99
ACTH, see Adrenocorticotropin SUR1 75, 87
Adrenal hypoplasia therapeutic implications 89
adrenocorticotropin resistance structure 71, 108
syndromes 40 sulphonylurea interactions 71
gene mutations 37, 38 tissue distribution and function 70, 71, 108
primary adrenal hypoplasia
autosomal adrenal hypoplasia 43, 44 Beta-cell, see ATP-sensitive potassium
overview 40, 41 channels
syndromic forms 44
X-linked adrenal hypoplasia 41–43 CAH, see Congenital adrenal hyperplasia
secondary adrenal hypoplasia Calcium-sensing receptor (CaR)
isolated adrenocorticotropin deficiency allosteric modulators 142, 143
38 mutation and disease 141, 142
multiple pituitary hormone deficiency structure 138–141
38, 39 therapeutic targeting 143
overview 37, 38 tissue distribution and function 137, 139
proopiomelanocortin synthesis and CaR, see Calcium-sensing receptor
release disorders 40 CHI, see Congenital hyperinsulinism
Adrenocorticotropin (ACTH) Chromatin immunoprecipitation, noncoding
deficiency and secondary adrenal region analysis 4, 5
hypoplasia 38 cMyc, embryonic stem cell function 148
resistance syndromes 40 COL1A2, chromosomal translocations and
Akt, knockout mice 22, 23 gene expression 2, 3
ALS, see Acid labile subunit Congenital adrenal hyperplasia (CAH)
ATP-sensitive potassium channels (KATP) classification 58
beta-cell function 71, 108–110 frequency 59
congenital hyperinsulinism pathophysiology gene mutations 58
regulation defects 112 preimplantation genetic diagnosis 61, 62
153
Congenital adrenal hyperplasia (CAH) surgical management 55, 56
(continued) prospects for study 56
prenatal diagnosis 59–61 DSD, see Disorders of sex development
treatment Dyslipidemia, management in severe
early postnatal treatment 65–67 insulin resistance 130
prenatal treatment outcomes and risks
EIF2AK3, neonatal diabetes mutations 85
62–65
Embryonic stem cell, see Stem cell
Congenital hyperinsulinism (CHI)
ATP-sensitive potassium channels FOXP3, neonatal diabetes mutations 86
pathophysiology
Gab-1, signaling 18
regulation defects 112
Genomics
trafficking defects 110, 111
function studies 1, 2
turnover of channels 110
noncoding region analysis 2–5
diagnosis 107
GH, see Growth hormone
diffuse versus focal disease 116, 117
GLIS3, neonatal diabetes mutations 87
exercise-induced hyperinsulinemic
Glucokinase
hypoglycemia 116
congenital hyperinsulinism gene
frequency 107
mutations 112–114
metabolopathies
neonatal diabetes gene mutations 72, 85,
glucokinase gene mutations 112–114
86, 97
glutamate dehydrogenase mutations
Glutamate dehydrogenase, congenital
112, 113
hyperinsulinism gene mutations 112, 113
overview 112
GPCRs, see G-protein-coupled receptors
short-chain L-3-hydroxyacyl-CoA
G-protein-coupled receptors (GPCRs), see
dehydrogenase mutations 114, 115
also specific receptors
sequelae 106, 107
calcium-sensing receptor, see Calcium-
severity 107
sensing receptor
DAX1 gain-of-function mutations 136, 137
domains 41 GTPase cycle 134, 135
gene locus 41 inborn errors of signal transduction 133,
mutation screening 43 134
sexual differentiation role 50 loss-of-function mutations 134–136
X-linked adrenal hypoplasia 41–43 Grb2, signaling 19
Dexamethasone, congenital adrenal Growth hormone (GH)
hyperplasia prenatal treatment 63–65 insulin-like growth factor-1 axis 6, 7
Diabetes, see Neonatal diabetes; Severe pharmacogenetics 30–33
insulin resistance; Stem cell therapy indications 29
Disorders of sex development (DSD) Growth hormone receptor
classification 51, 52 deficiency
frequency 47 insulin-like growth factor-1 therapy 8, 9
genetics and nomenclature 49–51 Laron syndrome 7, 29
management phenotype 8
consensus meeting gene structure and locus 29
aims 48 polymorphisms
structure 48, 49 distribution and frequency 30
early management 53, 54 growth hormone therapy response
psychological management 54, 55 effects 31–33
Subject Index 154

phenotypes 30–32 IRS, see Insulin receptor substrate
signaling 29 IUGR, see Intrauterine growth restriction
STAT 5b signaling, see STAT 5b
structure 8 KATP, see ATP-sensitive potassium
channels
Hydrocortisone, congenital adrenal Kir6.2, see ATP-sensitive potassium
hyperplasia early postnatal treatment channels
65–67
21-Hydroxylase deficiency, see Congenital Laron syndrome, see Growth hormone
adrenal hyperplasia receptor
Hyperinsulinism, see Congenital Leptin, replacement therapy 128–130
hyperinsulinism Luteinizing hormone receptor
gain-of-function mutations 137
IGF-1, see Insulin-like growth factor-1 loss-of-function mutations 134, 135
Inborn errors of signal transduction, see
G-protein-coupled receptors MAPK, see Mitogen-activated protein
Insulin-like growth factor-1 (IGF-1) kinase
acid labile subunit complex, see Acid Mitogen-activated protein kinase (MAPK),
labile subunit insulin-like growth factor-1 receptor
deficiency 9, 10 signaling alterations in placenta 23, 24
growth hormone axis 6, 7 MPHD, see Multiple pituitary hormone
missense mutation and altered receptor deficiency
interactions 19, 20 Multiple pituitary hormone deficiency
therapy for growth hormone receptor (MPHD), secondary adrenal hypoplasia
deficiency 8, 9 38, 39
Insulin-like growth factor-1 receptor
gene locus 22 Nanog, embryonic stem cell function 148
mutations and phenotypes 20–22 Neonatal diabetes
signaling ATP-sensitive potassium channel defects
human placenta alterations and in permanent neonatal diabetes
intrauterine growth restriction 23, 24 extra-pancreatic effects 76–78
knockout mouse studies of mediators Kir6.2 gene mutations and
22, 23 mechanisms 72–76, 86, 87, 97–99
overview 18, 19 SUR1 75, 87
structure 17 therapeutic implications 89
Insulin receptor classification 83, 84, 95, 96
signaling 18, 19 clinical significance of gene mutations
structure 17 89, 90
Insulin receptor substrate (IRS) EIF2AK3 mutations 85
knockout mice 22 FOXP3 mutations 86
signaling 18, 19 frequency 72, 95
Insulin resistance, see Severe insulin GLIS3 mutations 87
resistance glucokinase gene mutations 72, 85, 86,
Intersex, see Disorders of sex development 97
Intrauterine growth restriction (IUGR), history of study 83, 84
insulin-like growth factor-1 receptor imprinting defects in transient disease
signaling alterations in placenta 23, 24 84, 99–101
IPF1, neonatal diabetes mutations 85 IPF1 mutations 85
Subject Index 155

Neonatal diabetes (continued) insulin sensitizers 129
PTF1A mutations 87 leptin replacement therapy 128–130
sulfonylurea therapy 96 Sexual differentiation, see Disorders of sex
type 1 diabetes diagnosis and development
management 101, 102 SF1, see Steroidogenic factor-1
Wolcott-Rallison syndrome 101 Short-chain L-3-hydroxyacyl-CoA
NPPC, chromosomal translocations and dehydrogenase (SCHAD), congenital
gene expression 2, 3 hyperinsulinism gene mutations
NPS R-568, calcium-sensing receptor 114, 115
modulation 142, 143 Sox2, embryonic stem cell function 148
SRY, congenital adrenal hyperplasia
Oct4, embryonic stem cell function 148, 149 prenatal diagnosis 60, 61
STAT 5b
Parathyroid hormone receptor deficiency
gain-of-function mutations 137 domain localization of mutations 12
loss-of-function mutations 134 phenotypes 10–12
Peroxisome proliferator-activated receptor-␥ growth hormone receptor signaling 10
(PPAR␥), severe insulin resistance Stem cell (SC)
mutation 123 clinical applications 149–151
Pharmacogenetics defining properties 145, 146
definition 28 developmental function 146
growth hormone 30–33 diabetes management prospects 151
POMC, see Proopiomelanocortin embryonic stem cells 146, 147
Potassium channels, see ATP-sensitive engineering 147–149
potassium channels regeneration of organs and tissues 147
PPAR␥, see Peroxisome proliferator- somatic stem cells
activated receptor-␥ features 147
PPP1R3A, severe insulin resistance therapeutic potential 149, 150
mutation 123 Steroidogenic factor-1 (SF1), mutation in
Proopiomelanocortin (POMC), defects and adrenal hypoplasia 43
secondary adrenal hypoplasia 40 SUR1, see ATP-sensitive potassium
PTF1A, neonatal diabetes mutations 87 channels
Syp, signaling 18
RET, mutation and disease 3, 4
RSPO1, sexual differentiation role 50 Thyroid-stimulating hormone receptor
gain-of-function mutations 137
SCHAD, see Short-chain L-3-hydroxyacyl-
loss-of-function mutations 134
CoA dehydrogenase
Turner syndrome, growth hormone therapy
Severe insulin resistance
response 32, 33
case history 122–124
classification of syndromes 125–127 Vasopressin V2 receptor, loss-of-function
clinical features 124, 125 mutations 136
insulin sensitivity measurement 127–128
treatment WNT4, sexual differentiation role 50
acanthosis nigricans 130 Wolcott-Rallison syndrome (WRS),
dyslipidemia 130 neonatal diabetes 101
immunosuppression 128 WRS, see Wolcott-Rallison syndrome
Subject Index 156

Congenital Endocrinopathies

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Congenital Endocrinopathies

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Congenital Endocrinopathies

New Insights into Endocrine Diseases and Diabetes

Martin O. Savage London

Renata Lorini Genova

24 figures and 12 tables, 2007

Basel · Freiburg · Paris · London · New York ·

Library of Congress Cataloging-in-Publication Data

Congenital endocrinopathies : new insights into endocrine diseases and

© Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)

1 Genomic Approaches in Genetic Research for Endocrine Diseases

6 Genetic Defects of the Growth-Hormone-IGF Axis Associated with

16 Late Effects of Disturbed IGF Signaling in Congenital Diseases

28 Growth Hormone Receptor Polymorphisms

36 Genetic Disorders Involving Adrenal Development

47 Early Management and Gender Assignment in Disorders of Sexual

83 Diagnosis of Neonatal and Infancy-Onset Diabetes

94 Management of Neonatal and Infancy-Onset Diabetes Mellitus

106 Insights in Congenital Hyperinsulinism

122 A Clinical Approach to Severe Insulin Resistance

133 Inherited Endocrine Diseases Involving G Proteins and

145 Stem Cells

152 Author Index

153 Subject Index

This volume reports the proceedings of an outstanding symposium, held in

Martin O. Savage, London

Renata Lorini, Mohamad Maghnie, Genova

Genomic Approaches in Genetic Research

Sequencing of genomes has enabled the ‘genomic’ approach to research,

Fig. 1. Scheme of the genomic region upstream of COL1A2, in which 5 conserved

C-type natriuretic peptide, which in turn caused a marfanoid phenotype in the

Genomic Research in Noncoding Regions 3

Fig. 3. Example of differential histone acetylation level at 1 specific conserved ele-

Prof. Roberto Ravazzolo

Genomic Research in Noncoding Regions 5

Genetic Defects of the Growth-Hormone-IGF

Following binding to and activation of the cell-surface-bound growth hor-

effects). The relative contributions of circulating IGF-1, local IGF-1, and

Growth Hormone Receptor Gene Deficiency

Genetic Defects of the GH-IGF Axis 7

IGF-1 Gene Deficiency

In 1996, we described a 15-year-old boy with severe growth failure (height

Genetic Defects of the GH-IGF Axis 9

STAT-5b Gene Deficiency

The GH receptor activates 3 main signaling pathways: the signal transducer

Author Height SDS Age Sex IGF-1 IGFBP-3 Immunodeficiency and

Kofoed et al. [14], ⫺7.5 15.8 female ↓↓↓ ↓↓↓ ⫹

A630P: Kofoed et al. [14], 2003

N398fsX413: Hwa et al. [16], 2005

Q368fsX376: Vidarsottir et al. [17], 2006

R152X: Bernasconi et al. [18], 2006

Acid Labile Subunit Deficiency

ALS is a GH-dependent glycoprotein which stabilizes the IGF-IGFBP-3

In recent years, the causes of genetic GH insensitivity in man have broad-

Genetic Defects of the GH-IGF Axis 13

Katie Woods, MBBS, MRCP, MD

Genetic Defects of the GH-IGF Axis 15

Late Effects of Disturbed IGF Signaling

Stefano Cianfarani, Caterina Geremia, Antonella Puglianiello,

Structure of the Insulin and IGF-1 Receptors

IGF Signaling and Growth Retardation 17

PDK-2 PDK-1 PI-3,4-P2 PI-3,4,5-P3 PI-4,5-P 2 Plasma membrane

Akt inactive PI 3’-kinase

Signal Transduction via Insulin Receptor and IGF-1 Receptor

Many of the intracellular signaling events mediated by activation of the IR