Clinical and molecular analysis of human reproductive disorders in Brazilian patients

Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHß, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone- (LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade

Clinical and molecuar characterization of Brazilian patients with growth hormone gene deletions

Genomic DNA from 23 patients with isolated growth hormone (GH) deficiency (12 males and 11 females: heights -4.9 ± 1.4 SDS) was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one) of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (~13%) Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67%) with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletion

Genetic Predictors Of Long-term Response To Growth Hormone (gh) Therapy In Children With Gh Deficiency And Turner Syndrome: The Influence Of A Socs2 Polymorphism

Design and Patients: Genotypes were correlated with adult height data of 65 Turner syndrome (TS) and 47GHdeficiency (GHD) patients treated with rhGH, by multiple linear regressions. Generalized multifactor dimensionality reduction was used to evaluate gene-gene interactions.Background: There is great interindividual variability in the response to GH therapy. Ascertaining genetic factors can improve the accuracy of growth response predictions. Suppressor of cytokine signaling (SOCS)-2 is an intracellular negative regulator of GH receptor (GHR) signaling. CopyrightObjective: The objective of the study was to assess the influence of a SOCS2 polymorphism (rs3782415) and its interactive effect with GHR exon 3 and -202 A/C IGFBP3 (rs2854744) polymorphisms on adult height of patients treated with recombinant human GH (rhGH).Results: Baseline clinical data were indistinguishable among patients with different genotypes. Adult height SD scores of patients with at least one SOCS2 single-nucleotide polymorphism rs3782415-C were 0.7 higher than those homozygous for the T allele (P < .001). SOCS2 (P = .003), GHR-exon 3 (P=.016) and-202 A/C IGFBP3 (P=.013) polymorphisms, together with clinical factors accounted for 58% of the variability in adult height and 82% of the total height SD score gain. Patients harboring any two negative genotypes in these three different loci (homozygosity for SOCS2 T allele; the GHR exon 3 full-length allele and/or the -202C-IGFBP3 allele) were more likely to achieve an adult height at the lower quartile (odds ratio of 13.3; 95% confidence interval of 3.2-54.2, P = .0001).Conclusion: The SOCS2 polymorphism (rs3782415) has an influence on the adult height of children with TS andGHDafter long-term rhGH therapy. Polymorphisms located inGHR, IGFBP3, and SOCS2 loci have an influence on the growth outcomes of TS and GHD patients treated with rhGH. The use of these genetic markers could identify among rhGH-treated patients those who are genetically predisposed to have less favorable outcomes.999E1808E1813Geffner, M.E., Dunger, D.B., Future directions: Growth prediction models (2007) Horm Res., 68, pp. 51-56Flores-Morales, A., Greenhalgh, C.J., Norstedt, G., Rico-Bautista, E., Negative regulation of growth hormone receptor signaling (2006) Mol Endocrinol., 20, pp. 241-253Greenhalgh, C.J., Rico-Bautista, E., Lorentzon, M., SOCS2 negatively regulates growth hormone action in vitro and in vivo (2005) J Clin Invest., 115, pp. 397-406Wassenaar, M.J., Dekkers, O.M., Pereira, A.M., Impact of the exon 3-deleted GH receptor polymorphism on baseline height and the growth response to recombinant human growth hormone therapy in growth hormone deficient(GHD)andnon-GHDchildren with short stature: A systematic review and meta-analysis (2009) J Clin Endocrinol Metab., 94, pp. 3721-3730Renehan, A.G., Solomon, M., Zwahlen, M., Growth hormone receptor polymorphism and growth hormone therapy response in children: A Bayesian meta-analysis (2012) AmJ Epidemiol., 175, pp. 867-877Costalonga, E.F., Antonini, S.R., Guerra-Junior, G., Mendonca, B.B., Arnhold, I.J., Jorge, A.A., The-202 A allele of insulin-like growth factor binding protein-3 (IGFBP3) promoter polymorphism is associated with higher IGFBP-3 serum levels and better growth response to growth hormone treatment in patients with severe growth hormone deficiency (2009) J Clin Endocrinol Metab., 94, pp. 588-595Braz, A.F., Costalonga, E.F., Montenegro, L.R., The interactive effect of GHR-exon 3 and-202 A/C IGFBP3 polymorphisms on rhGH responsiveness and treatment outcomes in patients with Turner syndrome (2012) J Clin Endocrinol Metab., 97, pp. E671-E677Weedon, M.N., Lango, H., Lindgren, C.M., Genome-wide association analysis identifies 20 loci that influence adult height (2008) Nat Genet., 40, pp. 575-583Gudbjartsson, D.F., Walters, G.B., Thorleifsson, G., Manysequence variants affecting diversity of adult human height (2008) Nat Genet., 40, pp. 609-615Chan, Y., Holmen, O.L., Dauber, A., Common variants show predicted polygenic effects on height in the tails of the distribution, except in extremely short individuals (2011) PLoS Genet., 7, p. e1002439Lou, X.Y., Chen, G.B., Yan, L., A generalized combinatorial approach for detecting gene-by-gene and gene-by-environment interactions with application to nicotine dependence (2007) Am J Hum Genet., 80, pp. 1125-1137Lango Allen, H., Estrada, K., Lettre, G., Hundreds of variants clustered in genomic loci and biological pathways affect human height (2010) Nature., 467, pp. 832-838Lanktree, M.B., Guo, Y., Murtaza, M., Meta-analysis of dense genecentric association studies reveals common and uncommon variants associated with height (2011) Am J Hum Genet., 88, pp. 6-18Ranke, M.B., Lindberg, A., Albertsson-Wikland, K., Wilton, P., Price, D.A., Reiter, E.O., Increased response, but lower responsiveness, to growth hormone (GH) in very young children (aged 0-3 years) with idiopathicGHDeficiency: Analysis of data from KIGS (2005) J Clin Endocrinol Metab., 90, pp. 1966-1971Ranke, M.B., Lindberg, A., Chatelain, P., Prediction of long-term response to recombinant human growth hormone in Turner syndrome: Development and validation of mathematical models. KIGS International Board. Kabi International Growth Study (2000) J Clin Endocrinol Metab., 85, pp. 4212-4218Ranke, M.B., Lindberg, A., Cowell, C.T., Prediction of response to growth hormone treatment in short children born small for gestational age: Analysis of data from KIGS (Pharmacia International Growth Database) (2003) J Clin Endocrinol Metab., 88, pp. 125-131Clayton, P., Chatelain, P., Tato, L., Apharmacogenomic approach to the treatment of children withGHdeficiency or Turner syndrome (2013) Eur J Endocrinol., 169, pp. 277-289Stevens, A., Clayton, P., Tato, L., Pharmacogenomics of insulinlike growth factor-I generation during GH treatment in children with GH deficiency or Turner syndrome (2014) Pharmacogenomics J., 14, pp. 54-6

The Sitting Height/height Ratio For Age In Healthy And Short Individuals And Its Potential Role In Selecting Short Children For Shox Analysis

Aims: To determine the presence of abnormal body proportion, assessed by sitting height/height ratio for age and sex (SH/H SDS) in healthy and short individuals, and to estimate its role in selecting short children for SHOX analysis. Methods: Height, sitting height and weight were evaluated in 1,771 healthy children, 128 children with idiopathic short stature (ISS), 58 individuals with SHOX defects (SHOX-D) and 193 females with Turner syndrome (TS). Results: The frequency of abnormal body proportion, defined as SH/H SDS >2, in ISS children was 16.4% (95% CI 10-22%), which was higher than in controls (1.4%, 95% CI 0.8-1.9%, p 2 were less common in children (48%, 95% CI 37-59%) and in adults (28%, 95% CI 20-36%) with TS. Conclusion: Abnormal body proportions were observed in almost all individuals with SHOX-D, 50% of females with TS and 16% of children considered ISS. Defects in SHOX gene were identified in 19% of ISS children with SH/H SDS >2, suggesting that SH/H SDS is a useful tool to select children for undergoing SHOX molecular studies. © 2013 S. Karger AG, Basel.806449456Ellison, J.W., Wardak, Z., Young, M.F., Gehron Robey, P., Laig-Webster, M., Chiong, W., PHOG, a candidate gene for involvement in the short stature of turner syndrome (1997) Hum Mol Genet, 6, pp. 1341-1347Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., Mertz, A., Muroya, K., Rappold, G.A., Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome (1997) Nat Genet, 16, pp. 54-63Clement-Jones, M., Schiller, S., Rao, E., Blaschke, R.J., Zuniga, A., Zeller, R., Robson, S.C., Rappold, G.A., The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome (2000) Hum Mol Genet, 9, pp. 695-702Ross, J.L., Kowal, K., Quigley, C.A., Blum, W.F., Cutler Jr., G.B., Crowe, B., Hovanes, K., Zinn, A.R., The phenotype of short stature homeobox gene (SHOX) deficiency in childhood: Contrasting children with Leri-Weill dyschondrosteosis and Turner syndrome (2005) J Pediatr, 147, pp. 499-507Neufeld, N.D., Lippe, B.M., Kaplan, S.A., Disproportionate growth of the lower extremities. A major determinant of short stature in Turner's syndrome (1978) Am J Dis Child, 132, pp. 296-298Rongen-Westerlaken, C., Rikken, B., Vastrick, P., Jeuken, A.H., De Lange, M.Y., Wit, J.M., Van Der Tweel, L., Van Den Brande, J.L., Body proportions in individuals with Turner syndrome The Dutch growth hormone working group (1993) Eur J Pediatr, 152, pp. 813-817Gravholt, C.H., Weis Naeraa, R., Reference values for body proportions and body composition in adult women with Ullrich-Turner syndrome (1997) Am J Med Genet, 72, pp. 403-408Baldin, A.D., Armani, M.C., Morcillo, A.M., Lemos-Marini, S.H., Baptista, M.T., Maciel-Guerra, A.T., Guerra-Junior, G., Body proportions in a group of Brazilian patients with Turner syndrome (2005) Arq Bras Endocrinol Metabol, 49, pp. 529-535Jorge, A.A., Funari, M.F., Nishi, M.Y., Mendonca, B.B., Short stature caused by isolated SHOX gene haploinsufficiency: Update on the diagnosis and treatment (2010) Pediatr Endocrinol Rev, 8, pp. 79-85Ross, J.L., Scott Jr., C., Marttila, P., Kowal, K., Nass, A., Papenhausen, P., Abboudi, J., Zinn, A.R., Phenotypes associated with SHOX deficiency (2001) J Clin Endocrinol Metab, 86, pp. 5674-5680Binder, G., Ranke, M.B., Martin, D.D., Auxology is a valuable instrument for the clinical diagnosis of SHOX haploinsufficiency in schoolage children with unexplained short stature (2003) J Clin Endocrinol Metab, 88, pp. 4891-4896Rappold, G., Blum, W.F., Shavrikova, E.P., Crowe, B.J., Roeth, R., Quigley, C.A., Ross, J.L., Niesler, B., Genotypes and phenotypes in children with short stature: Clinical indicators of SHOX haploinsufficiency (2007) J Med Genet, 44, pp. 306-313Jorge, A.A., Souza, S.C., Nishi, M.Y., Billerbeck, A.E., Liborio, D.C., Kim, C.A., Arnhold, I.J., Mendonca, B.B., SHOX mutations in idiopathic short stature and Leri-Weill dyschondrosteosis: Frequency and phenotypic variability (2007) Clin Endocrinol (Oxf), 66, pp. 130-135Hirschfeldova, K., Solc, R., Baxova, A., Zapletalova, J., Kebrdlova, V., Gaillyova, R., Prasilova, S., Stekrova, J., SHOX gene defects and selected dysmorphic signs in patients of idiopathic short stature and Leri-Weill dyschondrosteosis (2012) Gene, 491, pp. 123-127Kant, S.G., Broekman, S.J., De Wit, C.C., Bos, M., Scheltinga, S.A., Bakker, E., Oostdijk, W., Losekoot, M., Phenotypic characterization of patients with deletions in the 3 ′ flanking SHOX region (2013) PeerJ, 1, pp. e35Fredriks, A.M., Van Buuren, S., Van Heel, W.J., Dijkman-Neerincx, R.H., Verloove-Vanhorick, S.P., Wit, J.M., Nationwide age references for sitting height, leg length, and sitting height/ height ratio, and their diagnostic value for disproportionate growth disorders (2005) Arch Dis Child, 90, pp. 807-812Kuczmarski, R.J., Ogden, C.L., Grummer-Strawn, L.M., Flegal, K.M., Guo, S.S., Wei, R., Mei, Z., Johnson, C.L., CDC growth charts: United States (2000) Adv Data, 314, pp. 1-27Silva, D.A., Pelegrini, A., Petroski, E.L., Gaya, A.C., Comparison between the growth of Brazilian children and adolescents and the reference growth charts: Data from a Brazilian project (2010) J Pediatr (Rio J), 86, pp. 115-120Funari, M.F., Jorge, A.A., Souza, S.C., Billerbeck, A.E., Arnhold, I.J., Mendonca, B.B., Nishi, M.Y., Usefulness of MLPA in the detection of SHOX deletions (2010) Eur J Med Genet, 53, pp. 234-238Lango Allen, H., Estrada, K., Lettre, G., Berndt, S.I., Weedon, M.N., Rivadeneira, F., Willer, C.J., Hirschhorn, J.N., Hundreds of variants clustered in genomic loci and biological pathways affect human height (2010) Nature, 467, pp. 832-838Shears, D.J., Vassal, H.J., Goodman, F.R., Palmer, R.W., Reardon, W., Superti-Furga, A., Scambler, P.J., Winter, R.M., Mutation and deletion of the pseudoautosomal gene SHOX cause Leri-Weill dyschondrosteosis (1998) Nat Genet, 19, pp. 70-73Belin, V., Cusin, V., Viot, G., Girlich, D., Toutain, A., Moncla, A., Vekemans, M., Cormier-Daire, V., SHOX mutations in dyschondrosteosis (Leri-Weill syndrome) (1998) Nat Genet, 19, pp. 67-69Rappold, G.A., Fukami, M., Niesler, B., Schiller, S., Zumkeller, W., Bettendorf, M., Heinrich, U., Ogata, T., Deletions of the homeobox gene SHOX (short stature homeobox) are an important cause of growth failure in children with short stature (2002) J Clin Endocrinol Metab, 87, pp. 1402-1406Jorge, A.A., Arnhold, I.J., Anthropometric evaluation of children with SHOX mutations can be used as indication for genetic studies in children of short stature (2007) J Med Genet, 44, pp. e90. , author reply e91Kosho, T., Muroya, K., Nagai, T., Fujimoto, M., Yokoya, S., Sakamoto, H., Hirano, T., Ogata, T., Skeletal features and growth patterns in 14 patients with haploinsufficiency of SHOX: Implications for the development of turner syndrome (1999) J Clin Endocrinol Metab, 84, pp. 4613-4621Fukami, M., Nishi, Y., Hasegawa, Y., Miyoshi, Y., Okabe, T., Haga, N., Nagai, T., Ogata, T., Statural growth in 31 Japanese patients with SHOX haploinsufficiency: Support for a disadvantageous effect of gonadal estrogens (2004) Endocr J, 51, pp. 197-200Blum, W.F., Cao, D., Hesse, V., Fricke-Otto, S., Ross, J.L., Jones, C., Quigley, C.A., Binder, G., Height gains in response to growth hormone treatment to final height are similar in patients with SHOX deficiency and Turner syndrome (2009) Horm Res, 71, pp. 167-172Scalco, R.C., Melo, S.S., Pugliese-Pires, P.N., Funari, M.F., Nishi, M.Y., Arnhold, I.J., Mendonca, B.B., Jorge, A.A., Effectiveness of the combined recombinant human growth hormone and gonadotropin-releasing hormone analog therapy in pubertal patients with short stature due to SHOX deficiency (2010) J Clin Endocrinol Metab, 95, pp. 328-33

Supplementary Material for: Molecular and Gene Network Analysis of Thyroid Transcription Factor 1 <b><i>(TTF1)</i></b> and Enhanced at Puberty <b><i>(EAP1)</i></b> Genes in Patients with GnRH-Dependent Pubertal Disorders

<b><i>Background/Aim:</i></b> TTF1 and EAP1 are transcription factors that modulate gonadotropin-releasing hormone expression. We investigated the contribution of <i>TTF1</i> and <i>EAP1</i> genes to central pubertal disorders. <b><i>Patients and Methods:</i></b> 133 patients with central pubertal disorders were studied: 86 with central precocious puberty and 47 with normosmic isolated hypogonadotropic hypogonadism. The coding region of <i>TTF1</i> and <i>EAP1</i> were sequenced. Variations of polyglutamine and polyalanine repeats in EAP1 were analyzed by GeneScan software. Association of <i>TTF1</i> and <i>EAP1</i> to genes implicated in timing of puberty was investigated by meta-network framework GeneMANIA and Cytoscape software. <b><i>Results:</i></b> Direct sequencing of the <i>TTF1</i> did not reveal any mutation or polymorphisms. Four EAP1 synonymous variants were identified with similar frequencies among groups. The most common EAP1 5′-distal polyalanine genotype was the homozygous 12/12, but the genotype 12/9 was identified in 2 central precocious puberty sisters without functional alteration in EAP1 transcriptional activity. TTF1 and EAP1 were connected, via genetic networks, to genes implicated in the control of menarche. <b><i>Conclusion:</i></b> No <i>TTF1 </i>or <i>EAP1 </i>germline mutations were associated with central pubertal disorders. <i>TTF1 </i>and <i>EAP1 </i>may affect puberty by changing expression in response to other members of puberty-associated gene networks, or by differentially affecting the expression of gene components of these networks

21-Hydroxylase deficiency in Brazil

We determined the frequency of large rearrangements and point mutations in 130 Brazilian patients with 21-hydroxylase deficiency and correlated genotype with phenotype. The frequency of CYP21 deletions was lower (4.4%) than in most of the previous series described, whereas the frequency of large gene conversions was similar to the frequency reported in the literature (6.6%). The most frequent point mutations were I2 splice (41.8% in salt wasting - SW), I172N (32.6% in simple virilizing - SV) and V281L (40.2% in the late onset form - LO). The frequency of the nine most common point mutations was similar to that reported for other countries. The 93 fully genotyped patients were classified into 3 mutation groups based on the degree of enzymatic activity (A<2%, B <FONT FACE="Symbol">@</FONT> 2%, C>20%). In group A, 62% of cases presented the SW form; in group B, 96% the SV form, and in group C, 88% the LO form. We diagnosed 80% of the affected alleles after screening for large rearrangements and 15 point mutations. To diagnose these remaining alleles we sequenced the CYP21 gene of one patient with the SV form and identified a heterozygous G->A transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region and was also found in a compound heterozygous state in 4 other patients. The mutation G424S presented a linkage disequilibrium with CYP21P and C4A gene deletions and HLA DR17, suggesting a probable founder effect. Search for the G424S mutation in other populations will reveal if it is restricted to the Brazilian patients or if it has a wider ethnic distribution