Ten novel mutations in the NR5A1 gene cause disordered sex development in 46,XY and ovarian insufficiency in 46,XX individuals.

CONTEXT: Steroidogenic factor-1 (SF-1/NR5A1) is a nuclear receptor that regulates adrenal and reproductive development and function. NR5A1 mutations have been detected in 46,XY individuals with disorders of sexual development (DSD) but apparently normal adrenal function and in 46,XX women with normal sexual development yet primary ovarian insufficiency (POI). OBJECTIVE: A group of 100 46,XY DSD and two POI was studied for NR5A1 mutations and their impact. DESIGN: Clinical, biochemical, histological, genetic, and functional characteristics of the patients with NR5A1 mutations are reported. SETTING: Patients were referred from different centers in Spain, Switzerland, and Turkey. Histological and genetic studies were performed in Barcelona, Spain. In vitro studies were performed in Bern, Switzerland. PATIENTS: A total of 65 Spanish and 35 Turkish patients with 46,XY DSD and two Swiss 46,XX patients with POI were investigated. MAIN OUTCOME: Ten novel heterozygote NR5A1 mutations were detected and characterized (five missense, one nonsense, three frameshift mutations, and one duplication). RESULTS: The novel NR5A1 mutations were tested in vitro by promoter transactivation assays showing grossly reduced activity for mutations in the DNA binding domain and variably reduced activity for other mutations. Dominant negative effect of the mutations was excluded. We found high variability and thus no apparent genotype-structure-function-phenotype correlation. Histological studies of testes revealed vacuolization of Leydig cells due to fat accumulation. CONCLUSIONS: SF-1/NR5A1 mutations are frequently found in 46,XY DSD individuals (9%) and manifest with a broad phenotype. Testes histology is characteristic for fat accumulation and degeneration over time, similar to findings observed in patients with lipoid congenital adrenal hyperplasia (due to StAR mutations). Genotype-structure-function-phenotype correlation remains elusive.

SRD5A2 gene mutations and polymorphisms in Spanish 46,XY patients with a disorder of sex differentiation.

One hundred and forty-six index patients with 46,XY DSD in whom gonads were confirmed as testes were consecutively studied for a molecular diagnosis during the period 2002-2010. AR gene was analysed in all patients as the first candidate gene, yielding a mutation in 42.5% of cases and SRD5A2 gene was analysed as the second candidate gene, resulting in the characterization of 10 different mutations (p.Y91D, p.G115D, p.Q126R, p.R171S, p.Y188CfsX9, p.N193S, p.A207D, p.F219SfsX60, p.R227Q and p.R246W) in nine index patients (6.2% of the total number of 46,XY DSD patients). One of the mutations (p.Y188CfsX9) has never been reported. In addition, we genotyped SRD5A2 gene p.V89L and c.281+15T>C polymorphisms in 46,XY DSD and in 156 normal adult males and found that patients with SRD5A2 mutations or without a known molecular diagnosis presented a higher frequency of homozygous p.L89, homozygous TT and combined CCTT genotypes compared with controls. This result suggests that 46,XY DSD patient phenotypes may be influenced by SRD5A2 polymorphism genotypes. SRD5A2 gene mutations may not be as infrequent as previously considered in 46,XY DSD patients with variable degrees of external genitalia virilization at birth and normal T production and appears to be the second aetiology in our series.

Novel (60%) and recurrent (40%) androgen receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development.

BACKGROUND: Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS). OBJECTIVE: The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients. SETTING: We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis. METHODS: The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients. RESULTS: AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel. CONCLUSIONS: AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.

Vitamin D and growth hormone regulate growth hormone/insulin-like growth factor (GH-IGF) axis gene expression in human fetal epiphyseal chondrocytes.

OBJECTIVE: Cell proliferation and gene expression regulation were studied in human fetal epiphyseal chondrocytes to ascertain the involvement of GH-IGF axis components in human fetal growth regulation by 1,25-dihydroxyvitamin D(3) (VitD) and growth hormone (GH). DESIGN: Chondrocytes from primary cultures were plated in serum-free medium for 48 h and incubated for a further 48 h with VitD (10(-11) to 10(-6)M) and/or IGF-I (100 ng/ml) and/or GH (500 ng/ml). We analyzed (3)H-thymidine incorporation into DNA and IGF-I, IGFBP-3, GHR, SOX9, COL2A1, aggrecan and COMP gene expression by real-time quantitative PCR. RESULTS: VitD dose-dependently and significantly inhibited (3)H-thymidine incorporation whereas GH had no effect on proliferation and, when combined with VitD, the same inhibition was observed as with VitD alone. IGF-I (100 ng/ml) significantly stimulated proliferation and opposed inhibition by VitD. VitD dose-dependently stimulated IGF-I (11.1+/-19.8 at VitD10(-6)M), IGFBP-3 (2.6+/-0.9), GHR (3.8+/-2.8) and COMP (1.5+/-0.6) expression whereas it inhibited SOX9 (0.7+/-0.2), COL2A1 (0.6+/-0.3) and aggrecan (0.6+/-0.2) expression and had no significant effect on IGF-II. IGF-I stimulated IGF-I, IGFBP-3, SOX9, COL2A1 and aggrecan expression and opposed COL2A1 and aggrecan gene expression inhibition by VitD. GH alone had no effect on gene expression whereas, in the presence of VitD, significantly-increased IGF-I expression stimulation was observed above values obtained with VitD alone (17.5+/-7.4). CONCLUSIONS: Our results suggest that VitD regulation of fetal growth cartilage could have consisted of parallel enhancing of cell differentiation and conditioning to a phenotype more sensitive to regulation by other hormones such as GH as shown by increased GHR and IGF-I expression, but not by IGF-II expression which was not regulated.

IGF-I and not IGF-II expression is regulated by glucocorticoids in human fetal epiphyseal chondrocytes.

OBJECTIVE: To elucidate the involvement of IGF axis components and the potential effects of glucocorticoids (GCs) in human fetal growth regulation. DESIGN: We studied the regulation by dexamethasone (Dx) and IGF-I of proliferation and IGF axis components and matrix protein gene expression in human fetal epiphyseal chondrocytes. RESULTS: High Dx concentration (10(-7)-10(-6)M) inhibited (3)H-thymidine incorporation, mifepristone (MF) 10(-6)M limited inhibition by Dx, and IGF-I (100 ng/ml) significantly stimulated proliferation and completely opposed inhibition by Dx. Dx dose-dependently (10(-9)-10(-6)M) inhibited IGF-I, IGFBP3 and SOX9 gene expression and expression of GHR, COL2A1 and aggrecan from 10(-7)M to 10(-6)M whereas it stimulated IGF-IR expression. By contrast, Dx had no significant effect on IGF-II expression. IGF-I stimulated IGF-I, IGFBP3, SOX9, COL2A1 and aggrecan expression whereas it inhibited IGF-IR expression. IGF-I could oppose COL2A1 and aggrecan gene expression inhibition by Dx. CONCLUSIONS: We demonstrated for the first time by real-time quantitative PCR that human fetal epiphyseal chondrocytes expressed IGF axis components, such as IGF-I, IGF-II, IGFBP3, IGF-IR and GHR and SOX9, COL2A1 and aggrecan, and that their expression was regulated by Dx and IGF-I. Among IGFs, IGF-I and not IGF-II expression was demonstrated to be down-regulated by GCs whereas IGF-I expression was up-regulated by itself.

The exon 3-deleted/full-length growth hormone receptor polymorphism does not influence the effect of puberty or growth hormone therapy on glucose homeostasis in short non-growth hormone-deficient small-for-gestational-age children: results from a two-year controlled prospective study.

CONTEXT: The exon 3-deleted/full-length (d3/fl) GH receptor polymorphism (d3/fl-GHR) has been associated with responsiveness to GH therapy in short small-for-gestational-age (SGA) patients, although consensus is lacking. However, its influence on glucose homeostasis, at baseline or under GH therapy, has not been investigated. OBJECTIVE: Our objective was to evaluate whether the d3/fl-GHR genotypes influence insulin sensitivity in short SGA children before or after puberty onset or during GH therapy. DESIGN: We conducted a 2-yr prospective, controlled, randomized trial. SETTING: Thirty Spanish hospitals participated. Auxological, GH secretion, and glucose homeostasis evaluation was hospital based, whereas molecular analyses and data computation were centralized. PATIENTS: Patients included 219 short SGA children [body mass index sd score (SDS) < or = 2.0]; 159 were prepubertal (group 1), and 60 had entered puberty (group 2). INTERVENTION: Seventy-eight patients from group 1 were treated with GH (66 microg/kg.d) for 2 yr (group 3). MAIN OUTCOME MEASURES: Previous and 2-yr follow-up auxological and biochemical data were recorded, d3/fl-GHR genotypes determined, and data analyzed. RESULTS: In groups 1 and 2, fasting glucose, insulin, homeostasis model assessment (HOMA), and quantitative insulin sensitivity check index (QUICKI) were similar in each d3/fl-GHR genotype. Group 2 glucose, insulin, and HOMA were significantly higher and QUICKI lower than in group 1. In group 3 GH-treated patients, height SDS, growth velocity SDS, fasting glucose, insulin, and HOMA significantly increased as did body mass index SDS at the end of the second year, and QUICKI decreased during the first and second years, with no differences among the d3/fl-GHR genotypes. CONCLUSION: In short SGA patients, the d3/fl-GHR genotypes do not seem to influence prepubertal or pubertal insulin sensitivity indexes or their changes over 2 yr of GH therapy (66 mug/kg.d).

The exon 3-deleted/full-length growth hormone receptor polymorphism did not influence growth response to growth hormone therapy over two years in prepubertal short children born at term with adequate weight and length for gestational age.

CONTEXT: Consensus is lacking as to whether the exon 3-deleted (d3)/full-length (fl) GH receptor (GHR) polymorphism is associated with responsiveness to GH therapy. OBJECTIVE: Our objective was to evaluate, in short, prepubertal, appropriate-for-gestational age (AGA) patients, 2-yr growth response to GH therapy (31.7+/-3.5 microg/kg.d) according to exon 3-deleted/full-length GHR genotypes. DESIGN: We conducted a retrospective study. PATIENTS: We studied 106 short AGA children, 58 boys and 48 girls, 7.8+/-2.3 yr, (d3/d3 n=18, d3/fl n=42, and fl/fl n=46). The GH response to two provocative stimuli were under 10 ng/ml in 65 and one or both over 10 ng/ml in 41 patients. MAIN OUTCOME MEASURES: Patients were followed by a single clinical team and remained prepubertal during the study. The exon 3-deleted/full-length GHR genotypes were determined and analyzed in the same hospital. RESULTS: Growth velocity significantly (P<0.0001) increased during the first and second years of therapy, as did height sd score (SDS). These increases were similar in each exon 3-deleted/full-length GHR genotype. Total 2-yr height gain (SDS) did not differ statistically among genotypes: 15.5+/-2.2 cm and 1.2+/-0.5 SDS in d3/d3, 15.9+/-2.0 cm and 1.3+/-0.4 SDS in d3/fl, and 15.4+/-2.1 cm and 1.1+/-0.3 SDS in fl/fl. No significant differences among the three genotypes were found in both sexes or in patients with different GH peak response to provocative stimuli for these parameters. An analysis of previously published studies was also performed. CONCLUSIONS: These results confirm in AGA patients those previously found by us and others in small-for-gestational-age patients and suggest that neither sex nor GH peaks after provocative stimuli might influence significantly the responsiveness to GH therapy according to the exon 3-deleted/full-length GHR genotypes.

The d3/fl-growth hormone (GH) receptor polymorphism does not influence the effect of GH treatment (66 microg/kg per day) or the spontaneous growth in short non-GH-deficient small-for-gestational-age children: results from a two-year controlled prospective study in 170 Spanish patients.

CONTEXT: The d3/fl-GH receptor (d3/fl-GHR, exon 3-deleted/full-length GHR) has recently been associated with responsiveness to GH therapy. OBJECTIVE: The objective of the study was to evaluate whether the d3/fl-GHR genotypes influence the intensity of spontaneous and/or GH therapy-stimulated growth in small-for-gestational-age (SGA) patients. DESIGN: This was a 2-yr prospective, controlled, randomized trial. SETTING: Thirty Spanish hospitals participated. Auxologic and GH secretion evaluation was hospital based, whereas molecular analyses and auxologic data computation were centralized. PATIENTS: Patients included 170 short SGA children: 140 remained prepubertal and 30 entered puberty during the second follow-up year. INTERVENTION: Eighty-six were treated with GH (66 microg/kg.d) for 2 yr and 84 were not treated. MAIN OUTCOME MEASURES: Previous and 2-yr follow-up auxologic data were recorded at each hospital, d3/fl-GHR genotypes determined, and data analyzed for patients who remained prepubertal (group 1, 68 GH treated and 72 non-GH treated) and for all the patients (group 2). RESULTS: In group 1 GH-treated patients, growth velocity, and height-sd score during the first and second years, total 2-yr height gain (18.5 +/- 2.4 cm in d3/d3; 18.4 +/- 2.6 in d3/fl; 19.5 +/- 2.3 in fl/fl), Delta 2-yr height increase (9.1 +/- 2.4 cm in d3/d3; 9.4 +/- 3.0 in d3/fl; 10.4 +/- 2.1 in fl/fl), first-year growth prediction and studentized residual values (0.08 +/- 1.26 in d3/d3; 0.28 +/- 1.21 in d3/fl; 0.67 +/- 0.95 in fl/fl) did not differ among the d3/fl-GHR genotypes. In group 1 non-GH-treated patients, neither growth velocity nor height-sd score changed significantly, and values were similar in each d3/fl-GHR genotype. Results in all patients (group 2) were similar to those in group 1. CONCLUSIONS: In short non-GH-deficient SGA children, both spontaneous growth rate and responsiveness to 66 microg/k.d GH therapy were similar for each d3/fl-GHR genotype carried.

Fractura de clavícula en recién nacidos: factores de riesgo y morbilidad asociada / Clavicle fracture in newborns: risk factors and associated morbidities

Introducción: La fractura de clavícula es el traumatismo obstétrico óseo más frecuente en el recién nacido; se asocia a partos con distocia de hombros y a múltiples factores biomédicos relacionados. Objetivo: Analizar los factores de riesgo y morbilidad asociada con la fractura de clavícula en el recién nacido de parto normal. Pacientes y método: Estudio retrospectivo, tipo caso control, realizado en el Hospital Herminda Martín de Chillán (Chile), entre los meses de enero a junio de 2004. El grupo de estudio estuvo constituido por el total de recién nacidos con fractura de clavícula (44 casos), el grupo control se constituyó en razón de 1:2, que correspondió a los 2 partos vaginales siguientes al caso estudio (88 casos). Resultados: La incidencia fue del 4,1 por ciento; resultaron estadísticamente significativos con la presencia de fractura de clavícula el período de dilatación y expulsivo en primíparas, el peso y talla del recién nacido y la presencia de complicaciones durante el parto, como distocia de hombros (el 9,1 frente al 2,3 por ciento), el descontrol materno (el 6,8 frente al 1,1 por ciento) y laterocidencia de mano, que sólo se presentó en el grupo estudio (6,8 por ciento). Conclusiones: La fractura de clavícula en el recién nacido se asocia principalmente a factores del trabajo de parto y expulsivo

Introduction: Clavicle fractures are caused by injury during the birth process. It is associated to childbirth with difficulty in the exit of shoulders and to multiple related biomedical factors. Objective: To evaluate risk factors and associated morbidities the clavicle facture in newborn of normal childbirth. Patients and method: Retropective study, type case control in the Hospital Herminda Martín, Chillán (Chile), January to June of 2004. The group study was constituted by 44 cases of newborn with clavicle fracture, the group control constituted itself in regard to 1:2, both corresponded to following vaginal childbirths to the case study (88 cases). Results: The incidence was of 4.1 percents, were statistically significant with the time of dilatation and expulsive in primiparas, weight and size of the newborns and presence of complications in the attention of the childbirth, shoulder dystocia (9.1 percents versus 2.3 percents), maternal uncontrol (6.8 percents versus 1.1 percents) and hand-dystocia (6.8 percents). Conclusions: Clavicle fracture in newborn is associated to factors of the normal labor and delivery

Vascular endothelial growth factor is expressed in human fetal growth cartilage.

Angiogenesis is a crucial event in endochondral ossification. Chemoattractants and mitogens for endothelial cells (such as basic fibroblast growth factor [bFGF] and transforming growth factor beta [TGF-beta]), which act as local regulators of the process, are synthesized by chondrocytes under several stimuli and in relation to the differentiation stage of the cartilage. Vascular endothelial growth factor (VEGF) is a 44-kDa protein well known as a potent angiogenic molecule owing to its mitogenic and permeability-causing properties. In this work, VEGF was located by immunohistochemistry in growth plate cartilage of human fetuses (20-22 weeks old) and its expression was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR). Primary culture of human fetal epiphyseal chondrocytes (HFEC) maintained VEGF expression at protein and messenger RNA (mRNA) levels and this expression was stimulated by cartilage-promoting growth factors incorporated into the culture media (rFGF-b, rTGF-beta1, and insulin-like growth factor [rFGF-b] at 50 ng/ml). The conditioned medium (CM) of HFEC stimulated the proliferation of endothelial cells, and this was partially blocked by anti-VEGF antibody. These studies showed VEGF production by chondrocytes of the epiphyseal growth cartilage and suggested a role of this factor in cartilage physiology and the angiogenic process.

Effects of TGF-beta1 on proliferation and IGFBP-3 production in a primary culture of human fetal epiphyseal chondrocytes (HFEC).

Proliferation and differentiation of chondrocytes from growth cartilage are modulated by hormones and growth factors, among which TGF-betas have been recognized as some of the more potent regulators although their specific cell effects on cartilage physiology are not fully understood. Primary human fetal epiphyseal chondrocytes (HEFC) constitutively produce TGF-beta1 at different times of culture progression. Treatment of 48-h. serum-deprived semiconfluent HFEC with 0.1-50 ng/ml of TGF-beta1 for 48-h. decreased (3H)Thymidine incorporation by 25-50 % and cell number by 25 %. In addition, IGFBP-3, the main insulin-like bonding protein produced by HFEC, showed a slight increase by TGF-beta1 in culture media. The changes in IGFBP-3 protein levels correlated well with its mRNA, indicating that TGF-beta1 is able to up-regulate IGFBP-3 synthesis in chondrocytes. Nevertheless, the IGFBP-3 accumulation in culture media does not produce a clear growth inhibitory effect on chondrocytes. Thus, we conclude that even though TGF-beta1 is able to up-regulate IGFBP-3, the growth inhibitory action produced by TGF-beta1 is not mediated by IGFBP-3 increase and appears to be mainly a direct TGF-beta1 effect on HFEC.