INVITED REVIEW
Recent Advances in the Molecular and Genetic
Understanding of Congenital Gastrointestinal
Malformations
Véronique Dauvé and Valérie A. McLin
ABSTRACT
Major developmental paradigms are highly conserved among vertebrates.
The contribution of developmental biology to the understanding of human
disease and regeneration has soared recently. We review advances in the
molecular and genetic understanding of gastrointestinal development
using evidence from both mammalian and nonmammalian models. When
appropriate, we highlight relevance and applicability to human disease.
Key Words: development, gastrointestinal malformations, molecular
regulation
(JPGN 2013;57: 4–13)
M olecular embryology has been instrumental in advancing
liver stem cell differentiation in vitro. In contrast,
its contribution to gastrointestinal (GI) development has been to
unravel the genetic underpinnings of GI malformations. Clinically,
this is significant for the purposes of antenatal screening and genetic
counselling. In addition, from an embryologist’s perspective,
the study of developmental anomalies offers insight into normal
development. Thus, this review focuses on malformations of the GI
tract along the craniocaudal axis from the oesophagus to the rectum.
It aims to correlate molecular and genetic regulation of develop-
ment with known clinical phenotypes of developmental defects.
BRIEF OVERVIEW OF GI DEVELOPMENT
Vertebrate development is characterized by the formation of
the 3 primitive germ layers (endoderm, mesoderm, and ectoderm)
during gastrulation. The multipotent endoderm gives rise to the liver,
epithelial lining of the GI tract, lung, thyroid, and pancreas, whereas
the mesoderm is the precursor of the intestinal mesenchyme and
Received October 11, 2012; accepted January 24, 2013.
From the Département de Pédiatrie, Faculté de Médecine, Hôpitaux
Universitaires de Genève et Université de Genève, Genève, Switzer-
land.
Address correspondence and reprint requests to Dr Valérie A. McLin,
Département de Pédiatrie, Hôpitaux Universitaires de Genève, 1211
Genève 14, Switzerland (e-mail: valerie.mclin@hcuge.ch).
Supplemental digital content is available for this article. Direct URL
citations appear in the printed text, and links to the digital files are
provided in the HTML text of this article on the journal’s Web site
(www.jpgn.org).
The present work was supported by the Swiss National Science Foundation
(grant 31003A-132721) and the Gertrude von Meissner Foundation to
V.A.M. and Hans Wilsdorf Foundation (Geneva, Switzerland) to V.D.
The authors report no conflicts of interest.
Copyright # 2013 by European Society for Pediatric Gastroenterology,
Hepatology, and Nutrition and North American Society for Pediatric
Gastroenterology, Hepatology, and Nutrition
DOI: 10.1097/MPG.0b013e3182922b49
4 JPGN
Volume 57, Number 1, July 2013
Copyright 2013 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
visceral musculature. Starting at gastrulation, an array of trans-
cription factors and signalling pathways in the endoderm and the
adjacent mesoderm contribute to cell-fate decisions, which direct the
fate of endoderm and mesoderm progenitors (Table 1).
Development of the GI tract is governed by a vast molecular
network comprising signalling pathways and transcription factors,
which are still incompletely characterized. The basic paradigm of
GI development is that cell-fate decisions occur along the following
4 axes: dorsoventral, anterioposterior, left–right, and radial (Fig. 1).
Anterior–posterior patterning is necessary for the regional speci-
fication of the gut and a prerequisite for differential function from
mouth to anus. Second, lumen formation is essential for epithelial
morphogenesis. In other words, lumen formation is a prerequisite
for the development of the large and specialized surface area
required for sufficient nutrient absorption to sustain life. Many
of these processes are directed by complex molecular and physical
interactions between the developing endoderm and mesoderm.
Furthermore, the vertebrate GI tract is characterized by its extensive
coiling. These morphogenetic movements are ‘‘necessary’’ for
spatial organization in the abdominal cavity, but their contribution
to intestinal function per se is unclear.
The weakness of such a complex system is that a single,
erroneous cell-fate decision can lead to severe developmental defects,
often incompatible with extrauterine life. Mutations in ubiquitous
signalling pathways are commonly assumed to underlie systemic
syndromes. It follows that many other signalling cascades and
transcription factors are probably involved in GI development as
evidenced by the number of syndromes comprising GI malformations
(1–3).
The purpose of this review is to illustrate this principle using
examples from human, mouse, Xenopus laevis (African clawed frog),
and zebrafish because it is accepted that developmental paradigms are
conserved across vertebrate species. We will not expand on early
specification and regional patterning for 2 reasons: they have been
well summarized by experts in the field (4,5) and signalling defects at
these early stages are likely associated with embryonic lethality.
Rather, the focus is on the developing luminal digestive system along
the craniocaudal axis from the oesophagus to the rectum after the
initial patterning events. The reader is referred to recent reviews
for molecular advances of liver and pancreas development (6–8).
Conventional gene and protein nomenclature and symbols are
used throughout. Figure 2 summarises genes and pathways that
are implicated in malformations according to anatomic origin.
GI MALFORMATIONS
Oesophagus
Oesophageal atresia is diagnosed either prenatally on
ultrasound or promptly noticed in the first hours of life. It occurs
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TABLE 1. Glossary

Primary germ layers

Endoderm The endoderm consists of cells which form the epithelial lining of the whole of the digestive tract (except part of the mouth
and pharynx and the terminal part of the rectum) and the lungs.
Mesoderm The mesoderm germ layer consists of cells having migrated between the endodermic and the ectodermic layers. It forms
skeletal muscle, the skeleton, the dermis of skin, connective tissue, the urogenital system, the urinary bladder, the lining
of the urethra, the heart, blood (lymph cells), the kidney, and the spleen.
Ectoderm The ectoderm forms from the outer layer of germ cells. It forms the nervous system (spine, peripheral nerves and brain),
tooth enamel, and the epidermis (the outer part of integument).
Axes
Dorsoventral axis The dorsoventral axis extends from spinal column (back, dorsal) to belly (front, ventral).
Craniocaudal axis The craniocaudal axis extends from the head end to the tail.
Anteroposterior axis The anteroposterior axis is similar to the craniocaudal axis but is mostly used when referring to embryonic development.
Left–right axis The left–right axis extends from the left to the right sides of the body.
Radial axis Radial axis refers to tubular segments.
Developmental and molecular terms
Specification Specification term is used to describe a region of a future organ capable of differentiating autonomously when placed in
a neutral environment.
Differentiation Differentiation characterized by cellular specialization.
Ontogeny Ontogeny refers the origin and the development of an individual organism from embryo to adult.
Morphogenesis Morphogenesis refers to the evolution and development of form.
Morphogen A morphogen is a signalling molecule which governs the pattern of tissue development, by acting directly on cells to
produce specific cellular responses depending on the local concentration of the morphogen.
Primordium Primordium is defined as an organ or tissue in its earliest stage of development.
Patterning Patterning refers the series of molecular signals generated that result in the spatial identity of regions along the axes.
Signalling pathway Signalling pathways (cascades) refer the relaying of molecular signals and are involved in control of many cell functions,
such as cell division and programmed cell death.
Transcription factor A transcription factor is a protein that binds to specific DNA sequences. The binding leads to gene transcription thereby
controlling the flow of genetic information from DNA to mRNA.

in 1 in 3500 births, and several anatomic variants have been
described (9).
Oesophageal and tracheal development are tightly linked.
During human gestation, early embryonic events lead to the
development of the respiratory primordium at approximately
4 weeks of development (9). Then, the early foregut endoderm
and mesenchyme give rise to the oesophagus and trachea.
Therefore, the advent of abnormal communications between the
2 neighbouring, tubular organs is a logical corollary. Typically,
anomalous communications present as 1 of the following:
oesophageal atresia with a distal tracheooesophageal fistula
(TOF) (85% of cases), oesophageal atresia without fistula (10%
of cases), oesophageal atresia with a proximal TOF (1% of cases),
oesophageal atresia with both proximal and distal TOF (<1%
of cases), and H-type fistula characterised by the absence of
oesophageal atresia but the isolated presence of TOF (4% of cases).
It is widely accepted that although genetic defects may
underlie familial cases of oesophageal atresia, few candidate genes
have been identified. In a mouse model, the absence of Nkx2.1
expression in the ventral foregut and/or altered or absent Sox2
expression in the dorsal foregut (10) leads to incomplete separation
of the oesophagus and trachea (11). Furthermore, the sonic
hedgehog (Shh) signalling pathway likely exerts independent or
additional effects, explaining some cases of oesophageal atresia/
TOF (9,12). It is unclear whether these defects originate in the
endoderm via SHH signalling or in the mesenchyme where SHH
receptors GLI2, GLI3 (13), and SHH-target genes are expressed
(12,14).
In humans, SOX2 mutations are associated with anophthal-
mia, oesophageal, and genital syndromes (15,16). Oesophageal
atresia may be associated with Coloboma, Heart anomaly, choanal
Atresia, Retardation, Genital and Ear anomalies (CHARGE)
syndrome, possibly implicating the chromodomain helicase
DNA-binding protein 7 gene (CHD7), a relatively ubiquitous
nuclear protein (17–19). Patients with Feingold syndrome
have also been described as presenting with oesophageal atresia.
Genetically, Feingold syndrome is characterized by a microdeletion
in the Shh target myelocytomatosis-viral-related-oncogene-
neuroblastoma-derived (MYCN). The fact that this gene is known
to be downstream of Shh suggests that akin to what is known in the
mouse, SHH is likely involved in human oesophageal development
and malformations (20).
Stomach and Pylorus
Gastric and pyloric malformations occur either as an isolated
finding or as part of a syndrome. Obstructions or malformations
of this anatomical region usually lead to unmistakable, obstructive
clinical symptoms in the neonate.
The first such malformation is extremely rare: agastria.
Only 1 case report was identified in the literature and the outcome
was fatal (21). Congenital microgastria is a little more common
and can be associated with other foregut malformations (22).
The only genetic association identified in humans to date is with
22q11.2 deletion and ciliary dyskinesia in a newborn with DiGeorge
syndrome (23).
Abnormalities of the pylorus are much more common.
The pyloric sphincter is an anatomic region composed of charac-
teristic epithelial and mesodermal structures, which, under normal
conditions, are slightly hypertrophied to limit luminal flow into the
duodenum. When hypertrophy is more severe, it obstructs gastric
emptying and the infant develops refractory vomiting. Obstructions
can be more or less complete. A total of 1 to 3/1000 live births
in Europe display a partial obstruction known to be infantile
hypertrophic pyloric stenosis (IHPS) (24). New genetic approaches

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 Dauvé and McLin JPGN
Volume 57, Number 1, July 2013

Cranial
(proximal)
A B
Mid
t gut
egu
For
Right Left

Hin
dgu
Posterior

t
Anterior Dorsal

C
Radial

Ventral

Caudal
(distal)
FIGURE 1. The 4 axes governing GI development, form, and function. The anteroposterior axis is characterized by an axis from head end
to opposite end of body of a human embryo at stage 13 of development (A). Others axes are illustrated in (B) (courtesy of Dr Michael Bates).
Note that the craniocaudal axis and the anteroposterior axis are similar, but the term anteroposterior is typically used to refer to embryonic
development. The radial axis is illustrated by a schematic of a transverse section of the jejunum (C).

in humans have demonstrated that the underlying defect is
a mutation of muscleblind-like (MBNL1) gene product (24). The
muscleblind protein family of zinc finger proteins is known for
its role in myotonic dystrophy. Its function is to regulate splicing in
the early postnatal period, which is essential for extensive muscle
remodelling during the first 3 weeks of extrauterine life. This timing
fits with the clinical presentation of IHPS, which occurs almost
exclusively between 2 and 8 weeks postnatally.
As may be expected, no single gene is implicated in the
morphogenesis of such a functionally important structure. Rather,
several other signalling pathways and transcription factors may
contribute to the onset of IHPS. First, mutations in nitric oxide
synthase (nNOS) have been identified in a small number of human
cases IHPS (3,25), in keeping with observations in Nos-1–deficient
mice (26). Nitric oxide mediates transient pyloric relaxation; thus,
defects in NOS-1, and consequently in smooth muscle relaxation,
could explain some cases of IHPS.
Another locus has recently been shown to be associated with
cases of IHPS: the SHH target and homeobox-containing transcrip-
tion factor NKX2.5, located on chromosome 5q35.2 (24). During
chick embryonic development, NKX2.5 is expressed during a finite
developmental window and is necessary for normal pyloric muscle
(27). Its expression is limited to the pyloric sphincter (27). NKX2.5
may play a role in pyloric development in 1 of 2 ways: either
directly through its effect on differentiating muscle precursors or
because it is a target of SHH signalling, known to be implicated in
the development of the foregut and pylorus.
Sox9 is another early SHH target located in the mouse
pylorus. Mesodermal bone morphogenetic protein-4 (BMP4) sig-
nalling leads to activation of Sox9-Gremlin, shown to be necessary
and sufficient in mice for normal development of the pylorus.
To date, however, SOX9 mutations have not been identified in
humans with IHPS (28).
Duodenum
At the morphological level, duodenal development is charac-
terized by alternating windows of cell proliferation and cell death,
in keeping with a fundamental theory of lumen formation (12,29).
The proliferative phase is associated with a decrease in lumen
diameter, ultimately leading to occlusion, whereas waves of
programmed cell death (apoptosis) lead to vacuole formation,
recanalization of the lumen, and villi formation (30).
Duodenal malformations diagnosed in neonates include
duodenal atresia, duodenal web, annular pancreas, duodenal
stenosis, and duodenal diverticulum. As many as half of these
malformations are associated with other anomalies (31). The esti-
mated incidence of congenital duodenal abnormalities varies
between 1:4000 and 1:15,000 live births (32).
Based on studies in mice, it is probable that abnormal
epithelial–mesenchymal interactions are at the root of many
duodenal malformations. There is some consensus that the SHH
pathway is an important player in this process by regulating gene
expression in the developing duodenum (2,33). In Shh/ mice, villi
overgrowth leads to luminal occlusion similar to human duodenal
stenosis (2,33,34).
In contrast, little data from humans are available. Duodenal
atresia has been identified in Feingold syndrome, described in the
setting of oesophageal atresia (20), thus implicating MYCN and

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 JPGN
Volume 57, Number 1, July 2013 Genetics of GI Malformations

Normal GI tract Malformations Molecular regulators

Mesodermal Endodermal Other

Oesophagus
Oesophageal atresia:
Sox2,
A Pure oesophageal atresia
Nkx2.1, Shh
GLI2, GLI3,
B Tracheo-oesophageal
Foxf1
fistula
A B

C Partial visceral organs C Heterotaxy CFC1, NKX2.5 NODAL, CX43 ZIC3, ACVR2B
CX43, LEFTYA
D Total visceral organs D Situs inversus DNAI1, DNAH5,
DNAH11

NKX2.5,

duodenum
E Infantile hypertrophic MNBL1,
Stomach E Pyloric stenosis Sox9 nNOS, Nos-1

F Duodenal atresia FOXF1, Fgfr2b MYCN

Fgf10
F

G Jejunal and ileal atresia RFX6

G H
H Colonic atresia Fgf10 Fgfr2b, Cdx2
Small bowel/colon

I Gastroschisis ICAM1, ADD1, NPPA

NOS3, Shroom3
J Omphalocele PITX2 TCN2, TCblR,
K L MTHFR
K Meckel’s diverticulum CDX2, SHH

L Hirschsprung’s disease SOX10, GDNF,

ET-3, PHOX2B
M Intestinal malrotation FOXF1, NKX2.5 ZIC3, ACVR2B
M
CFC1, LEFTYA

Anorectal malformations:
Rectum

N Cloaca HLXB9,
Gli2, Gli3,
Shh,
Gdf11
N O O Imperforate anus Pcsk5

FIGURE 2. Summary of genes and pathways implicated in gastrointestinal (GI) malformations and syndromes presented according to organ and
germ layer. International gene nomenclature is used. Mutations in the underlined genes have been identified in humans.

SHH in human duodenal development. Duodenal atresia has also
been identified in human infants with alveolar capillary dysplasia
who display deletions in the FOXC1/FOXL1/FOXF1 cluster (35).
The fibroblast growth factor (FGF) family is known
to participate in epithelial–mesenchymal interactions and as such
is another likely signalling pathway in duodenal (mal) develop-
ment. Fgfr2b/ or Fgf10/ mutant mice display variable pheno-
types of duodenal atresia, resembling what is observed in humans
(36,37). No case of FGF mutation has yet been reported in human
duodenal malformations.
Defects in genes regulating vacuolization and lumen
formation may be the cause of duodenal diverticula. This rare
anatomical anomaly mostly occurs along the pancreatic or
mesenteric border in the second or third portions of the duodenum.
Rarely, the diverticulum can be intraluminal, presenting as upper GI
obstruction (38,39). Although little is known in humans and mice
about lumen formation, it has been shown in a zebrafish model that
genes regulating ion flux participate in normal lumen formation.
Mutations in this cascade lead to multiple lumina (40). Looking for
mutations in human homologues seems the obvious next step.
Jejunum and Ileum
The midgut also displays an array of anatomical anomalies
accepted to occur secondary to developmental events. The 2 broad
categories are atresias and duplications.
Jejunal and Ileal Atresias
Jejunal or ileal atresias may present as GI obstruction early in
postnatal life (41). Their incidence varies from 1:300 to 1:3000 live
births. Atresias are defined according to their morphology and
blood supply. The morphological description refers to the nature
of the obstruction between bowel segments (diaphragm vs fibrous
cord) and to the blood supply via the mesentery (41). The import-
ance of emphasizing blood supply is that a defect in blood supply
may be the primum movens of several of these neonatal defects.
Although jejunal and ileal atresias are usually bundled
together as one and the same disease, their clinical presentation
and postoperative course often differ, suggesting 1 of 2 possibilities:
the aetiologies are different or the 2 segments respond differently to
obstruction and altered blood supply owing to regional, anatomical,
and functional differences (42,43).
The aetiology of jejunal and ileal atresia is unclear.
Historically, there are 2 theories. The first and older of the
2 proposes that imperfect recanalization during development is
the root of the obstruction (44). Today, the obvious question is
‘‘what are the genes governing this recanalization defect?’’ The
other theory proposed by Louw and Barnard suggests that vascular
events underlie these malformations (45,46). Canine models favour
the vascular theory, something that is also supported in a reported
case of ileal atresia associated with vascular band anomaly (47).
Recently, it has been suggested that the gene regulatory
factor X6 (RFX6) may be involved in intestinal atresias. The

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 Dauvé and McLin
rationale is that several patients with Mitchell-Riley syndrome,
characterised by jejunal or ileal atresia and others malformations
(pancreatic hypoplasia, gallbladder aplasia, or hypoplasia, extra-
hepatic biliary atresia with or without TOF and neonatal diabetes),
have mutations of the RFX6 gene (48–50). (References 51–158 are
available online only at http://links.lww.com/MPG/A216.) Because
RFX6 is expressed in the early pancreatic endoderm it is unclear
how it may be linked to intestinal atresias (50–52). An obvious
candidate partner is the pancreatic duodenal homeobox 1 (PDX1),
which is an important transcription factor in early foregut specifi-
cation (53,54). Gene profiling technologies should assist in
unravelling the molecular network governing duodenal develop-
ment (54).
Ileal and Jejunal Duplications
Atresias and duplications also occur in the midgut: jejunum
and ileum, both presenting with obstructive symptoms. Usually, GI
duplications presents before age 2 years. They can occur anywhere
along the digestive tract and typically present in 1 of 2 forms: cystic
(80%) or tubular. The frequency of their distribution is as follows:
ileum 30%, ileocecal valve 30%, duodenum 10%, stomach 8%,
jejunum 8%, colon 7%, and rectum 5% (42).
Killpack described a cystic, ileal duplication as a double
organ ‘‘with distinct mucosa composed of stratified polygonal
almost squamous, cells, the most superficial of which formed a
continuous layer of ciliated epithelium’’ (55). Thus, although little
is known in mammals about the molecular mechanisms underlying
duplications, it appears that they likely recapitulate most steps of
GI development. Again, one may postulate that genes involved
in lumen formation via vacuolization would be likely candidates
to explore.
Meckel Diverticulum
During human embryonic development, the yolk sac and
the developing foetus are joined by the vitelline duct. This duct
generally narrows and disappears by the ninth week of gestation.
If this fails to occur, the proximal part of the vitelline duct persists
as Meckel diverticulum (56). It is the most frequent congenital
anomaly of the GI tract, affecting as much as 2% of the general
population (57). Although this malformation does not compromise
life expectancy, as many as 25% of individuals will present with
a complication. Complications include inflammation (diverticuli-
tis), haemorrhage, intussusception, small bowel obstruction, stone
formation, and malignant transformation (57).
The molecular mechanisms governing the involution of the
vitelline duct are incompletely understood; however; evidence
from human samples suggests that CDX2 may be one of the actors
(58). In human samples of Meckel diverticulum associated
with gastric heterotopias, CDX2 expression was absent from the
epithelium (59). Likewise, diverticula and gastric heterotopia were
observed in Cdx2þ/ mice, suggesting that this association arises in
the absence of 1 wild-type allele (60). Finally, SHH overexpression
in human surgical specimens is in support of a role for CDX2
because CDX2 is known to repress SHH signalling (61,62).
Colon
Colonic Atresia
Colonic atresia is a rarer event than congenital luminal
obstruction of the small bowel. The incidence is estimated to be
approximately 1 in 20,000 live births (63). Colonic atresia is often
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JPGN
Volume 57, Number 1, July 2013
associated to other developmental anomalies such as jejunoileal
atresias, Hirschsprung disease, and genitourinary malformations.
The most common hypothesis for colonic atresia is that
a vascular accident leads to decreased intestinal perfusion and
ischaemia (46), resulting in tissue necrosis and, in severe cases,
complete luminal obstruction. This hypothesis was verified for the
small bowel, but no study has demonstrated a role for vascular
problems in the advent of colonic atresia (45,46).
Recent animal studies challenge this theory by suggesting
that developmental, genetic mechanisms may play a role.
Both Fgf10/ and Fgfr2b/ animals display colonic atresia in
addition to duodenal atresia. The proposed mechanism is that
altered mesoderm–endoderm signalling leads to decreased
proliferation and increased apoptosis of epithelial cells (36,64).
Another mouse model offering insight into the heritable
origins of colonic atresia is the Cdx2 null mouse. CDX2 is a
homeobox-containing transcription factor, which is expressed in
the developing foregut and hindgut, where it regulates proliferation
and differentiation along the crypt–villus axis (58,65,66). Again,
this model suggests that misregulated epithelial proliferation
contributes to maldevelopment of the gut lumen, which is in
keeping with the recanalization theory (12); however, to date,
CDX2 has been shown to participate in human GI neoplasias,
but has not been identified in samples from infants with colonic
atresia (67).
Based on this and other studies, it is safe to conclude that
molecular regulation of epithelial–mesenchymal interactions in the
gut is essential in controlling epithelial proliferation. Intestinal
atresias may be the results of localized misregulation of epithelial
proliferation in utero. What is unclear is why most atresias are focal
or localized rather extending over long segments. One postulate
is that multiple, redundant signalling pathways overlap to ensure
lumen patency and that maldevelopment ensues in areas where they
fail to overlap.
Colonic Duplication
Colonic and rectal duplications are among the rarest
forms of intestinal duplication. Most are silent and detected during
the investigation of genitourinary malformation. Because colonic
duplications have the potential for malignant transformation,
detection is important (68).
The cystic form (80%) is more common than the tubular form
(20%), and both types may communicate with the lumen. They are
located on the mesenteric border of the bowel and may affect any
segment of the colon, including the rectum (69). By histology,
mucosa, submucosa, and muscularis propria are readily identified
(42). From a developmental point of view, it is remarkable that
all intestinal segments are prone to atresias and duplications,
although at varying degrees, because molecular regulation and
plasticity of the intestine differs from one segment to another (70).
Little is known about the genetic causes of colonic dupli-
cation. Defects in vacuolization and recanalization are the over-
riding theory, much like what is assumed for the jejunum and ileum
(38,39). Others have suggested that caudal syndromes may affect
the distal bowel together with the notochord and genitourinary
organs (split notochord, embryonic diverticula) (68).
Anorectal Malformations
Anorectal malformations (ARMs) are a frequent clinical
problem for paediatric surgeons and gastroenterologists alike.
The spectrum of malformations extends from anal stenosis to
imperforate anus with or without fistula (rectovesical, prostatic,
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Volume 57, Number 1, July 2013
urethral, or vaginal) (71). The incidence is 1:2500 to 1:5000 (72).
More than 50% of ARMs are associated with other malformations
affecting the trachea, oesophagus, duodenum, colon, spinal cord,
kidneys, urinary tract, and heart (71). That ARMs are associated
with multiple other anomalies and syndromes speaks to the
molecular complexity regulating the development of the distal
GI tract in concert with neighbouring structures.
Understanding of the molecular network governing the
coordinated organogenesis of the caudal part of the embryo is still
in its infancy. Nonetheless, numerous chromosomal abnormalities
have been identified, the most significant of which is the 7q36
deletion, for its effect on the function of the homeobox gene HLXB9
(73). HLXB9 is now recognized as causally linked to the Currarino
triad, which includes a presacral mass, an anterior meningocele, and
rectal duplications (71,74,75). Previously, HLXB9 was known for
its role in pancreatic development (74). By virtue of the fact that
HLXB9 is a homeobox gene, its role in anterior–posterior patterning
is intuitive; however, it is only 1 actor in an intricate ontogenic
process regulating the complex, caudal development of the embryo.
Indeed, it is not causally linked to other ARM such as caudal
regression syndrome (75). Furthermore, the role of HLXB9 in the
Currarino triad remains unclear, and presently there is no
available mouse model to explore its function because the
Hlxb9/ mouse does not exhibit vertebral or ARMs (73,76).
It is possible that the molecular regulation of a sphincter region
differs between lower mammals and humans.
SHH and HLXB9 are in proximity at the 7q36 locus (73);
however, to date, no SHH mutation has been identified in human
ARM. Animal models are plentiful to demonstrate the critical role
of SHH signalling in hindgut development. Shh/ mice show
ARM resembling human disease. Gli2/or Gli3/ knockouts
phenocopy the Shh/ mouse (77); thus, SHH signalling, discussed
for its role in proximal GI development, is also a likely determinant
of spatial and temporal morphogenesis of the anorectal region.
Studies in zebrafish and chick have, respectively, identified
proprotein convertase subtilisin kexin 5 ( pcsk5) (78) and its target,
the growth differentiation factor (GDF11) (79), a member of the
transforming growth factor-b (TGF-b) signalling cascade, as
potential actors in the intricate morphogenetic events occurring
in the genitourinary and anorectal regions of the developing
embryo. Rodent models for pcsk5 or Gdf11 loss-of-function display
ARM and altered Hlxb9 expression, resembling what is observed in
humans (80,81). Although to date no mutation in these genes has
been identified in humans, it is likely that a PCSK5–GDF11
cascade participates in distal Hox gene expression, which in
turn, coordinates early distal patterning of urological, skeletal,
genitourinary, and anorectal development primordia (81).
Persistent cloaca is a rare malformation affecting 1:250,000
live-born girls, in which the urinary tract, vagina, and rectum
are fused, creating a single channel, the cloaca. Although this
malformation is at the crossroads of GI, genital, and urinary
development, and thus slightly beyond the scope of this review,
it is worth expanding on briefly because it is a poignant illustration
of an intricate developmental process gone awry.
In brief, development of the distal part of the embryo requires
a coordinated orchestration for normal morphogenesis of 3 tubular
systems: urinary, GI, and genital. It is accepted that the caudal
lateral mesenchyme is central to this process. Based on mouse and
zebrafish studies, the SHH signalling cascade is essential (77,82–
84), although no mutation has been identified in human cases.
How SHH interacts with the PCSK5–GDF11–HLXB9 sequence is
unclear. Finally, SHH is central to so many embryonic processes,
which may explain why no mutations have been identified to date in
humans; it is likely that in humans any such mutation leads to
embryonic or perinatal mortality.
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Genetics of GI Malformations
Finally, from an ontogenic perspective, it is tempting to
suggest that distal anorectal and genitourinary development is a
sort of ‘‘developmental Achilles heel’’ of the vertebrate embryo.
Indeed, developmental expression patterns of Hox genes and other
transcription factors, as well as signalling pathways, are enriched in
this region (Fig. 3), as they are in the foregut. Yet, the molecular and
cellular processes involved are so complex that minor variations of
the normal developmental course lead to significant morphological
(and clinical) events, suggesting that any redundant signalling
mechanism is at least in part insufficient to prevent against
ARM. In other words, one may hypothesize that areas of little or
no overlap between signalling cascades or morphogens may be
particularly vulnerable to developmental errors (Fig. 3).
Hirschsprung Disease
Hirschprung disease (HD) affects approximately 1 in
5000 infants. It is a congenital disease characterized by colonic
dysmotility owing to the absence of myenteric innervation necess-
ary for normal motility. The enteric nervous system (ENS) is
derived from neural crest cells (NCCs), which migrate to constitute
the vagal and sacral plexuses during development. Improper
migration through the sacral ganglia leads to impaired distal
motility, characterized on imaging by a thin distal segment and a
dilated proximal colon. Furthermore, NCC interact with neighbour-
ing cells: both interactions with the epithelium and the mesenchyme
are essential for NCC maintenance and function. Naturally, such a
complex system is regulated by numerous signalling cascades and
transcription factors, making for a vast array of candidate genes
involved in defective NCC migration, differentiation, and homeo-
stasis (85).
Briefly, NCC receptors recognize signalling molecules syn-
thesized by intestinal mesenchymal cells to which they respond by
activating cell-fate and cell-survival transcription factors (Fig. 4).
The 2 important pairs are the glial-cell-line–derived neurotrophic
factor (GDNF)-ret receptor tyrosine kinase (86) involved in
proliferation of progenitors located at the distal extremity of the
migrating ENS, and the endothelin-3 (ET-3)–endothelin-B receptor
pathway regulating the timing of NCC differentiation during colo-
nization of the gut (87). Mutations in both of these ligand-receptor
pairs have been identified in rodent models and human cases of HD
(88–90). Recently, mutations of RET ligands have also been
identified, probably associated with lack of receptor binding (91).
The 2 ligand-receptor pairs mentioned above are regulated
by 2 transcription factors: SRY-related HMG-box transcription
factor 10 (SOX10) and paired-like homeobox factor 2B (PHOX2B).
SOX10 is a key transcription factor expressed in NCC during early
human development, which contributes to peripheral nervous
system development including enteric neurons (92). The transcrip-
tion factor PHOX2B is involved in the development of several
noradrenergic neuron populations and autonomic nervous system
regulation (93). Mice heterozygous for the Sox10 locus exhibit a
phenotype similar to the human disease characterized by colonic
aganglionosis and hypopigmentation (92), and enteric ganglia are
absent in mice with a homozygous disruption of the PHOX2B gene
(71). Mutations in both SOX10 and PHOX2B have been identified in
human subjects with aganglionic megacolon (89,93). The molecular
network involved in NCC migration to the colonic ganglia, and
thus in colonic aganglionosis, is summarized in simple terms
in Figure 3.
Regulation and misregulation of NCC migration, prolifer-
ation, and differentiation illustrate the limits of the 1 gene–1
phenotype concept of heritable disease. Clearly, mutations at any
level in the cascade can lead to similar phenotypes. This point
9
 Dauvé and McLin JPGN
Volume 57, Number 1, July 2013

WNT pathway HOX genes

HOXA-3,
HOXA-2

HOXA-4
WNT8c

SOX2
SIX2,
WNT8b

SFRP1
SFRP2
FRZB1

BAPX1
WNT2
Proventriculus

FZ1
FZ8
Foregut
TCF4

NKX2.5
PDX1

HOXC-5

HOXC-8
Gizzard
WNT1
LEF1

Pyloric
sphincter

CDXA
Duodenum

HOXC-9
HOXC-9
Small Liver

HOXA-10, HOXD-10
HOXB-8
intestine
Midgut

HOXB-9

HOXA-13, HOXD-13
FRZB1

SFRP2
WNT1
LEF1

HOXD-12
HOXA-11, HOXD-11
FZ8

Caeca
SFRP1

BAPX1
Hindgut

Large
intestine

FIGURE 3. Molecular regulation is enriched in the extremities of the GI tract. Schematic representation of WNT ligands and receptors (left) and
HOMEOBOX gene expression (right) in stage 24 chick gut. Mesodermal expression is illustrated by dark bars, endodermal expression by light bars.
Modified from (155) and (156).

cannot be overemphasized at the bedside: a disease cannot be ruled
out on the sole basis of an absent mutation in 1 of the candidate
genes. As the unravelling of the molecular basis of disease evolves,
clinicians will increasingly think in terms of molecular networks
and cascades when approaching congenital malformations and
heritable diseases with variable phenotypes.
Abdominal Wall Defects
Gastroschisis
Gastroschisis is a defect of the anterior abdominal wall
affecting approximately 3/10,000 live births, in which the intestines
(and sometimes the stomach and other organs) of the newborn

Mesenchymal cell Neural crest cell

ET-3

ETB

ETB SOX10
RET PHOX2B

RET
GDNF, neurturin

FIGURE 4. Signalling between NCCs and surrounding mesenchyme. Intestinal mesenchymal cells secrete ET-3, GDNF, and neurturin. They
interact with NCC receptors, which in turn regulate migration, proliferation, and differentiation pathways. In NCCs, nuclear SOX10 and PHOX2B
regulate ETB and RET expression. RET encodes for the GDNF and neurturin receptor and is regulated by PHOX2B (157) and SOX10 (158). ETB is
regulated by SOX10 (158). ET-3 ¼ endothelin-3; ETB ¼ endothelin receptor; GDNF ¼ glial-cell-line–derived neurotrophic factor; RET ¼ RET
protooncogene. Modified from (85).

10 www.jpgn.org

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 JPGN
Volume 57, Number 1, July 2013
protrude into the amniotic cavity. The abdominal wall defect occurs
usually to the right of the umbilicus (94).
Gastroschisis is a typical clinical example of an early
developmental accident (between 5 and 10 weeks’ gestation) (94).
It is a common birth defect and its incidence is on the rise (95).
Nonetheless, its aetiology is unknown and has generated numerous
hypotheses. The first is that the somatic layer of the mesoderm does
not form normally owing to teratogen exposure (96). Second, in 1975,
Shaw (97) proposed that gastroschisis may be caused by a rupture of
the amniotic membrane at the base of the umbilical cord secondary to
ring closure. This theory did not hold because the umbilical ring is
intact in nearly all patients with gastroschisis. Third, deVries (98)
suggested a vascular theory whereby premature atrophy or abnormal
persistence of the right umbilical vein leads to impaired growth and
viability of the surrounding mesenchyme; however, this interesting
hypothesis was incorrect because the umbilical vein does not supply
the intestinal mesenchyme. Another theory proposed that a vitelline
artery abnormality (contributing to ischaemia) leads to abdominal
wall weakness and subsequent rupture of the intestine through
this defective region, leading to gastroschisis (99); however, this
hypothesis is improbable because vitelline arteries supply the yolk sac
rather than the body wall. Furthermore, considering that the vitelline
artery supplies the yolk sack as a network, it is unclear why one area of
the abdominal wall would be more prone to ischaemia than another
(100). The most recent and plausible hypothesis is that an abnormal
body fold closure may be the cause (101). Improper closure of a body
fold could impede merging of the yolk stalk with the connecting
stalk. The result is then that the intestine rather than the umbilical
cord herniates into the amniotic cavity with the vitelline duct (101).
The genetic basis of gastroschisis is under investigation.
As in other developmental malformations, many of the studies
have been performed in lower mammals, limiting the general-
izability of the findings to humans. Today, polymorphisms
in 4 genes have been identified as possibly linked to human
cases of gastroschisis: intracellular adhesion molecule 1 (ICAM1),
endothelial nitric oxide synthase 3 (NOS3), atrial natriuretic peptide
(NPPA), and alpha-adducin (ADD1) (102).
ICAM1 induces and controls endothelial cell activation
(103), ADD1 is important in cell proliferation and epidermal
differentiation (104), NOS3 plays a critical role in neovasculariza-
tion (105), and NPPA is implicated in the control of extracellular
fluid volume and electrolyte homeostasis. Although the inter-
play among these genes is unclear, it has been shown that the
risk of gastroschisis is higher in children with ICAM1 and NOS3
single-nucleotide polymorphisms (SNPs) whose mothers smoked
during pregnancy (102). Lammer et al (106) hypothesized that
tobacco impairs VEGF–NOS3 and ICAM1 signalling, which in
turn leads to defective angiogenesis and vascular remodelling, in
keeping with earlier suggestions that vascular defects are at the root
of gastroschisis. This theory is an elegant illustration of the potential
role of environment on gene expression during morphogenesis.
It is noteworthy that to date, no association has been observed
between early somatic (IROQUOIS homeobox factor) and splanchnic
mesoderm (FOXF1) genes and abdominal wall defects reviewed by
McLin et al (70). Although there is no established animal model
of gastroschisis, Shroom3/ mice display massive gastroschisis
(107). Shroom3 is a PDZ (postsynaptic density protein [PSD95],
Drosophila disc large tumour suppressor [Dlg1], and zonula occlu-
dens-1 protein [zo-1]) protein that regulates cell shape.
Omphalocele
Omphalocele represents nearly 50% of congenital abdominal
wall defects (95). Unlike gastroschisis, which occurs independently
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Genetics of GI Malformations
of the umbilical cord, omphalocele is a hernia around the base of
the umbilical cord. Severity is proportional to the size of the
intestinal or liver protrusion through the opening (108).
The causes of omphalocele are mostly unknown. Omphalo-
cele is often associated with other birth defects, including
single gene disorders, neural tube defects, diaphragmatic defects,
and >20 syndromes of unknown aetiology (108). Among these
syndromes, 2 are noteworthy for their association with specific gene
defects. The first is the OtoPalatoDigital (OPD) syndrome-spectrum
disorder, which has been linked to filamin A (FLNA) mutations
(109); however, no FLNA mutation has ever been identified in
a patient presenting with omphalocele only (110). Next, Rieger
syndrome, which is characterized by eye and tooth abnormalities
together with abdominal wall defects (111), has been linked to both
haploinsufficiency and gain-of-function mutations of PITX2 (112).
PITX2 is a bicoid-related Hox gene expressed in the somites
and lateral plate mesoderm, where it plays an important role in
primary myogenesis (113). It has recently been incriminated in loss
of abdominal musculature in a mouse model (114). Interestingly,
a human study suggests that mutations in PITX2 may also rarely
be linked to isolated omphalocele (110). Unlike experimental
models of gastroschisis in which extrapolation to humans is
difficult, Pitx2/ mice demonstrate abnormal cellularity of the
body wall and failure of body wall closure similar to what is
observed in human omphalocele (115). Finally, in keeping with
the environmental exposure theory put forward for gastroschisis,
chicks exposed to cadmium show an increased incidence of
omphalocele and a decrease in PITX2 mRNA expression (116).
In summary, the role of PITX2 in GI development is
manifold. It is known for its role in regulating mesenchymal genes
that contribute to the first gut tilt to the left (117). Therefore, it is
tempting to speculate that PITX2 may act on other mesenchymal
components besides the dorsal mesentery. Finally, it has recently
been shown in X laevis to be upstream of shroom3 together with
other Pitx proteins whereby it regulates epithelial morphogenesis
and cell shape (118). Although Pitx proteins are likely actors
in the advent of omphalocele, they play several other roles in
gut development.
Recent studies show that SNPs in genes related to
homocysteine metabolism via vitamin B12 and folate have been
associated with omphalocele. These include transcobalamin
receptor (TCblR), transcobalamin 2 (TCN2), and methylenetetra-
hydrofolate reductase (MTHFR). On the one hand, it is reasonable
to assume that akin to what has been described in neural tube
defects, the administration of folate and vitamin B12 during
pregnancy may be justified to prevent omphalocele (119). On
the other hand, folate is of increasing interest for its epigenetic
footprint, raising the possibility of the likely contribution of
epigenetics.
Owing to the complexity of intestinal development and gut
coiling, it is reasonable to assume that no single gene is responsible
for any of these abdominal wall defects. Rather than a 1 gene–1
malformation situation, it is more likely that omphalocele, or
gastroschisis, is the product of any number of signalling defects
or cell-fate decisions, controlled by genes, the environment, and
the epigenome.
Malrotation and Other Syndromes
The midgut includes the duodenum, the jejunum, the ileum,
the cecum, the ascending colon and approximately two-thirds of the
transverse colon. Its development is characterized by 2 major
morphogenetic events: rotation and fixation of the colon and
dorsal mesentery (120). These events require complex and tightly
11
 Dauvé and McLin
orchestrated molecular and cellular events and as such, are
vulnerable to frequent developmental ‘‘glitches.’’ Malrotation
occurs in as many as 1:500 live births and is the presumed con-
sequence of abnormal GI rotation or reinsertion into the body cavity
(121,122). Its morphological characteristics include right-sided
small bowel, a cecum in the epigastrium, narrow mesenteric base,
and rightward shift of the ligament of Treitz (123,124). The clinical
consequences include obstructive symptoms owing to acute or
chronic volvulus and acute bowel necrosis (121). On occasion,
malrotation is diagnosed in isolation. More typically, however, it is
associated with heterotaxy and other congenital malformations of
the GI tract.
Recent studies show that intestinal rotation is initiated by key
ultrastructural changes in the dorsal mesentery. Pitx2 regulates cell-
shape changes, which in turn induce left–right asymmetries via
islet-1 (Isl-1), thereby determining the chirality of midgut looping
(117). This important study is the first to identify early molecular
regulators of this critical morphogenetic event in GI development.
To date, however, these findings have not been confirmed in human
subjects. Pitx proteins were discussed earlier with respect to
gastroschisis and their effect on Shroom 3, another regulator of
cell shape, raising the obvious question of the link between chirality
and the advent of gastroschisis.
In contrast, little data from humans are available. Duodenal
atresia has been identified in Feingold syndrome, described in the
setting of oesophageal atresia (35). In the first instance, the associ-
ated gene defects were identified by screening cases of malrotation
associated with other laterality defects. For example, mutations of
cryptic family 1 (CFC1) were identified in patients presenting with
heart and stomach laterality or chirality defects (125). This gene is
known to be involved in the NODAL signalling pathway, critical in
early left–right patterning (125,126). The other mutations that have
been identified in syndromic malrotation (as opposed to isolated)
are the following: zinc finger protein ZIC3 (ZIC3), previously
shown to regulate left–right asymmetry in X laevis (127–130),
the growth and differentiation factors activin receptor type 2B
(ACVR2B) (131), and the highly homologous factor LEFTYA
(human homologue of the mouse LEFTY1), known for its role in
left–right patterning (131,132). Mutations in the gene encoding the
early mesoderm transcription factor NKX2.5 deserve special
mention because the NKX2.5 promoter has PITX2 binding sites,
further highlighting the many functions of PITX2 during early
vertebrate gut development (133); however, at present, none of these
are specific enough to be of any use for antenatal screening purposes.
Of note, Stankiewicz et al (35) showed that human neonates
experiencing the rare and fatal alveolar capillary dysplasia
with misalignment of pulmonary veins (ACDMPV), attributed to
FOXF1 heterozygosity, often display associated malrotation. These
findings are compelling because they implicate the key transcrip-
tion factor of the visceral mesoderm in a syndrome, which includes
malrotation. On the contrary, it does not prove causality; rather,
it merely adds a candidate to the long list of partners involved in
this intricate process. Furthermore, Foxf1 loss-of-function is
well characterized in both mouse and X laevis, and malrotation
is not a predominant feature of the phenotype in either species
(14,134,135).
In conclusion, although a few genetic associations have been
established in human syndromes presenting with malrotation, it is
obvious that gut coiling is governed by a regulatory network
rather than a single gene. It follows that any number of clinical
syndromes can present with associated malrotation. In fact, it is
generally accepted in the field of GI embryology and patterning that
any number of genetic or environmental modifications can disrupt
the delicate sequence of events leading to normal gut looping,
something readily observed in the amphibian X laevis.
12
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JPGN
Volume 57, Number 1, July 2013
Heterotaxy and Situs Inversus
Malrotation presents as part of heterotaxy syndromes, which
stem from abnormal left–right cell-fate decisions. Heterotaxy
comprises situs inversus and situs ambiguous and may present as
any of a number of visceral malpositions above and below the
diaphragm (136). As detailed, the NODAL signalling pathway is
most often involved because of its role in the lateral plate mesoderm
in left–right decisions. Importantly, for the gastroenterologist, these
syndromes are often associated with malrotation (137), biliary
atresia (138), or heart defects (137). As may be predicted, mutations
in the following genes discussed in the section on malrotation were
identified in human subjects: NODAL (139), ZIC3 (140), ACVR2B
(141), LEFTYA (142), CFC1 (143), and NKX2.5 (133). Mutations of
these genes involved in the propagation of left–right asymmetry to
the left lateral plate mesoderm are causes of partial situs inversus,
whereas mutations of gene that generate left–right information in
the node are responsible of complete situs inversus.
Full situs inversus is associated with ciliary defects, which
are known to control left–right decisions in the early embryo.
Primary ciliary dyskinesia or infantile nephronophthisis (NPHP2) is
associated with situs inversus in 50% of patients. It has been shown
to be associated with mutations in the following dynein-related
genes (dynein is an important ciliary protein): the dynein inter-
mediate chain (DNAI1) (144) and the dynein heavy chains DNAH5
(145) or DNAH11 (146).
Gap junctions have also been incriminated in heterotaxy
because mutations in the connexin subunit CX43 were identified in
patients with laterality defects (147), something shown in X laevis
(148,149).
Congenital Short Bowel Syndrome
Short bowel syndrome is a frequent cause of intestinal failure
in infancy and the most common indication for intestinal trans-
plantation in children. It is usually an acquired condition following
massive intestinal resection. A few cases of congenital short bowel
have been described (150–153). Recently, van der Werf et al (154)
linked mutations in coxsackie and adenovirus receptor–like mem-
brane protein (CLMP) to familial short bowel. They showed that
CLMP is a membrane protein expressed in the intestinal crypts of
human foetal samples. Using a zebrafish model, they showed that
clmp loss-of-function is associated with multiple abnormalities,
including short bowel. Although it is unclear whether the shortened
intestinal length in these morphant embryos is primary or secondary
to global developmental abnormalities, it nonetheless is an
interesting finding for 2 reasons: first, it is the first such description
in an amphibian model, and second, it implicates an epithelial
protein in gut elongation, something classically attributed to the
mesoderm (Fig. 2).
CONCLUSIONS
Much of the molecular underpinnings of GI malformations
and syndromes are being understood through the use of mammalian
and nonmammalian developmental biology models. The mechan-
isms governed by the genes identified to date are still incompletely
understood, although genetic mutations are increasingly recognized
in humans. Comparative embryology should pave the way to
deciphering the molecular network governing the complexity
of GI development and how environmental influences affect
phenotypic expression. The hope is also that these advances will
ultimately facilitate in vitro cellular differentiation for the purposes
of cell-based therapies and tissue engineering, in the footsteps of
recent accomplishments in the field of hepatic embryology.
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 JPGN
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