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.

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.

Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism.

Serum insulin-like growth factor (IGF) -1 is secreted mainly by the liver and circulates bound to IGF-binding proteins (IGFBPs), either as binary complexes or ternary complexes with IGFBP-3 or IGFBP-5 and an acid-labile subunit (ALS). The purpose of this study was to genetically dissect the role of IGF-1 circulatory complexes in somatic growth, skeletal integrity, and metabolism. Phenotypic comparisons of controls and four mouse lines with genetic IGF-1 deficits-liver-specific IGF-1 deficiency (LID), ALS knockout (ALSKO), IGFBP-3 (BP3) knockout, and a triply deficient LID/ALSKO/BP3 line-produced several novel findings. 1) All deficient strains had decreased serum IGF-1 levels, but this neither predicted growth potential or skeletal integrity nor defined growth hormone secretion or metabolic abnormalities. 2) IGF-1 deficiency affected development of both cortical and trabecular bone differently, effects apparently dependent on the presence of different circulating IGF-1 complexes. 3) IGFBP-3 deficiency resulted in increased linear growth. In summary, each IGF-1 complex constituent appears to play a distinct role in determining skeletal phenotype, with different effects on cortical and trabecular bone compartments.

A study on strategies for improving growth and body composition after renal transplantation.

Allograft function and metabolic effects of four treatment regimens, namely, methylprednisone (MP) standard dose (MP-STD), deflazacort (DFZ), MP-late steroid withdrawal (MP-LSW), and MP-very low dose (MP-VLD), were evaluated in prepubertal patients. MP was decreased by month 4 post-transplantation to 0.2 mg/kg/day in MP-STD and DFZ patients and to <0.1 mg/kg/day in MP-LSW and MP-VLD patients. Starting in month 16 post-transplant, MP was switched to DFZ in the DFZ group and totally withdrawn in the MP-LSW group. Creatinine clearance diminished in the MP-STD and MP-LSW groups from 77 +/- 6 to 63 +/- 6 ml/min/1.73 m(2)and from 103 +/- 5 to 78 +/- 3 ml/min/1.73 m(2), respectively (p < 0.01 and p < 0.001, respectively). Height increased >0.5 SDS only in the MP-LSW and MP-VLD groups. The body mass index and fat body mass for height-age increased only in the MP-STD patients (p < 0.05 and p < 0.01, respectively). Fat body mass decreased in the DFZ group (p < 0.05), total cholesterol and LDL-cholesterol increased in the MP-STD group, while LDL-cholesterol and total cholesterol/HDL-cholesterol ratio decreased in the DFZ group (p < 0.01). Lumbar spine bone mineral density (BMD) for height-age showed an increase in the MP-LSW and MP-VLD groups (p < 0.01). Our data suggest that MP-LSW and MP-VLD strategies improve linear growth, BMD, the peripheral distribution of fat, and preservation of the bone-muscle unit and maintain the normal lipid profile. The MP-LSW patients had a concerning rate of acute rejections and graft function deterioration in prepubertal patients.

Deficiency of the insulin-like growth factor-binding protein acid-labile subunit (ALS) of the circulating ternary complex in children with short stature.

The acid-labile subunit (ALS) protein is a key component of the circulating 150-kDa IGF ternary complex. The main role of ALS is the extension of IGF-I half life by protecting it from degradation and preventing the passage of IGF-I to the extravascular compartment. In humans, complete ALS deficiency is characterized by severe reduction of IGF-I and IGFBP-3 that remain low after GH treatment, associated with mild growth retardation, much less pronounced than the IGF-I deficit. Pubertal delay in boys and insulin insensitivity are common findings. At least 21 patients with ALS deficiency have been described presenting 16 different homozygous or compound heterozygous inactivating mutations of the IGFALS gene. Although the effect of ALS deficiency on prenatal growth is still uncertain, postnatal growth is clearly affected, with the majority of the patients presenting a height between -2 to -3 SDS before and during puberty. In the assessment of a child with short stature ALS deficiency should be considered in those patients presenting: 1) a normal response to GH stimulation test, 2) low IGF-I levels associated with more profoundly reduced IGFBP-3 levels, 3) a mild growth retardation, apparently out of proportion to the degree of IGF-I and IGFBP-3 deficits, 4) lack of response to an IGF generation test and 5) insulin insensitivity. The relatively mild growth retardation in relation to the severe IGF-I deficit might be related to the preserved autocrine/paracrine action of locally produced IGF-I. The observation that in families of ALS deficient patients, heterozygous carriers for IGFALS gene mutations are shorter than their wild type relatives and the relatively high frequency of heterozygosity for this gene in children with idiopathic short stature suggests a requirement of normal levels of ALS for the attainment of maximal growth potential.

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).

Human acid-labile subunit deficiency: clinical, endocrine and metabolic consequences.

The majority of insulin-like growth factor (IGF)-I and IGF-II circulate in the serum as a complex with the insulin-like growth factor binding protein (IGFBP)-3 or IGFBP-5, and an acid-labile subunit (ALS). The function of ALS is to prolong the half-life of the IGF-I-IGFBP-3/IGFBP-5 binary complexes. Fourteen different mutations of the human IGFALS gene have been identified in 17 patients, suggesting that ALS deficiency may be prevalent in a subset of patients with extraordinarily low serum levels of IGF-I and IGFBP-3 that remain abnormally low upon growth hormone stimulation. Postnatal growth was clearly affected. Commonly, the height standard deviation score before puberty was between -2 and -3, and approximately 1.4 SD shorter than the midparental height SDS. Pubertal delay was found in 50% of the patients. Circulating IGF-II, IGFBP-1, -2 and -3 levels were reduced, with the greatest reduction observed for IGFBP-3. Insulin insensitivity was a common finding, and some patients presented low bone mineral density. Human ALS deficiency represents a unique condition in which the lack of ALS proteins results in the disruption of the entire IGF circulating system. Despite a profound circulating IGF-I deficiency, there is only a mild impact on postnatal growth. The preserved expression of locally produced IGF-I might be responsible for the preservation of linear growth near normal limits.

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.

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.

Phenotypic effects of null and haploinsufficiency of acid-labile subunit in a family with two novel IGFALS gene mutations.

CONTEXT: IGF-I deficiency may result from impairment of GH secretion or action, or from defects in IGF-I synthesis, transport, or action. Complete deficiency of the acid-labile subunit (ALS), previously described in two male patients, the only known inherited alteration in IGF-I transport, is characterized by severe circulating IGF-I and IGF binding protein (IGFBP)-3 deficiency with only mild growth retardation. OBJECTIVE: Our objective was to study the characterization, at biochemical and molecular levels, of the cause for severe circulating IGF-I and IGFBP-3 deficiency in a male patient with mild growth retardation. PATIENTS: We report an adolescent male with delayed growth and pubertal development (Tanner stage I, -2.00 sd score for height at the age of 15.3 yr), profound circulating IGF-I and IGFBP-3 deficiency, and poor response to GH treatment. RESULTS: The index case, as well as one of his brothers, and his sister were found to be compound heterozygotes for two novel IGFALS gene mutations: C540R, a missense point mutation; and S195_197Rdup, a 9-bp duplication. The parents and youngest brother were found to be carriers for one of these two mutations. The three affected siblings had marked reduction of IGF-I and IGFBP-3 levels, undetectable serum levels of ALS, inability to form ternary complexes, and moderate insulin resistance. All of them attained a normal near-adult height (between -1.0 and -0.5 sd score), which was nonetheless lower than that of their heterozygous brother. The IGF system was only modestly affected in the heterozygous carriers. CONCLUSIONS: This study confirms the critical role of ALS in forming ternary complexes and the maintenance of normal levels of IGF-I and IGFBP-3. Insulin resistance, pubertal delay in male patients, and poor GH responsiveness seem to be frequent findings in ALS deficiency. However, haploinsufficiency of the IGFALS gene has no discernible clinical effects with only modest impact on the IGF system.

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.

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.

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.

Differential impact of simple childhood obesity on the components of the growth hormone-insulin-like growth factor (IGF)-IGF binding proteins axis.

Simple childhood obesity is characterized by normal or even accelerated growth in spite of reduced growth hormone (GH) secretion. There are conflicting reports on the effects of obesity upon components of the GH-insulin-like growth factor-I (IGF-I)-IGF binding proteins (IGFBPs) system. In the present study we aimed to determine GH, IGF-I, IGFBP-3 and IGFBP-2 as well as some of the less explored components of this axis (IGFBP-3 proteolytic activity, IGFBP-3 plasma fragments, and total acid labile subunit [ALS]) in 22 obese and 17 age-matched control children. We also evaluated not only total GH binding protein (GHBP) serum levels but also GHBP bound to GH (complexed) in both groups. Obese and control groups strongly differed in BMI (obese: 4.7 +/- 0.36 vs control: 0.37 +/- 0.25 SDS, p <0.0001). In the obese group, we found lower GH serum levels, but normal serum levels of GH-GHBP complex, IGF-I, IGFBP-3, IGF-I/IGFBP-3 molar ratio, IGFBP-3 proteolytic activity, IGFBP-3 plasma fragments and total ALS. Obese children presented higher total circulating GHBP (6.0 +/- 0.44 vs 2.9 +/- 0.29 nmol/l, p <0.001) and insulin levels (10.5 +/- 1.5 vs 5.1 +/- 0.8 mU/l, p <0.001), while IGFBP-2 (4.6 +/- 0.5 vs 6.6 +/- 0.7%, p <0.05) and the ratio IGFBP-2/IGF-I (0.032 +/- 0.019 vs 0.095 +/- 0.01, p = 0.013) were lower than in controls. BMI and insulin were directly, and IGFBP-2 serum levels inversely, correlated to total GHBP serum levels when multiple regression analysis was performed (r = 0.74, p <0.001). By stepwise regression analysis, insulin (r = -0.37, p <0.05) and BMI (r = -0.52, p <0.01) inversely determined IGFBP-2. In summary, obese children present normal growth in spite of reduced GH secretion, probably because the combination of increased total GHBP and normal GH-GHBP complex serum levels (suggesting increased GH receptor [GHR] number and a normal serum GH reservoir, respectively) allow for the achievement of normal levels of IGF-I, IGFBP-3, IGFBP-3 proteolytic activity, IGFBP-3 plasma fragments and total ALS. Reduced IGFBP-2 serum levels and a lower ratio of IGFBP-2/IGF-I in obese children may suggest an increase of tissue IGF-I bioavailability, thus promoting its action. Normal IGF-I and GH availability may be contributing to maintain normal growth in obese children.