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.

Genetic Mutations in the GH/IGF Axis.

The GH/IGF axis plays an important role in the control of pre and postnatal growth. At least 48 monogenic defects have been described affecting the production, secretion, and action of GH and IGFs. Molecular defects of the GH/IGF axis resulting in short stature were arbitrarily classified into 4 groups: 1. Combined pituitary hormone deficiency (CPHD) (a. syndromic CPHD and b. non-syndromic CPHD), 2. Isolated GH deficiency (IGHD), 3. GH insensitivity, and 4. IGF-I insensitivity. Genetic diagnosis is obtained in about 30-40% of children with growth retardation, severe IGHD, CPHD, apparent GH or IGF-I insensitivity, and small for gestational age. Increased accessibility to next generation sequencing (NGS) techniques resulted in a significant number of likely pathogenic variants in genes previously associated with short stature as well as in completely novel genes. Functional in vitro assays and in vivo animal models are required to determine the real contribution of these findings.

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.

Novel Insulin-Like Growth Factor 1 Gene Mutation: Broadening of the Phenotype and Implications for Insulin Resistance.

CONTEXT: Insulin-like growth factor (IGF)1 gene mutations are extremely rare causes of pre- and postnatal growth retardation. Phenotype can be heterogenous with varying degrees of neurosensory deafness, cognitive defects, glucose metabolism impairment and short stature. OBJECTIVE: This study describes a 12.6-year-old girl presenting with severe short stature and insulin resistance, but with normal hearing and neurological development at the lower limit of normal. METHODS: DNA was obtained from the proband and both parents for whole exome sequencing (WES). In silico analysis was performed to predict the impact of the IGF1 variant on IGF1 and insulin receptors (IGF1R and IR) signaling. Phosphorylation of the IGF1R at activating Tyr residues and cell proliferation analyses were used to assess the ability of each subject's IGF1 to bind and activate IGF1R. RESULTS: The proband had low immunoreactive IGF1 in serum and WES revealed a novel homozygous IGF1 missense variant (c.247A>T), causing a change of serine 83 for cysteine (p.Ser83Cys; p.Ser35Cys in mature peptide). The proband's parents were heterozygous for this mutation. In silico analyses indicated the pathogenic potential of the variant with electrostatic variations with the potential of hampering the interaction with the IGF1R but strengthening the binding to IR. The mutant IGF1 protein had a significantly reduced activity on in vitro bioassays. CONCLUSION: We describe a novel IGF1 mutation leading to severe loss of circulating IGF1 immunoreactivity and bioactivity. In silico modeling predicts that the mutant IGF1 could interfere with IR signaling, providing a possible explanation for the severe insulin resistance observed in the patient. The absence of significant hearing and neurodevelopmental involvement in the present case is unusual and broadens the clinical spectrum of IGF1 mutations.

Latin American consensus: children born small for gestational age.

BACKGROUND: Children born small for gestational age (SGA) experience higher rates of morbidity and mortality than those born appropriate for gestational age. In Latin America, identification and optimal management of children born SGA is a critical issue. Leading experts in pediatric endocrinology throughout Latin America established working groups in order to discuss key challenges regarding the evaluation and management of children born SGA and ultimately develop a consensus statement. DISCUSSION: SGA is defined as a birth weight and/or birth length greater than 2 standard deviations (SD) below the population reference mean for gestational age. SGA refers to body size and implies length-weight reference data in a geographical population whose ethnicity is known and specific to this group. Ideally, each country/region within Latin America should establish its own standards and make relevant updates. SGA children should be evaluated with standardized measures by trained personnel every 3 months during year 1 and every 6 months during year 2. Those without catch-up growth within the first 6 months of life need further evaluation, as do children whose weight is ≤ -2 SD at age 2 years. Growth hormone treatment can begin in SGA children > 2 years with short stature (< -2.0 SD) and a growth velocity < 25th percentile for their age, and should continue until final height (a growth velocity below 2 cm/year or a bone age of > 14 years for girls and > 16 years for boys) is reached. Blood glucose, thyroid function, HbA1c, and insulin-like growth factor-1 (IGF-1) should be monitored once a year. Monitoring insulin changes from baseline and surrogates of insulin sensitivity is essential. Reduced fetal growth followed by excessive postnatal catch-up in height, and particularly in weight, should be closely monitored. In both sexes, gonadal function should be monitored especially during puberty. SUMMARY: Children born SGA should be carefully followed by a multidisciplinary group that includes perinatologists, pediatricians, nutritionists, and pediatric endocrinologists since 10% to 15% will continue to have weight and height deficiency through development and may benefit from growth hormone treatment. Standards/guidelines should be developed on a country/region basis throughout Latin America.

Applying Bioinformatic Platforms, In Vitro, and In Vivo Functional Assays in the Characterization of Genetic Variants in the GH/IGF Pathway Affecting Growth and Development.

Heritability accounts for over 80% of adult human height, indicating that genetic variability is the main determinant of stature. The rapid technological development of Next-Generation Sequencing (NGS), particularly Whole Exome Sequencing (WES), has resulted in the characterization of several genetic conditions affecting growth and development. The greatest challenge of NGS remains the high number of candidate variants identified. In silico bioinformatic tools represent the first approach for classifying these variants. However, solving the complicated problem of variant interpretation requires the use of experimental approaches such as in vitro and, when needed, in vivo functional assays. In this review, we will discuss a rational approach to apply to the gene variants identified in children with growth and developmental defects including: (i) bioinformatic tools; (ii) in silico modeling tools; (iii) in vitro functional assays; and (iv) the development of in vivo models. While bioinformatic tools are useful for a preliminary selection of potentially pathogenic variants, in vitro-and sometimes also in vivo-functional assays are further required to unequivocally determine the pathogenicity of a novel genetic variant. This long, time-consuming, and expensive process is the only scientifically proven method to determine causality between a genetic variant and a human genetic disease.

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.

The role of acid-labile subunit (ALS) in the modulation of GH-IGF-I action.

Acid-labile subunit (ALS) deficiency (ACLSD) constitutes the first monogenic defect involving a member of the Insulin-like Growth Factor (IGF) binding protein system. The lack of ALS completely disrupts the circulating IGF system. Autocrine/paracrine action of local produced IGF-I could explain the mild effect on growth. In the present work we have revised the more relevant clinical and biochemical consequences of complete ACLSD in 61 reported subjects from 31 families. Low birth weight and/or length, reduced head circumference, height between -2 and -3 SD, pubertal delay and insulin resistance are commonly observed. Partial ACLSD could be present in children initially labeled as idiopathic short stature, presenting low IGF-I levels, suggesting that one functional IGFALS allele is insufficient to stabilize ternary complexes. Dysfunction of the GH-IGF axis observed in ACLSD may eventually result in increased risk for type-2 diabetes and tumor progression. Consequently, long term surveillance is recommended in these patients.

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.

Genetic disorders of GH action pathway.

While insensitivity to GH (GHI) is characterized by low IGF-I levels, normal or elevated GH levels, and lack of IGF-I response to GH treatment, IGF-I resistance is characterized by elevated IGF-I levels with normal/high GH levels. Several genetic defects are responsible for impairment of GH and IGF-I actions resulting in short stature that could affect intrauterine growth or be present in the postnatal period. The genetic defects affecting GH and/or IGF-I action can be divided into five different groups: GH insensitivity by defects affecting the GH receptor (GHR), the intracellular GH signaling pathway (STAT5B, STAT3, IKBKB, IL2RG, PIK3R1), the synthesis of insulin-like growth factors (IGF1, IGF2), the transport/bioavailability of IGFs (IGFALS, PAPPA2), and defects affecting IGF-I sensitivity (IGF1R). Complete GH insensitivity (GHI) was first reported by Zvi Laron and his colleagues in patients with classical appearance of GH deficiency, but presenting elevated levels of GH. The association of GH insensitivity with several clinical sings of immune-dysfunction and autoimmune dysregulation are characteristic of molecular defects in the intracellular GH signaling pathway (STAT5B, STAT3, IKBKB, IL2RG, PIK3R1). Gene mutations in the IGF1 and IGF2 genes have been described in patients presenting intrauterine growth retardation and postnatal short stature. Molecular defects have also been reported in the IGFALS gene, that encodes the acid-labile subunit (ALS), responsible to stabilize circulating IGF-I in ternary complexes, and more recently in the PAPPA2 gen that encodes the pregnancy-associated plasma protein-A2, a protease that specifically cleaves IGFBP-3 and IGFBP-5 regulating the accessibility of IGFs to their target tissues. Mutations in the IGF1R gene resulted in IGF-I insensitivity in patients with impaired intrauterine and postnatal growth. These studies have revealed novel molecular mechanisms of GH insensitivity/primary IGF-I deficiency beyond the GH receptor gene. In addition, they have also underlined the importance of several players of the GH-IGF axis in the complex system that promotes human growth.

Same Phenotype in Children with Growth Hormone Deficiency and Resistance.

By definition, about 2.5% of children show a short stature due to several causes. Two clinical conditions are characterized by serum IGF-I low levels, idiopathic GH deficiency (IGHD), and GH insensitivity (GHI), and the phenotypic appearance of these patients may be very similar. We studied two children with short stature and similar phenotypes. The first case showed frontal bossing, doll face, acromicria, and truncal obesity, with a GH peak <0.05 ng/ml after stimuli and undetectable serum IGF-I levels. After PCR amplification of the whole GH1 gene, type IA idiopathic GHD was diagnosed. The second case had cranium hypoplasia, a large head, protruding forehead, saddle nose, underdeveloped mandible, and a micropenis. Basal GH levels were high (28.4 ng/ml) while serum IGF-I levels were low and unchangeable during the IGF-I generation test. Laron syndrome was confirmed after the molecular analysis of the GH receptor (GHR) gene. IGHD type IA and Laron syndrome is characterized by opposite circulating levels of GH, while both have reduced levels of IGF-I, with an overlapping clinical phenotype, lacking the effects of IGF-I on cartilage. These classical cases show the importance of differential diagnosis in children with severe short stature.

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.

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.

Treatment With Recombinant Human Insulin-Like Growth Factor-1 Improves Growth in Patients With PAPP-A2 Deficiency.

CONTEXT: Pregnancy-associated plasma protein-A2 (PAPP-A2) is a metalloproteinase that specifically cleaves IGFBP-3 and IGFBP-5. Mutations in the PAPP-A2 gene have recently been shown to cause postnatal growth failure in humans, with specific skeletal features, due to the resulting decrease in IGF-1 bioavailability. However, a pharmacological treatment of this entity is yet to be established. CASE DESCRIPTION: A 10.5-year-old girl and a 6-year-old boy, siblings from a Spanish family, with short stature due to a homozygous loss-of-function mutation in the PAPP-A2 gene (p.D643fs25*) and undetectable PAPP-A2 activity, were treated with progressive doses (40, 80, 100, and 120 µg/kg) of recombinant human IGF-1 (rhIGF-1) twice daily for 1 year. There was a clear increase in growth velocity and height in both siblings. Bioactive IGF-1 was increased, and spontaneous GH secretion was diminished after acute administration of rhIGF-1, whereas serum total IGF-1 and IGFBP-3 levels remained elevated. No episodes of hypoglycemia or any other secondary effects were observed during treatment. CONCLUSION: Short-term treatment with rhIGF-1 improves growth in patients with PAPP-A2 deficiency.

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.

Congenital secondary hypothyroidism due to a mutation C105Vfs114X thyrotropin-beta mutation: genetic study of five unrelated families from Switzerland and Argentina.

We identified five patients with congenital secondary hypothyroidism with isolated thyrotropin (TSH) deficiency originating from three and two unrelated Argentinean and Swiss families, respectively. The affected patients presented with both low TSH as well as low thyroid hormone levels. Further, TSH-releasing hormone (TRH) stimulation failed to increase serum TSH, whereas prolactin increased adequately. These affected children were homozygous for a 1-bp deletion (822delT) in the TSH-beta subunit gene leading to a cysteine 105 to valine conversion (C105V) and to a frameshift with a premature stop codon at position 114 (C105Vfs114X). In a total of 22 families five different mutations located within the coding region of the TSH-beta subunit gene responsible for congenital secondary hypothyroidism have been reported so far (E12X; G29R; Q49X; IVS2 +5, G --> A; C105Vfs114X). Importantly, out of 13 families, including our five families, the C105Vfs114X mutation has been described in 12 unrelated and non-consanguineous families, whereas the remaining four TSH-beta subunit gene mutations have been described in consanguineous families only. Therefore the C105Vfs114X mutation within the TSH-beta subunit gene is the most frequent alteration causing congenital secondary hypothyroidism (13 of 22; 59%) and occurs mainly in unrelated and non-consanguineous families (12 of 13; 92%). As we could exclude a common ancestry by microsatellite marker analysis in our five independent families we concluded that the codon 105 in the TSH-beta subunit gene might be a "hot spot," although a founder effect has been reported in certain cases clustered in a highly specific and restricted geographical area.