Growth Hormone & IGF Research 50 (2020) 61–70

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Growth Hormone & IGF Research

journal homepage: www.elsevier.com/locate/ghir

A novel heterozygous STAT5B variant in a patient with short stature and T

partial growth hormone insensitivity (GHI)
Laura Ramíreza, Nora Sanguinetia, Paula Scagliaa, Ana Keselmana, María Gabriela Ballerinia,
Liliana Karabatasa, Estefanía Landia, Julia Castroa, Sabina Domenéa, Patricia Pennisia,
Héctor Jaspera, Rodolfo A. Reya, Martín Vázquezb, Horacio Domenéa, Ignacio Bergadáa,
⁎
Mariana Gutiérreza,
a
Centro de Investigaciones Endocrinológicas ‘Dr César Bergadá’ (CEDIE), CONICET, FEI, División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires,
Argentina
b
HERITAS, CONICET, Rosario, Argentina

A R T I C LE I N FO A B S T R A C T

Keywords: Background: The most frequent monogenic causes of growth hormone insensitivity (GHI) include defects in
Growth hormone insensitivity genes encoding the GH receptor itself (GHR), the signal transducer and activator of transcription (STAT5B), the
Whole-exome analysis insulin like-growth factor type I (IGF1) and the acid-labile subunit (IGFALS). GHI is characterized by a con-
STAT5B tinuum of mild to severe post-natal growth failure.
IGFALS
Objective: To characterize the molecular defect in a patient with short stature and partial GHI.
Patient and methods: The boy was born at term adequate for gestational age from non-consanguineous normal-
stature parents. At 2.2 years, he presented proportionate short stature (height −2.77 SDS), wide forehead and
normal mental development. Whole-exome analysis and functional characterization (site-directed mutagenesis,
dual luciferase reporter assay, immunofluorescence and western immunoblot) were performed.
Results: Biochemical and endocrinological evaluation revealed partial GH insensitivity with normal stimulated
GH peak (7.8 ng/mL), undetectable IGF1 and low IGFBP3 levels. Two heterozygous variants in the GH-signaling
pathway were found: a novel heterozygous STAT5B variant (c.1896G > T, p.K632N) and a hypomorphic IGFALS
variant (c.1642C > T, p.R548W). Functional in vitro characterization demonstrated that p.K632N-STAT5b is an
inactivating variant that impairs STAT5b activity through abolished phosphorylation. Remarkably, the patient's
immunological evaluation displayed only a mild hypogammaglobulinemia, while a major characteristic of
STAT5b deficient patients is severe immunodeficiency.
Conclusions: We reported a novel pathogenic inactivating STAT5b variant, which may be associated with partial
GH insensitivity and can present without severe immunological complications in heterozygous state. Our results
contribute to expand the spectrum of phenotypes associated to GHI.

1. Introduction
Growth hormone insensitivity (GHI) is characterized by a con-
tinuum of mild to severe post-natal growth failure, normal to elevated
GH levels, and low serum concentrations of insulin like growth factor I
(IGF1) and IGF binding protein 3 (IGFBP3) [1]. Complete GHI was in-
itially associated to defects in the GH receptor gene (GHR) [2,3].
However, due to technical advances in DNA sequencing and high-re-
solution genotyping, the number of monogenic causes of GHI has in-
creased and include defects not only along the GHR-IGF1 axis (IGF1,
IGFALS, STAT5B and STAT3) but also in other growth-related genes,
such as IKBKB, PAPPA2 and PRKCA [4,5]. Mutations in heterozygous
state in some of these genes have been associated to a milder clinical
phenotype, contributing to broadening the GHI spectrum. For instance,
heterozygosity for IGFALS variants causes a 1 SD loss in height com-
pared to wild type first degree relatives [6,7] and most heterozygous
carriers of mutations in the IGF1 and STAT5B genes were shown to be
significantly shorter than non-carriers [8,9]. Interestingly, hetero-
zygous carriers of STAT5b variants present diverse clinical features,
including in some cases absence of overt immunodeficiency, a major

⁎
Corresponding author at: Centro de Investigaciones Endocrinológicas ‘Dr César Bergadá’ (CEDIE), CONICET, FEI, División de Endocrinología, Hospital de Niños
Ricardo Gutiérrez, Gallo 1360, Buenos Aires CP1425EFD, Argentina.
E-mail address: marianagutierrez@cedie.org.ar (M. Gutiérrez).

https://doi.org/10.1016/j.ghir.2019.12.005
Received 5 November 2019; Received in revised form 13 December 2019; Accepted 26 December 2019
Available online 27 December 2019
1096-6374/ © 2019 Elsevier Ltd. All rights reserved.
L. Ramírez, et al.
characteristic of STAT5b deficient patients [10,11]. In addition, com-
binations of heterozygous gene variants in STAT5B and IGFALS, asso-
ciated or not with copy number variations, have been previously re-
ported in children with apparent GHI, suggesting that this condition
could result from digenic, oligogenic or polygenic defects [10]. In the
present study, we report two heterozygous variants in the GH/IGF1
signaling pathway, a novel missense variant in STAT5b, c.1896G > T
(p.K632N) and a previously described missense variant in IGFALS,
c.1642C > T (p.R548W), in a patient with short stature and a mild
immunological phenotype. Clinical presentation and functional studies
of the novel heterozygous p.K632N mutation in the SH2 domain of
STAT5b are presented.
2. Patient and methods
2.1. Patient: Case report
The proband is an Argentine boy born at 40 weeks of gestational age
from non-consanguineous healthy parents. His birth weight was 3750 g
(+0.78 SDS) and his birth length 49.5 cm (−0.18 SDS). His mother's
and father's heights were 155 cm (−0.93 SDS) and 165.2 cm (−1.11
SDS), respectively. His younger sister and brother had normal height
(Fig. 1).
He was referred to our pediatric-endocrinology unit at 2.2 years of
age for short stature. His height was 80.0 cm (−2.77 SDS), weight, 9.10
Kg (−2.52 SDS), and head circumference, 48.5 cm (0.01 SDS), ac-
cording to Argentine reference growth charts [12]. Physical examina-
tion showed proportionate short stature (Sitting height: 48.8 cm, 3rd-
10th percentile), wide forehead and high-pitched voice. Developmental
milestones were normal. The patient had normal fasting routine la-
boratory studies and normal prolactin and thyroid and adrenal func-
tion: serum hemoglobin (11.6 g/dL), glucose (71 mg/dL), creatinine
(0.28 mg/dL), AST (55 U/L), ALT (22 U/L), albumin (5 g/dL), calcium
(10 mg/dL), phosphate (5.7 mg/dL), insulin (0.8 μU/mL), TSH (5.3 μU/
L), free T4 (1.36 ng/dL), prolactin (12.2 ng/mL), cortisol (15,7 μg/dL),
anti-thyroperoxidase antibodies (< 20 UI/mL), ACTH 43 (pg/mL). He
presented low levels of IGF1 (< 25 ng/mL, −2.59 SDS) and IGFBP3
(1.34 μg/mL, −2.01 SDS), and a normal arginine clonidine GH pro-
vocative test (maximum peak of growth hormone: 7.8 ng/mL). At the
age of 2.3 years, two IGF1 generation tests (IGF-GT) were performed:
one with low (11 μg/kg·day) and another with high (38 μg/kg·day) dose
of recombinant human growth hormone (rhGH) (Omnitrope, Sandoz).
No response was observed in the low-dose IGF-GT, while for the high-
dose IGF-GT, at least 1 SDS gain was observed for IGF1, IGFBP3 and
ALS after 7 days of GH treatment (Table 1). At 2.9 years of age with a
62
Growth Hormone & IGF Research 50 (2020) 61–70
height of 84.5 cm (−2.98 SDS), rhGH treatment was started at a daily
dose of 45 μg/kg·d. After 2 years of treatment, increments in height
(+1.0 SDS) and in IGF1 levels (+3.2 SDS) were observed (Table 1 and
Supplementary Fig. 1).
2.2. Methods
2.2.1. Endocrinological and immunological tests
Serum levels of GH, IGF1, IGFBP3 and anti-thyroperoxidase anti-
bodies were determined by chemiluminescent immunometric assays
(Immulite 2000, Siemens Healthcare Diagnostics, Llamberis, Gwynedd,
UK). TSH, FT4, ACTH, cortisol, prolactin, and insulin were measured by
electrochemiluminescence (Cobas e411 analyzer; Roche Diagnostics
GmbH, Mannheim, Germany). Serum ALS levels were evaluated by
enzyme-linked immunosorbent assay (ELISA, Mediagnost, Reutlingen,
Germany). Total serum immunoglobulin (IgG, IgM and IgA) were
measured by kinetic nephelometry using commercially available kits
(Array 360, Beckman Coulter Inc). IgE was measured by ELFA
(VIDAS®).
2.2.2. Molecular studies
Whole-exome sequencing of genomic DNA, obtained from the per-
ipheral blood of the patient [13], was performed using Illumina's
Nextera Exome V1.2 kit (Illumina, San Diego, USA) for library pre-
paration and exome capture and the Illumina HiSeq1500 sequencer at
the Instituto de Agrobiotecnología de Rosario (INDEAR)-CONICET
(Rosario, Argentina). Alignments and variant annotations were made as
previously described [14]. Variants were prioritized according to po-
pulation frequency (GnomAD, 1000Genome project), variant con-
sequence and a 376-candidate gene list associated to short stature (see
Supplementary Table 1). The identified IGFALS and STAT5B variants
were confirmed by Sanger sequencing, as well as segregation of these
variants in other family members (primers used are available upon
request).
2.2.3. In silico bioinformatics analysis
The pathogenicity of the STAT5b variant identified during genetic
analysis was evaluated in silico with PolyPhen-2 [15], SIFT [16], Mu-
tation Taster [17], SNAP2 [18] and CADD [19], using the following
NCBI reference sequences: NG_007271.1 (gene), NM_012448.3 (mRNA)
and NP_036580.2 (protein) and it was classified according to the
American College of Medical Genetics and Genomics (ACMG) [20].
Multiple sequence alignments among STAT5b from different species
and among STAT proteins were performed with PRALINE program
(http://www.ibi.vu.nl/programs /pralinewww/) using reference
Fig. 1. Pedigree of the family. Open symbols
(squares: males, circles: females) indicate unaffected
individuals; red solid symbols: heterozygous IGFALS
c.1642C > T (p.R548W) carriers; blue solid symbols:
carriers for STAT5B c.1896G > T (p.K632N) hetero-
zygous variant. (For interpretation of the references
to colour in this figure legend, the reader is referred
to the web version of this article.)
L. Ramírez, et al. Growth Hormone & IGF Research 50 (2020) 61–70

Table 1 Change II XL Site-Directed Mutagenesis Kit (Agilent Technologies,

Clinical and biochemical characteristics of the patient. Santa Clara, CA, USA) and the following primers: Fw 5′-attctttcctga-
Patient (male) gaatcaaaattccaagcaatggtgatgcc-3′ and Rv: 5′-ggcatcaccattgcttggaatttt-
gattctcaggaaagaat-3′. All constructs were verified by sequencing.
Birth Gestational Age, weeks 40
Birth weight, g (SDS) 3750 (−0.78)
Birth length, cm (SDS) 49.5 (−0.18) 2.2.5. Cell culture and transfection experiments
First visit Chronological Age, years 2.2
HEK293-T cells were routinely grown in Dulbecco's modified Eagle's
Bone Age, years 0.75
Height, cm (SDS) 80.0 (−2.77) medium (DMEM) supplemented with 10% fetal calf serum, penicillin
Weight, kg (SDS) 9.10 (−2.50) (100 units/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM) at
Head circumference, cm (SDS) 48.5 (0.01) 37 °C in a humidified atmosphere with 5% CO2. For transfection ex-
Sitting Height, cm (Percentile) 48.8 (3rd – 10th) periments, HEK293-T cells were seeded at a density of 2 × 105 cells/
Clinical features Physical exam Wide forehead
Mental development High-pitched voice
well in a 24-multi well plate, grown to approximately 70% confluence,
Normal and transiently transfected with 500 ng Empty-pCMV6, or with a
History of severe infections Negative combination of 250 ng pcDNAI_Amp-GHRfl [24] and 250 ng vector
Endocrine evaluation GH basal, ng/mL 2.1 pCMV6 carrying WT-STAT5b or K632 N variant using Lipofectamine
maximum peak (GHPT), ng/mL 7.8
3000 reagent (Invitrogen, Carlsbad, CA, USA). After 24 h transfection,
Generation Test (rhGH 11 μg/
kg.day, 7 days): cells were washed, and serum starved for 6 h before a 30-min treatment
IGF1 basal, ng/mL (SDS) < 25 (−2.59) with 200 ng/mL rhGH (Sandoz, Olivos, Argentina). Transfection ex-
post IGF-GT, ng/mL (SDS) < 25 (−2.59) periments were performed in duplicates, at least three independent
IGFBP3 basal, μg/mL (SDS) 1.48 (−1.83) times.
post IGF-GT, μg/mL (SDS) 1.03 (−2.41)
ALS basal, mU/mL (SDS) 380 (−2.14)
post IGF-GT, mU/mL (SDS) 388 (−2.11)
Generation Test (rhGH 38 μg/
2.2.6. Luciferase reporter assays
kg.day, 7 days): HEK293-T cells were seeded as described above and transfected
IGF1 basal, ng/mL (SDS) < 25 (−2.59) with a total input of 500 ng per well: 390 ng of the luciferase reporter
post IGF-GT, ng/mL (SDS) 40 (−1.52) construct carrying 8xGH response element (GHRE) from the rat Spi2.1
IGFBP3 basal, μg/mL (SDS) 0.85 (−2.62)
gene in pGL2 (pGHRE-LUC), 50 ng of pcDNAI_Amp-GHRfl [24], 50 ng
post IGF-GT, μg/mL (SDS) 2.15 (−0.97)
ALS basal, mU/mL (SDS) 269 (−2.53) of WT or mutant STAT5b containing plasmids and 10 ng of pRL-TK
post IGF-GT, mU/mL (SDS) 671 (−1.13) -renilla luciferase control reporter vector- (Promega Corp., Madison,
Prolactin (ng/mL) (RR: 2–15) 12.2 WI) using the Lipofectamine 3000 transfection reagent. After 24 h
TSH (mIU/mL) (RR: 0.5–6.5) 5.3 transfection, cells were washed and serum starved for 6 h before 18-h
FT4 (ng/dL) (RR: 0.8–2.0) 1.36
TPO-Ab/TG-Ab (IU/mL) < 10/ < 20
treatment with 20, 50, 100 or 200 ng/mL rhGH, as indicated in each
(RR: < 20/ < 20) experiment. STAT5b reporter activity was evaluated using a dual luci-
rhGH treatment Dose, μg/kg.day 45 ferase reporter assay system (Promega, Madison, WI, USA) according to
Evaluation at 1st year of the manufacturer's instructions. To study the heterozygous effect and
treatment:
the impact of p.K632N on the activity of WT-STAT5b protein, co-
Height gain, (SDS) (+0.80)
IGF1, ng/mL (SDS) 59 (−0.63) transfection experiments were conducted as described above but using
IGFBP3, μg/mL (SDS) 1.9 (−1.29) equal amounts of WT and mutant STAT5b containing plasmids (25 ng of
Evaluation at 2nd year of each, ratio 1:1) or combinations with increasing concentration of
treatment: K632N-STAT5b expression plasmid (25 ng WT and 50 or 100 ng K632N-
Height gain, (SDS) (+0.20)
IGF1, ng/mL (SDS) 103 (+0.65)
STAT5b, ratios 1:2 and 1:4, respectively). Cell lysates were prepared as
IGFBP3, μg/mL (SDS) 2.4 (−0.65) described above. Results represent the ratio of reporter (firefly) to
Molecular studies Heterozygous IGFALS variant c.1642C > T control (renilla) luciferase or are normalized as fold-change in the ratio
(p.R548W) as compared with WT-STAT5b plasmid. Data are presented as the
Heterozygous STAT5b variant c.1896G > T
mean ± SEM of five independent experiments.
(p.K632N)

2.2.7. Western immunoblot (WIB)

sequences obtained from Uniprot.
STAT5b phosphorylation in HEK293-T cells expressing GHR was
Homology model was prepared based on the crystal structure of the
examined by WIB analysis. Cells were starved for 6 h in serum-free
unphosphorylated mouse Stat5a core [21] or human unphosphorylated
medium and then stimulated with 200 ng/mL rhGH at 37 °C for 30 min.
STAT1 [22] (PDB accession codes 1Y1U and 1YVL, respectively) using
After treatment, cells were washed with PBS and lysed in RIPA lysis
I-TASSER structure homology-modeling server [23]. The images were
buffer (1× phosphate-buffered saline, 1% v/v Nonidet P-40, 0.1% w/v
prepared using the molecular graphics program PyMOL (PyMOL Mo-
SDS, 10 mg/mL phenylmethylsulfonyl fluoride, 1 mM sodium orthova-
lecular Graphics System, Version 1.8.4.0, Schrödinger, LLC, http://
nadate and protease inhibitor mixture). Extracts containing equal
www.pymol.org/) which was also used to generate in silico the K632N
amounts of proteins, determined by the Bradford method (Bio-Rad,
mutation. Side chain conformation of asparagine was selected from the
Michigan, USA), were separated by SDS-PAGE (12% acrylamide) and
PyMOL backbone-dependent rotamer library that resulted in less steric
transferred to polyvinylidenedifluoride membranes (EMD Millipore,
clashes with surrounding residues and with the greatest energy mini-
Billerica, MA, USA). Phosphorylation was detected using an anti-
mization out of the total number of rotamers possible for the sub-
phospho-STAT5 rabbit monoclonal antibody (Cell Signaling
stitution.
Technology, Danvers, MA, USA) and protein abundance of STAT5b,
using a rabbit monoclonal antibody against STAT5b (Cell Signaling
2.2.4. Site-directed mutagenesis Technology, Danvers, MA, USA). The signal was developed with donkey
STAT5B gene variant was introduced into a commercial plasmid anti-rabbit IgG-horseradish peroxidase (GE Healthcare Life Sciences,
(pCMV6-Entry, RC209429, Origene, Rockville, MD, USA) containing Freiburg, Germany) by chemiluminescence using 20× LumiGLO
the wild-type (WT) STAT5B cDNA (NM_012448), using the Quick Reagent (Cell Signaling Technology, Danvers, MA, USA).

63
L. Ramírez, et al.
2.2.8. Immunofluorescence
HEK293-T cells were grown on poly-D-lysine-coated glass coverslips
in 24-well plates and transfected with constructs encoding the WT- and
p.K632N-STAT5b proteins as previously described. Next day, the cells
were washed and serum starved for 24 h before a 30-min treatment
with 200 ng/mL of rhGH (or vehicle) at 37 °C. Cells were washed with
PBS and immediately fixed with methanol:glacial acetic acid (3:1) for
30 min at room temperature and rinsed sequentially with 70% ethanol
and 100% ethanol. Slides were air-dried and then, rehydrated with PBS
and blocked with PBS/1% horse serum for 30 min at RT. The cells were
incubated overnight at 4 °C with primary antibody for STAT5b (Cell
Signaling) 1:1000 in blocking solution. After two washing steps with
PBS, cells were incubated for 1 h at room temperature in the dark with
the secondary antibody (Alexa Fluor 488 goat-α-mouse; 1:500;
Invitrogen), washed with PBS and incubated with 1 μg/mL of Hoechst
33258 dye (Sigma-Aldrich) for 15 min at room temperature. Slides were
then washed with PBS and mounted using Glycerol 90% in PBS.
Pictures were taken using a fluorescence microscope (Carl Zeiss; Axio-
Scope A1) and the ZEN 2009 software. Images of STAT5b immuno-
fluorescence staining and Hoechst nuclear staining were analyzed using
ImageJ.
2.2.9. Statistical analysis
All experiments were repeated at least three times. Mean ± SEM of
results from multiple experiments of the same study are reported.
Results were analyzed with the Mann-Whitney test for non-parametric
data using Prism 5 (GraphPad Software, San Diego, CA) and sig-
nificance was set at P < 0.05.
3. Results
3.1. Genetic analysis
The patient presented growth failure with normal stimulated GH,
undetectable IGF1, and low IGFBP3 and ALS levels (Table 1). These
observations are compatible with a diagnosis of GH insensitivity.
Therefore, a WES analysis was conducted to determine the presence of
potentially pathogenic genetic variants. Initially, we obtained 63,643
variants passing quality filters with depth > 10× in 15,113 genes. Our
filtering strategy included the following criteria: population frequency
(< 1% in GnomAD and 1000Genomes Project: 5949 variants in 3270
genes), mutation consequence (nonsense, missense, deletion or inser-
tion in coding sequence and intron/exon boundaries: 1125 variants in
645 genes), and a list of 376 candidate genes associated to short stature
(20 variants in 18 genes). After a final step of manual curation, a novel,
heterozygous missense variant in STAT5B, c.1896G > T (p.K632N) was
assumed as the most likely candidate gene variant and confirmed by
Sanger sequencing. The variant was not listed in the NCBI SNP database
(www.ncbi.nlm.nih.gov/snp), the Genome Aggregation Database
(gnomAD) (https://gnomad.broadinstitute.org/) or the 1000 Genomes
(www.1000genomes.org/data) population-variation data sets and it
was predicted to be pathogenic by most of the bioinformatics prediction
tools (Table 2). It was classified as Variant of Unknown Significance
(VUS) according to ACMG guidelines. Genetic testing by direct se-
quencing of STAT5B in the other unaffected family members revealed
that the patient's mother and sister were heterozygous for the same
Table 2
Variant pathogenicity prediction by different softwares.
PolyPhen2 (score) SIFT (score) Mutation taster (probability)
IGFALS Benign Tolerated Polymorphism
p.R548W (0.045) (0.16) (0.830)
STAT5B Possibly damaging Tolerated Disease causing
p.K632N (0.523) (0.40) (0.998)
64
Growth Hormone & IGF Research 50 (2020) 61–70
variant, suggesting maternal inheritance. Further detailed im-
munological background assessment of the patient and his family did
not show any immunological clinical features suggestive of a STAT5B
defect, such as eczema, recurrent infections, history of pulmonary or
immunologic-related problems. However, the patient's immunological
evaluation showed IgG levels below −2 SDS for age (554 mg/dL), IgA
in the lower range of normality for age (50 mg/dL), and normal IgM
(130 mg/dL) and IgE (11 UI/mL). Generation of anti-pneumococcal and
tetanus toxoid antibodies were normal. Lymphocytes subsets CD3+,
CD4+, CD8+, CD19+, CD56+ and regulatory T cells (CD127low
FOXP3+ Tregs) were normal. Autoantibodies workout (ANA/AMA/
LKM/antiendomisium) was negative in the patient (Data not shown).
WES analysis also revealed a heterozygous c.1642C > T (p.R548W)
variant in the IGFALS gene (Table 2). This variant has a relatively high
frequency in the population (about 3.60% according to GnomAD, see
Table 2) and is classified as benign according ACMG recommendations
and most of the in silico prediction tools. However, this variant has al-
ready been reported in a cohort of children with idiopathic short stature
[25] and it was later classified as a hypomorphic variant by in vitro
studies from our group [26]. Segregation analysis showed that this
variant was maternally inherited (Fig. 1). No additional variants with
potential pathological effects were identified in other growth associated
genes including GHR, GH1, IGF1 or IGF1R.
3.2. In silico molecular modeling of p.K632N-STAT5b variant
The mutation p.K632N is located in the SH2 domain of STAT5b, a
region highly conserved among STAT5a and STAT5b proteins (Fig. 2A).
Other STAT proteins have a valine in the equivalent position, none-
theless among different species the residue is highly conserved
(Fig. 2B). There is no availability of structural data of human STAT5b,
thus we performed a homology model using the crystal structures of
human, unphosphorylated STAT1 (PDB ID 1YVL) [22] or mouse
STAT5a (PDB ID 1Y1U) [21]. The SH2 domain has a structurally con-
served core-architecture consisting in a mixed α-helical/β-sheeted do-
main that triggers dimer formation through hydrophobic interactions
after Tyr699 phosphorylation. According to the molecular model, the
hydrophobic part of Lys632 side-chain is buried, and only the charged
portion is exposed (Fig. 3). This residue can be involved in salt-bridges
with the negatively charged Asp612 and Aps634. These interactions are
disrupted upon mutation to Asn, and could lead to altered flexibility
around the site, destabilizing locally the structure, including the hy-
drophobic core involved in STAT5b dimerization (Fig. 3).
3.3. Functional in vitro characterization of p.K632N-STAT5b
To study functional pathogenicity, p.K632N variant was generated
by site-directed mutagenesis and transiently transfected into HEK293-T
cells overexpressing GHR. The transcriptional activities of WT- and
p.K632N-STAT5b variant were evaluated using a dual reporter assay.
Under unstimulated conditions, expression of p.K632N mutant resulted
in a significant decrease in reporter activity (P < .01) in comparison to
WT-STAT5b (Fig. 4A). Co-transfection of equimolar amounts pf WT-
and p.K632N-STAT5b, mimicking heterozygosity, also caused a de-
crease in the transcriptional activity (P < .05) compared to WT-
STAT5b (Fig. 4A). In response to GH, p.K632N was not able to drive
SNAP2 (score) CADD (score) GnomAD (Freq) HGMD
Effetc 14.0 0.0359 –
(80)
Neutral 23.6 – –
(−70)
L. Ramírez, et al. Growth Hormone & IGF Research 50 (2020) 61–70

Fig. 2. Schematics of human STAT5b protein domains and multiple sequence alignments performed with PRALINE software. A) The position of the mutation K632N
is boxed above the SH2 domain. The secondary structure of the SH2 domain, based on the crystal structure of human STAT1 [22], is indicated above the scheme. The
amino acid Lys632 is located at the end of ßC and it is conserved among STAT5 proteins. B) The Lys632 (boxed in red) is highly conserved among different species.
The colour scheme indicates the least conserved alignment position (dark blue), to the most conserved alignment position (red). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

transcriptional activity (P < .001). By contrast, GH induced reporter
activity for cells co-transfected with both WT- and p.K632N-STAT5b
proteins was similar to that detected for cells transfected with only WT-
STAT5b (Fig. 4B). To further characterize the response to GH of co-
transfected cells compared to WT-STAT5b alone, we performed a GH-
dose response analysis. We observed that reporter activity levels in-
duced by GH, were not significantly different for WT-STAT5b trans-
fected cells and for cells co-transfected with WT- and p.K632N-STAT5b
(Fig. 4C). Next, we reconstituted the in vitro system with a fixed con-
centration of the WT-STAT5b encoding plasmid and different con-
centrations of mutant STAT5b expression vector (ratios 1:2 and 1:4, WT
to mutant). As shown in Fig. 4D, increasing amounts of p.K632N-
STAT5b dose-dependently reduced the level of reporter activity in re-
sponse to GH, in comparison to cells co-transfected with equal quan-
tities of WT and mutant STAT5b (ratio 1:1), whereas no major change
in activity upon addition of the empty vector was detected (Data not
shown).
In addition, we analyzed the expression of p.K632N by Western
Blot. p.K632N was shown to be expressed at comparable levels to those
of WT-STAT5b in reconstitution studies (Fig. 5A). However, GH-in-
duced phosphorylation of p.K632N variant was not detected. Moreover,
cells co-expressing both WT and p.K632N proteins showed reduced
levels of STAT5b phosphorylation (Fig. 5A). p.K632N expression and
cellular localization were also investigated after transfection using
immunofluorescence. As expected, after GH stimulation we observed
that WT protein localization was mainly nuclear, but p.K632N-STAT5b
remained in the cytoplasm (Fig. 5B).
4. Discussion
We describe a patient with short stature, decreased serum IGF1 and
ALS levels, and partial GH insensitivity carrying a combination of two
heterozygous gene variants in the GH/IGF1 signaling pathway:
p.K632N-STAT5b and p.R548W-ALS variants. Our first though was that
both variants may act synergistically to cause short stature in this pa-
tient. However, we found by targeted molecular genetic analysis both
heterozygous variants in the patient's unaffected mother and the het-
erozygous STAT5B mutation in his unaffected sister, as well. Similar to
the patient, they both showed normal IgE levels and a mild hypo-
gammaglobulinemia (Supplementary Table 2) without clinical mani-
festations of immune dysregulation. In the patient's exome data ana-
lysis, we could not find any additional clear variant/gene candidate
that pass out the analytic criteria used. Unfortunately, WES analysis
was not performed for the patient's relatives and no growth data during
childhood were available for the mother. Although we cannot con-
clusively rule out the possibility of an additional genetic variant con-
tributing to the short stature in this patient, incomplete penetrance
could also be considered as a possible explanation for this discrepancy.
Furthermore, as in many hereditary diseases, the onset of clinical
manifestation could be sex related. GH and STAT5b have been im-
plicated in the regulation of a number of liver genes that are expressed
in a sexually dimorphic pattern [27–30]. In Stat5b knock out mouse
model, the levels of IGF1 were reduced only in male Stat5b mutant
mice, but not in females, and mutant males were 20%–30% smaller
than their Stat5b wild-type littermates [31]. Authors attribute these
sex-related differences in the phenotypes to differences in the produc-
tion of IGF1: while in males the primary IGF1 source is the liver, in
females, both the liver and the ovaries contribute. Although a sexual
dimorphism pattern for STAT5b has not been demonstrated in humans,
this could be due to the low number of reported cases of STAT5b mu-
tations carriers. Interestingly, two female relatives of a severe short
statured male patient carrying the heterozygous dominant-negative
p.A478V-STAT5b variant, presented height within normal limits (−1.5
SDS) [11], supporting the concept of incomplete penetrance for STAT5B

65
L. Ramírez, et al. Growth Hormone & IGF Research 50 (2020) 61–70

Fig. 3. Homology based structural model of unphosphorylated STAT5b. A) Monomer in cartoon representation. Domains are colored as in Fig. 2. Lys632 is indicated
and represented in stick mode in magenta. B) Expanded SH2 domain showing the exposed positive charged portion of Lys632 (stick). Red surface represents the
hydrophobic core favoring SH2 domains interactions for dimerization. C) On C-terminus of beta strand C, Lys632 (in magenta) is proposed to form an intramolecular
salt bridge with Asp634 and Asp612. Residues forming the hydrophobic system are represented in stick mode in this image. D) As in C, but the structure has been
rotated to show the predicted side-chain position of Asn (circled in black) in the variant p.K632N using the PyMOL backbone-dependent rotamer library. Substitution
of Lys632 by Asn is predicted to result in the loss of salt-bridge interactions Asp612/Aps634-Lys632 (yellow dotted lines). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

gene variants. Moreover, mutations in non-coding regions of the IG-
FALS and STAT5B genes and epigenetic factors, could be involved in
regulation of their expression.
The heterozygous STAT5b p.K632N mutation, is novel. Biallelic
STAT5B mutations cause a rare disorder reported in only a handful of
patients. It is characterized by IGF1 deficiency, severe growth failure,
and immune dysregulation which associates primary immunodeficiency
and autoimmunity. The patient shares some physical features, such as
short stature, a prominent forehead, normal body proportions and high-
pitched voice, with total STAT5b deficient patients. Of note, he pre-
sented only a mild hypogammaglobulinemia without immunological
complications. Similar to this patient, Klammt et al. reported three
cases of heterozygous STAT5B-mutation carriers (p.Q474R, p.A478V,
p.Q177P) who lacked the severe immune deficiency and pathological
autoimmunity typically associated with total STAT5b deficiency [11].
Authors hypothesized that cytokine signaling is less sensitive to de-
creased STAT5b activity than GH signaling. In agreement with this,
mice with half the normal dose of Stat5b seem almost unaffected by
immunodeficiencies [32,33].
In silico analysis for p.K632N-STAT5b variant suggested patho-
genicity. Consistent with this, the homology model predicted Lys632
can be involved in intramolecular salt bridges with Asp612 and Asp634,
which can be important for local protein structure stability. In addition,
salt bridges could have stabilizing electrostatic free energy
contributions, regardless of whether they are buried or solvent exposed,
or contained in hydrogen bonds or not [34,35]. Disruption of these
interactions may induce local protein remodeling and partial unfolding
[36]. Therefore, the change of Lys632 for Asn could promote a re-
stricted folding of the β-sheet C and neighbor fragments in STAT5b,
leading to altered phosphorylation and interaction with other proteins.
Previously reported homozygous inactivating missense mutations
(p.A630P, p.F646S) were also located close to this residue suggesting an
overall disturbing effect on the mutant proteins structure [37,38].
Moreover, experimental point replacements in this region (p.N639M,
p.L640F) promoted a significant destabilization of the helices αB-αC
close and stimulated the unfolding of the β-sheet C in STAT5b [39].
In vitro expression of p.K632N was comparable to WT-STAT5b.
Despite the local β-sheet C misfolding, this structural disruption would
not long-distance spread from the residue change and the protein ap-
pears to be normally synthesized and immunologically detected by
Western Blot. Therefore, this change would not grossly affect protein
levels of STAT5b. Our data demonstrate that mutation K632N di-
minishes STAT5b activity through impaired Tyr699 phosphorylation.
When the p.K632N-STAT5b variant was expressed in homozygous form,
we were not able to detect GH-induced phosphorylation, nuclear
translocation or luciferase activation. Furthermore, under unstimulated
conditions, transcriptional activity for HEK293-T cells expressing
p.K632N-STAT5b variant was reduced compared to that detected for

66
L. Ramírez, et al.
WT-STAT5b. Co-transfection of p.K632N and WT-STAT5b also resulted
in remarkably decreased basal transcriptional activity compared to WT-
STAT5b alone. However, in response to GH, WT-STAT5b in hetero-
zygous state with p.K632N is able to overcome this limitation and the
activity is induced ~20 fold. In addition, GH-dose response experiments
showed that a 10-fold lower dose of GH (20 ng/mL) stimulated STAT5b-
dependent gene transcription in co-transfected cells at levels compar-
able to those of cells expressing only WT-STAT5b. Nonetheless, the co-
67
Growth Hormone & IGF Research 50 (2020) 61–70
Fig. 4. Reconstitution studies of the heterozygous
STAT5B K632 N mutation in HEK293-T cells. To
study the heterozygous effects, co-transfections ex-
periments were conducted using equal amounts of
WT- and p.K632N-STAT5b containing plasmids. A)
STAT5b transcriptional activity determined by luci-
ferase reporter assay under unstimulated (basal)
conditions. B) STAT5b activity of WT and p.K632N
under GH treatment (200 ng/mL). C) Dose effect of
GH on STAT5b transcriptional activity. Transfected
HEK293-T cells were treated with various con-
centrations of GH for 30 min, collected, and lysed for
reporter gene assay. D) Luciferase reporter activities
detected in cell lysates from HEK293-T cells co-
transfected with WT-STAT5b and increasing
amounts of p.K632N-STAT5b, as indicated in the
graph. Data represent the mean ratio of firefly/renilla
control luciferase for each construct or are normal-
ized as fold-change in the ratio ± SEM, as compared
with WT-STAT5b transfected cells, which was arbi-
trarily assigned a value of 1 (Fig. 1A, B, and C) or
compared to activity detected in cells co-transfected
with equal amounts of WT-STAT5b and p.K632 N-
STAT5b (ratio 1:1) (Fig. 1D). *P < .05, **P < .01,
***P < .001 (n = 5, by duplicate); Mann-Whitney
test.
transfection of increasing amounts of p.K632N expression plasmid in
association with a fixed amount of WT plasmid, diminished GH-sti-
mulated luciferase activity compared to the activation obtained with
the equimolar ratio WT-mutant 1:1. Although further studies are re-
quired to completely elucidate the functional mechanisms underlying
these effects, it is possible to speculate that while one allele of WT-
STAT5b is not enough to achieve adequate growth, it would be suffi-
cient to respond to pharmacological doses of GH. Supporting this
L. Ramírez, et al. Growth Hormone & IGF Research 50 (2020) 61–70

Fig. 5. Analysis of p.K632N-STAT5b expression, phosphorylation and cellular localization. A) WT- and p.K632N-STAT5b were transfected into HEK293-T cells
expressing GHR and p-STAT5 and total STAT5b were determined by Western Immunoblot under basal (−) or GH-stimulated conditions (+, 30 min, 200 ng/mL). B)
Cellular localization of p.K632N-STAT5b mutant under GH-treatment (+). HEK293-T cells were transfected with WT- or p.K632N-STAT5b expressing plasmids and
then stimulated with GH (200 ng/mL, 30 min) followed by anti-STAT5b antibody immunostaining and Hoechst nuclear staining (scale bar, 10 μm). The fluorescent
signals were recorded by epi-fluorescent microscope. Representative images are presented.

observation, in vitro studies results are in line with the patient's re-
sponse to GH-treatment. Although GH therapy received USA Food and
Drug Administration (FDA) approval for idiopathic short stature pa-
tients, the degree of expected efficacy in such a heterogeneous group of
children remains undetermined. The consensus statement of the Lawson
Wilkins Pediatric Endocrine Society and the European Society for
Paediatric Endocrinology suggested criteria for poor first year response
as a height velocity SDS < 1 or change in height SDS < 0.3–0.5 [40].
This patient had an increment in height of 1.0 SDS in two years of
treatment and in addition, both IGF1 and IGFBP3 increased towards
normal range for age. However, long term responsiveness has still to be
determined.
The second variant found in the patient, p.R548W-ALS, has been
reported in children with idiopathic short stature and is described as
SNP in several databases [10,26]. However, studies with the serum of a
patient carrying this variant showed a lower 150-KDa ternary (IGF1/
IGFBP3/ALS) complex peak that was substantially increased in pre-
sence of a high concentration of purified hIGFBP3, suggesting that this
variant possibly shows decreased IGFBP3 affinity [25]. In addition,
former studies from our group demonstrated a significant reduction of
the secreted amount of p.R548W-ALS protein in vitro [26]. Nonetheless,
as we already mentioned, heterozygosity for IGFALS variants is known
to cause a milder short stature phenotype, although adult height usually
falls within the normal range [6,7]. Thus, it is not clear whether this
hypomorphic IGFALS variant could contribute to the clinical and bio-
chemical phenotype.
The diversity of the phenotype associated with GH insensitivity
underlines the importance of not only reporting novel variants, but also
to in vitro characterize them, to better delineate the pathophysiology
and define the therapeutic strategy, as well. In this regard, we would

68
L. Ramírez, et al.
like to emphasize the potential predictive value of functional char-
acterization assays for the outcome to the rhGH treatment. Although it
has the limitations inherent to in vitro assays, functional studies are
robust tools that may help to make clinical decisions in a personalized
medicine approach.
In conclusion, our results contribute to expand the spectrum of
phenotypes associated to GHI and provide valuable functional in vitro
characterization that supports p.K632 N-STAT5b as a novel pathogenic
inactivating STAT5b variant. This variant may be associated with par-
tial GH insensitivity and it can present without severe immunological
complications.
Acknowledgements
Thanks are due to the patient and relatives who agreed to take part
in this study. We are also grateful to all members of the UE Growth
Project for useful discussions and Dr. Marina Peluffo for her help on
immunofluorescence.
Statement of ethics
Subjects involved in this study have given their written informed
consent according to Buenos Aires City Government regulations (3301/
2009, 595/201, 58/2011, 595/2014).
The study protocol has been approved by the Hospital de Niños R.
Gutiérrez's committee on human research.
Disclosure statement
The authors declare no conflict of interest.
Funding sources
This work was supported by Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET)-UE131 and Fundación A. J.
Roemmers.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ghir.2019.12.005.
References
[1] A. David, V. Hwa, L.A. Metherell, I. Netchine, C. Camacho-Hubner, A.J.L. Clark,
R.G. Rosenfeld, M.O. Savage, Evidence for a continuum of genetic, phenotypic, and
biochemical abnormalities in children with growth hormone insensitivity, Endocr.
Rev. 32 (2011) 472–497, https://doi.org/10.1210/er.2010-0023.
[2] P.J. Godowski, D.W. Leung, L.R. Meacham, J.P. Galgani, R. Hellmiss, R. Keret,
P.S. Rotwein, J.S. Parks, Z. Laron, W.I. Wood, Characterization of the human
growth hormone receptor gene and demonstration of a partial gene deletion in two
patients with Laron-type dwarfism, Proc. Natl. Acad. Sci. U. S. A. 86 (1989)
8083–8087.
[3] R.G. Rosenfeld, A.L. Rosenbloom, J. Guevara-Aguirre, Growth hormone (GH) in-
sensitivity due to primary GH receptor deficiency, Endocr. Rev. 15 (1994) 369–390,
https://doi.org/10.1210/edrv-15-3-369.
[4] J.M. Wit, W. Oostdijk, M. Losekoot, H.A. van Duyvenvoorde, C.A.L. Ruivenkamp,
S.G. Kant, MECHANISMS IN ENDOCRINOLOGY: novel genetic causes of short sta-
ture, Eur. J. Endocrinol. (2015) 145–173, https://doi.org/10.1530/EJE-15-0937.
[5] A. Dauber, M.T. Munoz-Calvo, V. Barrios, H.M. Domene, S. Kloverpris, C. Serra-
Juhe, V. Desikan, J. Pozo, R. Muzumdar, G.A. Martos-Moreno, F. Hawkins,
H.G. Jasper, C.A. Conover, J. Frystyk, S. Yakar, V. Hwa, J.A. Chowen, C. Oxvig,
R.G. Rosenfeld, L.A. Perez-Jurado, J. Argente, Mutations in pregnancy-associated
plasma protein A2 cause short stature due to low IGF-I availability, EMBO Mol.
Med. 8 (2016) 363–374, https://doi.org/10.15252/emmm.201506106.
[6] O.V. Fofanova-Gambetti, V. Hwa, J.M. Wit, H.M. Domene, J. Argente, P. Bang,
W. Hogler, S. Kirsch, C. Pihoker, H.K. Chiu, L. Cohen, C. Jacobsen, H.G. Jasper,
G. Haeusler, A. Campos-Barros, E. Gallego-Gomez, R. Gracia-Bouthelier, H.A. van
Duyvenvoorde, J. Pozo, R.G. Rosenfeld, Impact of heterozygosity for acid-labile
subunit (IGFALS) gene mutations on stature: results from the international acid-
labile subunit consortium, J. Clin. Endocrinol. Metab. 95 (2010) 4184–4191,
https://doi.org/10.1210/jc.2010-0489.
69
Growth Hormone & IGF Research 50 (2020) 61–70
[7] H.M. Domene, P.A. Scaglia, A.S. Martinez, A.C. Keselman, L.M. Karabatas,
V.R. Pipman, S.V. Bengolea, M.C. Guida, M.G. Ropelato, M.G. Ballerini,
E.M. Lescano, M.A. Blanco, J.J. Heinrich, R.A. Rey, H.G. Jasper, Heterozygous
IGFALS gene variants in idiopathic short stature and normal children: impact on
height and the IGF system, Horm. Res. Paediatr. 80 (2013) 413–423, https://doi.
org/10.1159/000355412.
[8] M.J.E. Walenkamp, M. Karperien, A.M. Pereira, Y. Hilhorst-Hofstee, J. van Doorn,
J.W. Chen, S. Mohan, A. Denley, B. Forbes, H.A. van Duyvenvoorde, S.W. van Thiel,
C.A. Sluimers, J.J. Bax, J.A.P.M. de Laat, M.B. Breuning, J.A. Romijn, J.M. Wit,
Homozygous and heterozygous expression of a novel insulin-like growth factor-I
mutation, J. Clin. Endocrinol. Metab. 90 (2005) 2855–2864, https://doi.org/10.
1210/jc.2004-1254.
[9] R.C. Scalco, V. Hwa, H.M. Domene, H.G. Jasper, A. Belgorosky, R. Marino,
A.M. Pereira, C.A. Tonelli, J.M. Wit, R.G. Rosenfeld, A.A.L. Jorge, STAT5B muta-
tions in heterozygous state have negative impact on height: another clue in human
stature heritability, Eur. J. Endocrinol. 173 (2015) 291–296, https://doi.org/10.
1530/EJE-15-0398.
[10] J.M. Wit, W. Oostdijk, M. Losekoot, Spectrum of insulin-like growth factor defi-
ciency, Endocr. Dev. 23 (2012) 30–41, https://doi.org/10.1159/000341739.
[11] J. Klammt, D. Neumann, E.F. Gevers, S.F. Andrew, I.D. Schwartz, D. Rockstroh,
R. Colombo, M.A. Sanchez, D. Vokurkova, J. Kowalczyk, L.A. Metherell,
R.G. Rosenfeld, R. Pfaffle, M.T. Dattani, A. Dauber, V. Hwa, Dominant-negative
STAT5B mutations cause growth hormone insensitivity with short stature and mild
immune dysregulation, Nat. Commun. 9 (2018) 2105, , https://doi.org/10.1038/
s41467-018-04521-0.
[12] H. Lejarraga, M. del Pino, V. Fano, S. Caino, T.J. Cole, Growth references for weight
and height for Argentinian girls and boys from birth to maturity: incorporation of
data from the World Health Organisation from birth to 2 years and calculation of
new percentiles and LMS values, Arch. Argent. Pediatr. 107 (2009) 126–133,
https://doi.org/10.1590/S0325-00752009000200006.
[13] G. Del Sal, G. Manfioletti, C. Schneider, The CTAB-DNA precipitation method: a
common mini-scale preparation of template DNA from phagemids, phages or
plasmids suitable for sequencing, Biotechniques. 7 (1989) 514–520.
[14] M. Gutiérrez, P. Scaglia, A. Keselman, L. Martucci, L. Karabatas, S. Domené,
A. Martin, P. Pennisi, M. Blanco, N. Sanguineti, L. Bezrodnik, D. Di Giovanni,
M.S. Caldirola, M.E. Azcoiti, M.I. Gaillard, L.A. Denson, K. Zhang, A. Husami, N.-
H. Yayah Jones, V. Hwa, S. Revale, M. Vázquez, H. Jasper, A. Kumar, H. Domené,
Partial growth hormone insensitivity and dysregulatory immune disease associated
with de novo germline activating STAT3 mutations, Mol. Cell. Endocrinol. (2018),
https://doi.org/10.1016/j.mce.2018.01.016.
[15] I. a Adzhubei, S. Schmidt, L. Peshkin, V.E. Ramensky, A. Gerasimova, P. Bork,
A.S. Kondrashov, S.R. Sunyaev, A method and server for predicting damaging
missense mutations, Nat. Methods 7 (2010) 248–249, https://doi.org/10.1038/
nmeth0410-248.
[16] P. Kumar, S. Henikoff, P.C. Ng, Predicting the effects of coding non-synonymous
variants on protein function using the SIFT algorithm, Nat. Protoc. 4 (2009)
1073–1081, https://doi.org/10.1038/nprot.2009.86.
[17] J.M. Schwarz, D.N. Cooper, M. Schuelke, D. Seelow, MutationTaster2: mutation
prediction for the deep-sequencing age, Nat. Methods 11 (2014) 361–362, https://
doi.org/10.1038/nmeth.2890.
[18] M. Hecht, Y. Bromberg, B. Rost, Better prediction of functional effects for sequence
variants, BMC Genomics 16 (2015) S1, , https://doi.org/10.1186/1471-2164-16-
S8-S1.
[19] M. Kircher, D.M. Witten, P. Jain, B.J. O'Roak, G.M. Cooper, J. Shendure, A general
framework for estimating the relative pathogenicity of human genetic variants, Nat.
Genet. 46 (2014) 310–315, https://doi.org/10.1038/ng.2892.
[20] S. Richards, N. Aziz, S. Bale, D. Bick, S. Das, J. Gastier-Foster, W.W. Grody,
M. Hegde, E. Lyon, E. Spector, K. Voelkerding, H.L. Rehm, On behalf of the ACMG
laboratory quality assurance committee, standards and guidelines for the inter-
pretation of sequence variants: a joint consensus recommendation of the American
College of Medical Genetics and Genomics and the association for molecular pa-
thology, Genet. Med. 17 (2015) 405–424, https://doi.org/10.1038/gim.2015.30.
[21] D. Neculai, A.M. Neculai, S. Verrier, K. Straub, K. Klumpp, E. Pfitzner, S. Becker,
Structure of the unphosphorylated STAT5a dimer, J. Biol. Chem. 280 (2005)
40782–40787, https://doi.org/10.1074/jbc.M507682200.
[22] X. Mao, X. Chen, Crystallization and X-ray crystallographic analysis of human
STAT1, Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 61 (2005) 666–668,
https://doi.org/10.1107/S1744309105017392.
[23] A. Roy, A. Kucukural, Y. Zhang, I-TASSER: a unified platform for automated protein
structure and function prediction, Nat. Protoc. 5 (2010) 725–738, https://doi.org/
10.1038/nprot.2010.5.
[24] P. Fang, R. Girgis, B.M. Little, K.L. Pratt, J. Guevara-Aguirre, V. Hwa,
R.G. Rosenfeld, Growth hormone (GH) insensitivity and insulin-like growth factor-I
deficiency in Inuit subjects and an Ecuadorian cohort: functional studies of two
codon 180 GH receptor gene mutations, J. Clin. Endocrinol. Metab. 93 (2008)
1030–1037, https://doi.org/10.1210/jc.2007-2022.
[25] J.M. Wit, H.A. van Duyvenvoorde, S.A. Scheltinga, S. de Bruin, L. Hafkenscheid,
S.G. Kant, C.A.L. Ruivenkamp, A.C.J. Gijsbers, J. van Doorn, E. Feigerlova,
C. Noordam, M.J. Walenkamp, H. Claahsen-van de Grinten, P. Stouthart,
I.E. Bonapart, A.M. Pereira, J. Gosen, H.A. Delemarre-van de Waal, V. Hwa,
M.H. Breuning, H.M. Domene, W. Oostdijk, M. Losekoot, Genetic analysis of short
children with apparent growth hormone insensitivity, Horm. Res. Paediatr. 77
(2012) 320–333, https://doi.org/10.1159/000338462.
[26] L.C. Martucci, M.L. Gutiérrez, L.M. Karabatas, P.A. Scaglia, R.A. Rey, H.M. Domené,
H.G. Jasper, S. Domené, Assessment of pathogenicity of natural IGFALS gene var-
iants by in silico bioinformatics tools and in vitro functional studies, Mol. Cell.
L. Ramírez, et al.
Endocrinol. 429 (2016), https://doi.org/10.1016/j.mce.2016.03.031.
[27] J. Connerney, D. Lau-Corona, A. Rampersaud, D.J. Waxman, Activation of male
liver chromatin accessibility and STAT5-dependent gene transcription by plasma
growth hormone pulses, Endocrinology. 158 (2017) 1386–1405, https://doi.org/
10.1210/en.2017-00060.
[28] K. Oshida, D.J. Waxman, J.C. Corton, Chemical and hormonal effects on STAT5b-
dependent sexual dimorphism of the liver transcriptome, PLoS One 11 (2016)
e0150284, , https://doi.org/10.1371/journal.pone.0150284.
[29] K. Oshida, N. Vasani, D.J. Waxman, J.C. Corton, Disruption of STAT5b-regulated
sexual dimorphism of the liver transcriptome by diverse factors is a common event,
PLoS One 11 (2016) e0148308, , https://doi.org/10.1371/journal.pone.0148308.
[30] G.B. Udy, R.P. Towers, R.G. Snell, R.J. Wilkins, S.-H. Park, P.A. Ram, D.J. Waxman,
H.W. Davey, Requirement of STAT5b for sexual dimorphism of body growth rates
and liver gene expression, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 7239–7244
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC23803/.
[31] S. Teglund, C. McKay, E. Schuetz, J.M. van Deursen, D. Stravopodis, D. Wang,
M. Brown, S. Bodner, G. Grosveld, J.N. Ihle, Stat5a and Stat5b proteins have es-
sential and nonessential, or redundant, roles in cytokine responses, Cell. 93 (1998)
841–850, https://doi.org/10.1016/S0092-8674(00)81444-0.
[32] N. Abraham, M.C. Ma, J.W. Snow, M.J. Miners, B.G. Herndier, M.A. Goldsmith,
Haploinsufficiency identifies STAT5 as a modifier of IL-7-induced lymphomas,
Oncogene. 24 (2005) 5252–5257, https://doi.org/10.1038/sj.onc.1208726.
[33] Z. Yao, Y. Kanno, M. Kerenyi, G. Stephens, L. Durant, W.T. Watford, A. Laurence,
G.W. Robinson, E.M. Shevach, R. Moriggl, L. Hennighausen, C. Wu, J.J. O'Shea,
Nonredundant roles for Stat5a/b in directly regulating Foxp3, Blood. 109 (2007)
4368–4375, https://doi.org/10.1182/blood-2006-11-055756.
[34] S. Basu, D. Mukharjee, Salt-bridge networks within globular and disordered pro-
teins: characterizing trends for designable interactions, J. Mol. Model. 23 (2017)
206, , https://doi.org/10.1007/s00894-017-3376-y.
70
Growth Hormone & IGF Research 50 (2020) 61–70
[35] S. Kumar, R. Nussinov, Relationship between ion pair geometries and electrostatic
strengths in proteins, Biophys. J. 83 (2002) 1595–1612, https://doi.org/10.1016/
S0006-3495(02)73929-5.
[36] J.J. Skinner, S. Wang, J. Lee, C. Ong, R. Sommese, S. Sivaramakrishnan,
W. Koelmel, M. Hirschbeck, H. Schindelin, C. Kisker, K. Lorenz, T.R. Sosnick,
M.R. Rosner, Conserved salt-bridge competition triggered by phosphorylation reg-
ulates the protein interactome, Proc. Natl. Acad. Sci. U. S. A. 114 (2017)
13453–13458, https://doi.org/10.1073/pnas.1711543114.
[37] P.A. Scaglia, A.S. Martinez, E. Feigerlova, L. Bezrodnik, M.I. Gaillard, D. Di
Giovanni, M.G. Ballerini, H.G. Jasper, J.J. Heinrich, P. Fang, H.M. Domene,
R.G. Rosenfeld, V. Hwa, A novel missense mutation in the SH2 domain of the
STAT5B gene results in a transcriptionally inactive STAT5b associated with severe
IGF-I deficiency, immune dysfunction, and lack of pulmonary disease, J. Clin.
Endocrinol. Metab. 97 (2012) E830–E839, https://doi.org/10.1210/jc.2011-2554.
[38] B. Varco-Merth, E. Feigerlova, U. Shinde, R.G. Rosenfeld, V. Hwa, P. Rotwein,
Severe growth deficiency is associated with STAT5b mutations that disrupt protein
folding and activity, Mol. Endocrinol. 27 (2013) 150–161, https://doi.org/10.
1210/me.2012-1275.
[39] F. Langenfeld, Y. Guarracino, M. Arock, A. Trouve, L. Tchertanov, How intrinsic
molecular dynamics control intramolecular communication in signal transducers
and activators of transcription factor STAT5, PLoS One 10 (2015) e0145142, ,
https://doi.org/10.1371/journal.pone.0145142.
[40] P. Cohen, A.D. Rogol, C.L. Deal, P. Saenger, E.O. Reiter, J.L. Ross, S.D. Chernausek,
M.O. Savage, J.M. Wit, Consensus statement on the diagnosis and treatment of
children with idiopathic short stature: a summary of the Growth Hormone Research
Society, the Lawson Wilkins Pediatric Endocrine Society, and the European Society
for Paediatric Endocrinology Workshop, J. Clin. Endocrinol. Metab. 93 (2008)
4210–4217, https://doi.org/10.1210/jc.2008-0509.