An in vivo functional assay to characterize human STAT5B genetic variants during zebrafish development.

Growth hormone (GH) binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway, which stimulates transcription of insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3) and insulin-like growth factor acid-labile subunit (IGFALS). Although STAT5B deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants have been reported in patients with less severe growth deficit and milder immune dysfunction. We developed an in vivo functional assay in zebrafish to characterize the pathogenicity of three human STAT5B variants (p.Ala630Pro, p.Gln474Arg and p.Lys632Asn). Overexpression of human wild-type (WT) STAT5B mRNA and its variants led to a significant reduction of body length together with developmental malformations in zebrafish embryos. Overexpression of p.Ala630Pro, p.Gln474Arg or p.Lys632Asn led to an increased number of embryos with pericardial edema, cyclopia and bent spine compared with WT STAT5B. Although co-injection of WT and p.Gln474Arg and WT and p.Lys632Asn STAT5B mRNA in zebrafish embryos partially or fully rescues the length and the developmental malformations in zebrafish embryos, co-injection of WT and p.Ala630Pro STAT5B mRNA leads to a greater number of embryos with developmental malformations and a reduction in body length of these embryos. These results suggest that these variants could interfere with endogenous stat5.1 signaling through different mechanisms. In situ hybridization of zebrafish embryos overexpressing p.Gln474Arg and p.Lys632Asn STAT5B mRNA shows a reduction in igf1 expression. In conclusion, our study reveals the pathogenicity of the STAT5B variants studied.

Expression of acid-labile subunit (ALS) in developing and adult zebrafish and its role in dorso-ventral patterning during development.

Mammalian acid-labile subunit (ALS) is a serum protein that binds binary complexes between Insulin-like growth factors (IGFs) and Insulin-like growth factor-binding proteins (IGFBPs) extending their half-life and keeping them in the vasculature. Human ALS deficiency (ACLSD), due to homozygous or compound heterozygous mutations in IGFALS, leads to moderate short stature with reduced levels of IGF-I and IGFBP-3. There is only one corresponding zebrafish ortholog gene and it has not yet been studied. In this study we elucidate the role of igfals during zebrafish development. In zebrafish embryos igfals mRNA is expressed throughout development, mainly in the brain and subsequently also in the gut and swimbladder. To determine its role during development, we knocked down igfals gene product using morpholinos (MOs). Igfals morphant embryos displayed dorsalization in different degrees of severity, including a shortened trunk and loss of tail. Furthermore, co-injection of human IGFALS (hIGFALS) mRNA was able to rescue the MO-induced phenotype. Finally, overexpression of either hIGFALS or zebrafish igfals (zigfals) mRNA leads to ventralization of embryos including a reduced head and enlarged tail. These findings suggest that als plays an important role in dorso-ventral patterning during zebrafish development.

A novel heterozygous STAT5B variant in a patient with short stature and partial growth hormone insensitivity (GHI).

BACKGROUND: The most frequent monogenic causes of growth hormone insensitivity (GHI) include defects in genes encoding the GH receptor itself (GHR), the signal transducer and activator of transcription (STAT5B), the insulin like-growth factor type I (IGF1) and the acid-labile subunit (IGFALS). GHI is characterized by a continuum of mild to severe post-natal growth failure. 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.

p.R209H GH1 variant challenges short stature assessment.

OBJECTIVE: to describe the marked variability in clinical and biochemical patterns that are associated with a p.R209H GH1 missense variant in a large Argentinean pedigree, which makes the diagnosis of GHD elusive. DESIGN: We describe a non-consanguineous pedigree composed by several individuals with short stature, including 2 pediatric patients with typical diagnosis of isolated growth hormone deficiency (IGHD) and 4 other siblings with severe short stature, low serum IGF-1 and IGFBP-3, but normal stimulated GH levels, suggesting growth hormone insensitivity (GHI) in the latter group. RESULTS: Patients with classical IGHD phenotype carried a heterozygous variant in GH1: c.626G>A (p.R209H). Data from the extended pedigree suggested GH1 as the initial candidate gene, which showed the same pathogenic heterozygous GH1 variant in the four siblings with short stature and a biochemical pattern of GHI. CONCLUSIONS: We suggest considering GH1 sequencing in children with short stature associated to low IGF-1 and IGFBP-3 serum levels, even in the context of normal response to growth hormone provocative testing (GHPT).

A homozygous mutation in the highly conserved Tyr60 of the mature IGF1 peptide broadens the spectrum of IGF1 deficiency.

BACKGROUND: IGF1 is a key factor in fetal and postnatal growth. To date, only three homozygous IGF1 gene defects leading to complete or partial loss of IGF1 activity have been reported in three short patients born small for gestational age. We describe the fourth patient with severe short stature presenting a novel homozygous IGF1 gene mutation. RESULTS: We report a boy born from consanguineous parents at 40 weeks of gestational age with intrauterine growth restriction and severe postnatal growth failure. Physical examination revealed proportionate short stature, microcephaly, facial dysmorphism, bilateral sensorineural deafness and mild global developmental delay. Basal growth hormone (GH) fluctuated from 0.2 to 29 ng/mL, while IGF1 levels ranged from -1.15 to 2.95 SDS. IGFBP3 was normal-high. SNP array delimited chromosomal regions of homozygosity, including 12q23.2 where IGF1 is located. IGF1 screening by HRM revealed a homozygous missense variant NM_000618.4(IGF1):c.322T>C, p.(Tyr108His). The change of the highly conserved Tyr60 in the mature IGF1 peptide was consistently predicted as pathogenic by multiple bioinformatic tools. Tyr60 has been described to be critical for IGF1 interaction with type 1 IGF receptor (IGF1R). In vitro, HEK293T cells showed a marked reduction of IGF1R phosphorylation after stimulation with serum from the patient as compared to sera from age-matched controls. Mutant IGF1 was also less efficient in inducing cell growth. CONCLUSION: The present report broadens the spectrum of clinical and biochemical presentation of homozygous IGF1 defects and underscores the variability these patients may present depending on the IGF/IGF1R pathway activity.

Insensibilidad a la GH en pacientes con retraso de crecimiento (Participación del Dr. Juan Jorge Heinrich en la descripción de dos nuevas alteraciones genéticas) / Growth hormone (GH) insensitivity in patients with growth retardation (Dr. Juan Jorge Heinrich contribution to the characterization of two novel gene c altera ons)

Como homenaje a la actuación del Dr. Juan Jorge Heinrich en su carácter de docente, pediatra clínico e investigador, los autores del presente artículo consideran adecuada una revisión de su participación en el descubrimiento de dos nuevas patologías dentro del campo de la resistencia a la acción de la hormona de crecimiento. La primera es causada por una mutación en el gen que codifica para la STAT5b (transductor de señal y activador de transcripción 5b), proteína que participa en la transmisión intracelular de la señal de GH. La segunda es causada por una mutación en el gen que codifica para la subunidad ácido lábil (ALS), una proteína esencial para la formación de complejos ternarios con IGF-I e IGFBP-3, los que incrementan marcadamente la vida media del IGF-I en la circulación

In homage to Dr. Juan Jotge Heinrich actions as teacher, pediatrician, and researcher, we feel pertinent to remark his contributions to the discovery of two new pathologies within the field of resistance to the action of growth hormone. One is due to a mutation in the gene coding for a protein, STAT5b (signal transducer and activator of transcription 5b), involved in the intracellular chain of transmission of the growth hormone signal; the other due to a mutation in the gene coding for ALS (acid labile subunit) a protein essential for the ternary complex formation among IGF-I, IGFBP-3, and ALS, which markedly increases the half life of IGF-I in the circulation

Partial growth hormone insensitivity and dysregulatory immune disease associated with de novo germline activating STAT3 mutations.

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms.

Circulating IGF-I, IGFBP-3 and the IGF-I/IGFBP-3 Molar Ratio Concentration and Height Outcome in Prepubertal Short Children on rhGH Treatment over Two Years of Therapy.

OBJECTIVE: To investigate the occurrence of abnormally elevated values of biomarkers of growth hormone (GH) action in short children on recombinant human GH (rhGH) therapy. METHODS: Sixty-three prepubertal short children were examined: 31 with GH deficiency (GHD), 25 small for gestational age (SGA), and 9 with Turner syndrome (TS). The main outcomes were the following: standard deviation score (SDS) values of IGF-I, IGFBP-3, and IGF-I/IGFBP-3 molar ratio before, at the 1st and at the 2nd year on rhGH and Δheight (Ht)-SDS to evaluate GH treatment efficacy (adequate 1st-year ΔHt SDS: >0.4 SDS for GHD and >0.3 SDS for non-GHD). RESULTS: Seventy-eight percent of GHD, 78% of SGA and 55% of TS children had adequate 1st-year ΔHt SDS. In GHD, 88% of IGF-I SDS and IGFBP-3 SDS that were ≤-2.0 SDS at baseline normalized on treatment. Abnormal IGF-I values >+2.0 SDS were observed in 52% of SGA and in 55% of TS patients on rhGH. Within each group, the IGF-I/IGFBP-3 molar ratio increased significantly from pretreatment and throughout therapy, remaining within normal range for most patients. ΔIGF-I/IGFBP-3 molar ratio SDS were significantly higher in children with an adequate response (p < 0.01). CONCLUSION: Non-GHD groups presented markedly elevated concentrations of GH biomarkers on rhGH and normal IGF-I/IGFBP-3 molar ratio in most patients. Since there is a lack of consensus regarding the molar ratio usefulness, we think that interventions towards a more physiological IGF-I serum profile should be implemented.

Characterization of four Latin American families confirms previous findings and reveals novel features of acid-labile subunit deficiency.

OBJECTIVE: Acid-labile subunit deficiency (ACLSD), caused by inactivating mutations in both IGFALS gene alleles, is characterized by marked reduction in IGF-I and IGFBP-3 levels associated with mild growth retardation. The aim of this study was to expand the known phenotype and genetic characteristics of ACLSD by reporting data from four index cases and their families. DESIGN: Auxological data, biochemical and genetic studies were performed in four children diagnosed with ACLSD and all available relatives. METHODS: Serum levels of IGF-I, IGFBP-3, acid-labile subunit (ALS), and in vitro ternary complex formation (ivTCF) were determined. After sequencing the IGFALS gene, pathogenicity of novel identified variants was evaluated by in vitro expression in transfected Chinese hamster ovarian (CHO) cells. ALS protein was detected in patients' sera and CHO cells conditioned media and lysates by Western immunoblot (WIB). RESULTS: Four index cases and four relatives were diagnosed with ACLSD. The following variants were found: p.Glu35Glyfs*17, p.Glu35Lysfs*87, p.Leu213Phe, p.Asn276Ser, p.Leu409Phe, p.Ala475Val and p.Ser490Trp. ACLSD patients presented low IGF-I and low or undetectable levels of IGFBP-3 and ALS. Seven out of 8 patients did not form ivTCF. CONCLUSIONS: This study confirms previous findings in ACLSD, such as the low IGF-I and a more severe reduction in IGFBP-3 levels, and a gene dosage effect observed in heterozygous carriers (HC). In addition, father-to-son transmission (father compound heterozygous and mother HC), preservation of male fertility, and marginal ALS expression with potential involvement in preserved responsiveness to rhGH treatment, are all novel aspects, not previously reported in this condition.

Past, Present, and Future in the Relationship between Growth Retardation and the IGF System: Excerpts from the Cesar Bergada Lecture Given during the SLEP 2015 Annual Meeting.

This mini review presents a personal view about the past, the present and the future of the relationship between growth retardation and the IGF system. Looking back, it is pertinent to include a brief look at the evolution of the somatomedin hypothesis, the use of IGF-I determinations in the clinic, and a review of the literature beginning in the late 1980s with the description of mutations in the Growth Hormone Receptor (GHR) gene. The present possibly started in the mid-1990s with the description of mutations in the IGF-I gene, followed in 2003 by reports of mutations in the genes coding for the IGF-I receptor and in the signal transducer and activator of transcription 5b (STAT5b). Finally, in 2004, mutations in the IGFALS gene were described. A diffuse limit between the present and the future might have been reached (the author's arbitrary decision) with the clinical applications of whole exome sequencing, which rapidly showed mutations in genes coding for STAT3, PAPP-A2 (pregnancy-associated plasma protein A2), and IGF-II.

Concentración de insulina e índices de insulinosensibilidad en niños y adolescentes sanos / Insulin level and insulin sensitivity indices among healthy children and adolescents

Introducción. Existe escasa información acerca de los valores de referencia de la insulina y de los índices de insulinosensibilidad en pediatría. Objetivo. Describir la variación de insulina e índices subrogantes de insulinosensibilidad en la etapa pediátrica. Población y métodos. Variación de la concentración de insulina en ayuno y de los índices subrogantes, como el modelo de evaluación homeostática de resistencia a la insulina (homeostasis model assessment of insulin resistance; HOMA-IR, por sus siglas en inglés), en niños sanos con la edad, el índice de masa corporal, estadio puberal (EP), la concentración de IGF-I, colesterol total y triglicéridos. Resultados. Se incluyeron 226 niños sanos (1-18 años). La insulina aumentó con la edad, el índice de masa corporal, el EP, los niveles de IGF-I y triglicéridos (r²= 0,38; p 7,5 años presentaron mayores valores de insulina [mediana (Pc3 y Pc97) pUI/ mL: 5,0 (1,7-9,6)] que los prepuberales < 7,5 años [2,9 pUI/mL (1,3-10,9); p < 0,01]. En la pubertad (del EP II al EP V), la insulina fue mayor en las niñas que en los varones [(7,4 (1,8-16,9) versus 5,8 (1,8-12,9); p 7,5 años: 1,1 (0,3-2,0) versus niños < 7,5 años: 0,6 (0,3-1,4; p < 0,01). Los grupos puberales presentaron niveles más elevados de insulina y de HOMA-IR respecto de los niños prepuberales (p 2,0 y > 2,6 en prepúberes y púberes, respectivamente, podrían alertar a los pediatras sobre un posible estado de insulinorresistencia.

Introduction. Information on insulin reference values and insulin sensitivity indices in the field of pediatrics is scarce. Objective. To describe insulin range and insulin sensitivity surrogate indices during childhood. Population and methods. Fasting insulin level range and surrogate indices, such as the homeostasis model assessment of insulin resistance (HOMA-IR), among healthy children and adolescents by age, body mass index, pubertal stage (PS), insulin-like growth factor-1 (IGF-1), total cholesterol, and triglycerides. Results. Two hundred and twenty-six healthy children and adolescents (1-18 years old) were included. Insulin increased with age, body mass index, pubertal stage, IGF-1 and triglyceride levels (r²= 0.38, p 7.5 years old had higher insulin levels [median (P3 and P97) pIU/mL: 5.0 (1.7-9.6)] than prepubertal children < 7.5 years old [2.9 pIU/ mL (1.3-10.9), p < 0.01]. During puberty (from PS II to PS V), insulin was higher in girls than in boys [7.4 (1.8-16.9) versus 5.8 (1.8-12.9), p 7.5 years old: 1.1 (0.32.0) versus children < 7.5 years old: 0.6 (0.3-1.4, p < 0.01). The insulin level and HOMA-IR results were higher in pubertal children compared to the prepubertal group (p 2.0 and > 2.6 in prepubertal and pubertal children, respectively, may be considered a warning sign for pediatricians to further investigate insulin resistance.

Insulin level and insulin sensitivity indices among healthy children and adolescents. / Concentración de insulina e índices de insulinosensibilidad en niños y adolescentes sanos.

INTRODUCTION: Information on insulin reference values and insulin sensitivity indices in the field of pediatrics is scarce. OBJECTIVE: To describe insulin range and insulin sensitivity surrogate indices during childhood. MATERIALS AND METHODS: Fasting insulin level range and surrogate indices, such as the homeostasis model assessment of insulin resistance (HOMA-IR), among healthy children and adolescents by age, body mass index, pubertal stage (PS), insulin-like growth factor-1 (IGF-1), total cholesterol, and triglycerides. RESULTS: Two hundred and twenty-six healthy children and adolescents (1-18 years old) were included. Insulin increased with age, body mass index, pubertal stage, IGF-1 and triglyceride levels (r2= 0.38, p 〈 0.0001). Prepubertal children 〉 7.5 years old had higher insulin levels [median (P3 and P97) pIU/mL: 5.0 (1.7-9.6)] than prepubertal children 〈 7.5 years old [2.9 pIU/ mL (1.3-10.9), p 〈 0.01]. During puberty (from PS II to PS V), insulin was higher in girls than in boys [7.4 (1.8-16.9) versus 5.8 (1.8-12.9), p 〈 0.01]. The HOMA-IR index increased in the group of prepubertal children 〉 7.5 years old: 1.1 (0.32.0) versus children 〈 7.5 years old: 0.6 (0.3-1.4, p 〈 0.01). The insulin level and HOMA-IR results were higher in pubertal children compared to the prepubertal group (p 〈 0.001). CONCLUSIONS: Known physiological changes were observed inboth insulin levels and the HOMA-IR index among children and adolescents. A fasting blood insulin level of 10 pIU/mL in prepubertal children and of 17 pIU/mL and 13 pIU/mL in pubertal girls and boys, respectively, may be considered as an acceptable cut-off value in healthy children. A HOMA-IR value 〉 2.0 and 〉 2.6 in prepubertal and pubertal children, respectively, may be considered a warning sign for pediatricians to further investigate insulin resistance.

INTRODUCCIÓN: Existe escasa información acerca de los valores de referencia de la insulina y de los índices de insulinosensibilidad en pediatría. OBJETIVO: Describir la variación de insulina e índices subrogantes de insulinosensibilidad en la etapa pediátrica. MATERIALES Y MÉTODOS: Variación de la concentración de insulina en ayuno y de los índices subrogantes, como el modelo de evaluación homeostática de resistencia a la insulina (homeostasis model assessment of insulin resistance; HOMA-IR, por sus siglas en inglés), en niños sanos con la edad, el índice de masa corporal, estadio puberal (EP), la concentración de IGF-I, colesterol total y triglicéridos. RESULTADOS: Se incluyeron 226 niños sanos (1-18 años). La insulina aumentó con la edad, el índice de masa corporal, el EP, los niveles de IGF-I y triglicéridos (r2= 0,38; p 〈 0,0001). Los niños prepuberales 〉 7,5 años presentaron mayores valores de insulina [mediana (Pc3 y Pc97) pUI/ mL: 5,0 (1,7-9,6)] que los prepuberales 〈 7,5 años [2,9 pUI/mL (1,3-10,9); p 〈 0,01]. En la pubertad (del EP II al EP V), la insulina fue mayor en las niñas que en los varones [(7,4 (1,8-16,9) versus 5,8 (1,8-12,9); p 〈 0,01]. El índice HOMA-IR aumentó en el grupo prepuberal 〉 7,5 años: 1,1 (0,3-2,0) versus niños 〈 7,5 años: 0,6 (0,3-1,4; p 〈 0,01). Los grupos puberales presentaron niveles más elevados de insulina y de HOMA-IR respecto de los niños prepuberales (p 〈 0,001). CONCLUSIONES: La insulina y el índice HOMA-IR mostraron los cambios fisiológicos conocidos en niños y adolescentes. Valores de insulinemia en ayuno de 10 pUI/mL en prepúberes y 17 pUI/ mL y 13 pUI/mL en niñas y niños púberes respectivamente pueden ser considerados como valor límite aceptable en niños sanos. HOMA-IR 〉 2,0 y 〉 2,6 en prepúberes y púberes, respectivamente, podrían alertar a los pediatras sobre un posible estado de insulinorresistencia.

Assessment of pathogenicity of natural IGFALS gene variants by in silico bioinformatics tools and in vitro functional studies.

Acid-labile subunit (ALS) is essential for stabilization of IGF-I and IGFBP-3 in ternary complexes within the vascular system. ALS deficient (ALS-D) patients and a subset of children with idiopathic short stature (ISS), presenting IGFALS gene variants, show variable degree of growth retardation associated to IGF-I and IGFBP-3 deficiencies. The aim of this study was to evaluate the potential pathogenicity of eleven IGFALS variants identified in ALS-D and ISS children using in silico and in vitro approaches. We were able to classify seven of these variants as pathogenic since they present impaired synthesis (p.Glu35Lysfs*87, p.Glu35Glyfs*17, p.Asn276Ser, p.Leu409Phe, p.Ser490Trp and p.Cys540Arg), or partial impairment of synthesis and lack of secretion (p.Leu213Phe). We also observed significant reduction of secreted protein for variants p.Ala330Asp, Ala475Val and p.Arg548Trp, while still retaining their ability to form ternary complexes. These findings provide an approach to test the pathogenicity of IGFALS gene variants.

Mutations in pregnancy-associated plasma protein A2 cause short stature due to low IGF-I availability.

Mutations in multiple genes of the growth hormone/IGF-I axis have been identified in syndromes marked by growth failure. However, no pathogenic human mutations have been reported in the six high-affinity IGF-binding proteins (IGFBPs) or their regulators, such as the metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) that is hypothesized to increase IGF-I bioactivity by specific proteolytic cleavage of IGFBP-3 and -5. Multiple members of two unrelated families presented with progressive growth failure, moderate microcephaly, thin long bones, mildly decreased bone density and elevated circulating total IGF-I, IGFBP-3, and -5, acid labile subunit, and IGF-II concentrations. Two different homozygous mutations in PAPPA2, p.D643fs25* and p.Ala1033Val, were associated with this novel syndrome of growth failure. In vitro analysis of IGFBP cleavage demonstrated that both mutations cause a complete absence of PAPP-A2 proteolytic activity. Size-exclusion chromatography showed a significant increase in IGF-I bound in its ternary complex. Free IGF-I concentrations were decreased. These patients provide important insights into the regulation of longitudinal growth in humans, documenting the critical role of PAPP-A2 in releasing IGF-I from its BPs.

STAT5B mutations in heterozygous state have negative impact on height: another clue in human stature heritability.

CONTEXT AND OBJECTIVE: GH insensitivity with immune dysfunction caused by STAT5B mutations is an autosomal recessive condition. Heterozygous mutations in other genes involved in growth regulation were previously associated with a mild height reduction. Our objective was to assess for the first time the phenotype of heterozygous STAT5B mutations. METHODS: We genotyped and performed clinical and laboratory evaluations in 52 relatives of two previously described Brazilian brothers with homozygous STAT5B c.424_427del mutation (21 heterozygous). Additionally, we obtained height data and genotype from 1104 adult control individuals from the same region in Brazil and identified five additional families harboring the same mutation (18 individuals, 11 heterozygous). Furthermore, we gathered the available height data from first-degree relatives of patients with homozygous STAT5B mutations (17 individuals from seven families). Data from heterozygous individuals and non-carriers were compared. RESULTS: Individuals carrying heterozygous STAT5B c.424_427del mutation were 0.6 SDS shorter than their non-carrier relatives (P = 0.009). Heterozygous subjects also had significantly lower SDS for serum concentrations of IGF1 (P = 0.028) and IGFBP3 (P = 0.02) than their non-carrier relatives. The 17 heterozygous first-degree relatives of patients carrying homozygous STAT5B mutations had an average height SDS of -1.4 ± 0.8 when compared with population-matched controls (P < 0.001). CONCLUSIONS: STAT5B mutations in the heterozygous state have a significant negative impact on height (∼ 3.9 cm). This effect is milder than the effect seen in the homozygous state, with height usually within the normal range. Our results support the hypothesis that heterozygosity of rare pathogenic variants contributes to normal height heritability.

Heterozygous IGFALS gene variants in idiopathic short stature and normal children: impact on height and the IGF system.

BACKGROUND: In acid-labile subunit (ALS)-deficient families, heterozygous carriers of IGFALS gene mutations are frequently shorter than their wild-type relatives, suggesting that IGFALS haploinsufficiency could result in short stature. We have characterized IGFALS gene variants in idiopathic short stature (ISS) and in normal children, determining their impact on height and the IGF system. PATIENTS AND METHODS: In 188 normal and 79 ISS children levels of IGF-1, IGFBP-3, ALS, ternary complex formation (TCF) and IGFALS gene sequence were determined. RESULTS: In sum, 9 nonsynonymous or frameshift IGFALS variants (E35Gfs*17, G83S, L97F, R277H, P287L, A330D, R493H, A546V and R548W) were found in 10 ISS children and 6 variants (G170S, V239M, N276S, R277H, G506R and R548W) were found in 7 normal children. If ISS children were classified according to the ability for TCF enhanced by the addition of rhIGFBP-3 (TCF+), carriers of pathogenic IGFALS gene variants were shorter and presented lower levels of IGF-1, IGFBP-3 and ALS in comparison to carriers of benign variants. In ISS families, subjects carrying pathogenic variants were shorter and presented lower IGF-1, IGFBP-3 and ALS levels than noncarriers. CONCLUSIONS: These findings suggest that heterozygous IGFALS gene variants could be responsible for short stature in a subset of ISS children with diminished levels of IGF-1, IGFBP-3 and ALS.

Association of serum components of the GH-IGFs-IGFBPs system with GHR-exon 3 polymorphism in normal and idiopathic short stature children.

OBJECTIVE: To investigate the possible association of circulating components of GH-IGFs-IGFBPs system with the GHR-exon 3 genotype in normal and idiopathic short stature (ISS) children. DESIGN: Descriptive, cross-sectional study in normal and ISS children. SUBJECTS AND METHODS: 192 normal and 81 ISS children (age: 5-17 years) were included. Serum IGF-I, IGFBP3, ALS and GHBP levels were measured. GHR-exon 3 polymorphism (GHRd3) was analyzed by multiplex PCR assay. Normal and ISS children were divided according to GHR-exon 3 genotype: homozygous for the full-length GHR isoform (GHRfl) and carriers of one or two copies of the GHRd3 allele. RESULTS: GHRd3 genotype distribution (fl:fl/fl:d3/d3:d3,%) in normal (60:34:6) and ISS (64:32:4) was similar and reached Hardy-Weinberg equilibrium. ISS children had significantly reduced levels of GHBP and GH-dependent factors as compared to controls (p<0.0001). Within the normal group, homozygous carriers of the GHRfl allele had significantly higher GHBP serum levels than those with one or two copies of the GHRd3 allele (Mean ± SEM; GHRfl: 3.2 ± 0.2 vs GHRd3: 2.7 ± 0.2 nmol/L, p = 0.04). No other significant association with GHR exon 3 polymorphism was found in either the normal or the ISS groups. CONCLUSIONS: GHR exon 3 polymorphism is distributed similarly in normal and ISS children, however only normal homozygous children for GHRfl allele showed higher GHBP levels. The lack of association between GHBP and GHR polymorphism in ISS children might be related to the heterogeneity of this group, where potential defects in GH receptor action may result in partial GH insensitivity.

Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor.

States of growth hormone (GH) resistance, such those observed in Laron dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout [GHRKO]) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKO-HIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1, allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron syndrome does not achieve full skeletal growth.

Type IA isolated growth hormone deficiency (IGHD) consistent with compound heterozygous deletions of 6.7 and 7.6 Kb at the GH1 gene locus / Deficiência isolada de hormônio do crescimento tipo IA (DIGH) consistente com deleções heterozigotas compostas de 6,7 e 7,6 Kb no locus gênico GH1

Isolated growth hormone deficiency (IGHD) may result from deletions/mutations in either GH1 or GHRHR genes. The objective of this study was to characterize the molecular defect in a girl presenting IGHD. The patient was born at 41 weeks of gestation from non-consanguineous parents. Clinical and biochemical evaluation included anthropometric measurements, evaluation of pituitary function, IGF-I and IGFBP-3 levels. Molecular characterization was performed by PCR amplification of GH1 gene and SmaI digestion of two homologous fragments flanking the gene, using genomic DNA from the patient and her parents as templates. At 1.8 years of age the patient presented severe growth retardation (height 61.2 cm, -7.4 SDS), truncal obesity, frontal bossing, doll face, and acromicria. MRI showed pituitary hypoplasia. Laboratory findings confirmed IGHD. GH1 gene could not be amplified in samples from the patient while her parents yielded one fragment of the expected size. SmaI digestion was consistent with the patient being compound heterozygous for 6.7 and 7.6 Kb deletions, while her parents appear to be heterozygous carriers for either the 6.7 or the 7.6 Kb deletions. We have characterized type IA IGHD caused by two different GH1 gene deletions, suggesting that this condition should be considered in severe IGHD, even in non-consanguineous families. Arq Bras Endocrinol Metab. 2012;56(8):558-63.

A deficiência isolada do hormônio do crescimento (DIGH) pode ser resultado de deleções/mutações no gene GH1 ou no gene GHRHR. O objetivo deste estudo foi caracterizar o defeito molecular em uma menina que apresenta DIGH. A paciente nasceu às 41 semanas de gestação de pais não consanguíneos. As avaliações clínica e bioquímica incluíram medidas antropométricas, avaliação da função pituitária e concentrações de IGF-I e IGFBP-3. A caracterização molecular foi feita por meio de amplificação do GH1 por PCR e digestão com SmaI de dois fragmentos homólogos flanqueando o gene, usando-se DNA genômico da paciente e de seus pais como padrões. Com 1,8 ano de idade, a paciente apresentou atraso grave no crescimento (altura 61,2 cm, -7.4 DP), obesidade central, protuberância frontal, face de boneca e acromicria. A RM mostrou hipoplasia pituitária. Os achados laboratoriais confirmaram a DIGH. O gene GH1 não pôde ser amplificado nas amostras da paciente, enquanto as amostras de seus pais produziram um fragmento do tamanho esperado. A digestão com SmaI foi consistente com a paciente ser heterozigota composta para deleções para 6,7 e 7,6 Kb, enquanto seus pais parecem ser carreadores heterozigotos para deleções de 6,7 ou 7,6 Kb. Caracterizamos a DIGH tipo IA causada por duas deleções diferentes no gene GH1, sugerindo que essa condição pode ser considerada na DIGH grave, mesmo em famílias não consanguíneas. Arq Bras Endocrinol Metab. 2012;56(8):558-63.

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.

CONTEXT: Signal transducer and activator of transcription 5b (STAT5b) deficiency, first reported in a patient who carried a p.Ala630Pro missense mutation in the Src homology 2 (SH2) domain, results in a rare clinical condition of GH insensitivity (GHI), IGF-I deficiency (IGFD), and severe immune dysregulation manifesting as progressive worsening of pulmonary function. PATIENT: The new patient presented with severe cutaneous eczema, episodic infections in the first years of life, and autoimmune thyroiditis. Immunological evaluation revealed T lymphopenia, but severe pulmonary symptoms were notably absent. She concomitantly exhibited pronounced growth failure, reaching an adult height of 124.7 cm [-5.90 SD score (SDS)]. Endocrine evaluations (normal provocative GH tests; low serum IGF-I, -3.7 SDS, and IGF-binding protein-3, -4.5 SDS) were consistent with GHI and IGFD. RESULTS: Analysis of the STAT5B gene revealed a novel homozygous missense mutation, p.Phe646Ser, located within the ßD' strand of the SH2 domain. Reconstitution studies demonstrated expression of the p.Phe646Ser variant was less robust than wild type but, in contrast to the previously described STAT5B p.Ala630Pro SH2 mutation, could be phosphorylated in response to GH and interferon-γ. The phosphorylated p.Phe646Ser, however, could not drive transcription. CONCLUSION: A novel STAT5B p.Phe646Ser mutation has been identified in a patient with clinical characteristics of STAT5b deficiency. Only the second STAT5B missense mutation identified, its lack of transcriptional activities despite GH-induced phosphorylation, confirms the crucial role of STAT5b for regulating the expression of IGF1 and provides insights into the importance of the SH2 ßD' strand for full STAT5b transcriptional activities. Whether the phosphorylated p.Phe646Ser variant retained functions that prevented pulmonary distress remains unresolved.