FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies.

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca(2) (+) pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition

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 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

Analysis Of Anti-müllerian Hormone (amh) And Its Receptor (amhr2) Genes In Patients With Persistent Müllerian Duct Syndrome [pesquisa De Mutações Nos Genes Do Hormônio Antimülleriano (amh) E Do Seu Receptor (amhr2) Em Pacientes Com Síndrome De Persistência Dos Ductos Müllerianos]

Objective: To screen for mutations in AMH and AMHR2 genes in patients with persistent Müllerian duct syndrome (PMDS). Patients and method: Genomic DNA of eight patients with PMDS was obtained from peripheral blood leukocytes. Directed sequencing of the coding regions and the exon-intron boundaries of AMH and AMHR2 were performed. Results: The AMH mutations p.Arg95*, p.Arg123Trp, c.556-2A>G, and p.Arg502Leu were identified in five patients; and p.Gly323Ser and p.Arg407* in AMHR2 of two individuals. In silico analyses of the novel c.556-2A>G, p.Arg502Leu and p.Arg407* mutations predicted that they were harmful and were possible causes of the disease. Conclusion: A likely molecular etiology was found in the eight evaluated patients with PMDS. Four mutations in AMH and two in AMHR2 were identified. Three of them are novel mutations, c.556-2A>G, and p.Arg502Leu in AMH; and p.Gly323Ser in AMHR2. © ABE&M todos os direitos reservados.568473478Jost, A., Problems of fetal endocrinology: The gonadal and hypophyseal hormones (1953) Recent Prog Horm Res, 8, pp. 379-418Farikullah, J., Ehtisham, S., Nappo, S., Patel, L., Hennayake, S., Persistent Müllerian duct syndrome: Lessons learned from managing a series of eight patients over a 10-year period and review of literature regarding malignant risk from the Mullerian remnants (2012) BJU Int, , Epub 2012 Apr 30Rey, R., Picard, J.Y., Josso, N., Síndrome de Persistência dos Ductos de Müller (2010) Menino ou Menina? Distúrbios da Diferenciação do Sexo, pp. 279-295. , In: Guerra ATM, Guerra Júnior G, editors, 2. ed. RubioHutson, J.M., Davidson, P.M., Reece, L.A., Baker, M., Zhou, B., Failure of gubernacular development in the persistent Müllerian duct syndrome allows herniation of the testes (1994) Pediatr Surg Int, 9, pp. 544-546Abduljabbar, M., Taheini, K., Picard, J.Y., Cate, R.L., Josso, N., Mutations of the AMH Type II receptor in two extended families with persistent Müllerian duct syndrome: Lack of phenotype/genotype correlation (2012) Horm Res Paediatr, 77 (5), pp. 291-297Imbeaud, S., Faure, E., Lamarre, I., Mattei, M.G., Di Clemente, N., Tizard, R., Insensitivity to anti-Müllerian hormone due to a mutation in the human anti-Müllerian hormone receptor (1995) Nat Genet, 11 (4), pp. 382-388Cate, R.L., Mattaliano, R.J., Hession, C., Tizard, R., Farber, N.M., Cheung, A., Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells (1986) Cell, 45 (5), pp. 685-698Josso, N., Di Clemente, N., TGF-beta family members and gonadal development (1999) Trends Endocrinol Metab, 10 (6), pp. 216-222Josso, N., Belville, C., Di Clemente, N., Picard, J.Y., AMH and AMH receptor defects in persistent Müllerian duct syndrome (2005) Hum Reprod Update, 11 (4), pp. 351-356Menabo, S., Balsamo, A., Nicoletti, A., Gennari, M., Pirazzoli, P., Cicognani, A., Three novel AMH gene mutations in a patient with persistent Müllerian duct syndrome and normal AMH serum dosage (2008) Horm Res, 70 (2), pp. 124-128van der Zwan, Y.G., Bruggenwirth, H.T., Drop, S.L., Wolffenbuttel, K.P., Madern, G.C., Looijenga, L.H., A novel AMH missense mutation in a patient with persistent Müllerian duct syndrome (2012) Sex Dev, , Epub 2012 Jul 11Baarends, W.M., van Helmond, M.J., Post, M., van der Schoot, P.J., Hoogerbrugge, J.W., de Winter, J.P., A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the Müllerian duct (1994) Development, 120 (1), pp. 189-197Di Clemente, N., Wilson, C., Faure, E., Boussin, L., Carmillo, P., Tizard, R., Cloning, expression, and alternative splicing of the receptor for anti-Müllerian hormone (1994) Mol Endocrinol, 8 (8), pp. 1006-1020Belville, C., Marechal, J.D., Pennetier, S., Carmillo, P., Masgrau, L., Messika-Zeitoun, L., Natural mutations of the anti-Müllerian hormone type II receptor found in persistent Müllerian duct syndrome affect ligand binding, signal transduction and cellular transport (2009) Hum Mol Genet, 18 (16), pp. 3002-3013Miller, S.A., Dykes, D.D., Polesky, H.F., A simple salting out procedure for extracting DNA from human nucleated cells (1988) Nucleic Acids Res, 16 (3), p. 1215Imbeaud, S., Carre-Eusebe, D., Rey, R., Belville, C., Josso, N., Picard, J.Y., Molecular genetics of the persistent Müllerian duct syndrome: A study of 19 families (1994) Hum Mol Genet, 3 (1), pp. 125-131Josso, N., Picard, J.Y., Rey, R., Di Clemente, N., Testicular anti-Müllerian hormone: History, genetics, regulation and clinical applications (2006) Pediatr Endocrinol Rev, 3 (4), pp. 347-358Rey, R.A., Codner, E., Iniguez, G., Bedecarras, P., Trigo, R., Okuma, C., Low risk of impaired testicular Sertoli and Leydig cell functions in boys with isolated hypospadias (2005) J Clin Endocrinol Metab, 90 (11), pp. 6035-6040Imbeaud, S., Belville, C., Messika-Zeitoun, L., Rey, R., Di Clemente, N., Josso, N., A 27 base-pair deletion of the anti-Müllerian type II receptor gene is the most common cause of the persistent Müllerian duct syndrome (1996) Hum Mol Genet, 5 (9), pp. 1269-127

Novel Mutations In Cyp11b1 Gene Leading To 11β-hydroxylase Deficiency In Brazilian Patients

Background: Deficiency of 11β-hydroxylase results in the impairment of the last step of cortisol synthesis. In females, the phenotype of this disorder includes different degrees of genital ambiguity and arterial hypertension. Mutations in the CYP11B1 gene are responsible for this disease. Objective: The objective of the study was to screen the CYP11B1 gene for mutations in two unrelated Brazilian females with congenital adrenal hyperplasia due to 11β-hydroxylase deficiency. Design: The coding and intron-exon junction regions of CYP11B1 were totally sequenced. A putative splice mutation was further investigated by minigene transcription. Results: We report two novel CYP11B1 mutations in these Brazilian patients. An Arabian Lebanese descendent female was found to be homozygous for a cytosine insertion at the beginning of exon 8, changing the 404 arginine to proline. It alters the open reading frame, creating a putative truncated protein at 421 residue, which eliminates the domain necessary for the association of heme prosthetic group. A severely virilized female was homozygous for the g.2791G>A transition in the last position of exon 4. This nucleotide is also part of 5′ intron 4 donor splice site consensus sequence. Minigene experiments demonstrated that g.2791G>A activated an alternative splice site within exon 4, leading to a 45-bp deletion in the transcript. The putative translation of such modified mRNA indicates a truncated protein at residue 280. Conclusions: We describe two novel mutations, g.4671-4672insC and g.2791G>A, that drastically affects normal protein structure. These mutations abolish normal enzyme activity, leading to a severe phenotype of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency. Copyright © 2009 by The Endocrine Society.94934813485White, P.C., Curnow, K.M., Pascoe, L., Disorders of steroid 11β- hydroxylase isoenzymes (1994) Endocr Rev, 15, pp. 421-438White, P.C., Speiser, P.W., Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (2000) Endocrine Reviews, 21 (3), pp. 245-291. , DOI 10.1210/er.21.3.245Mornet, E., Dupont, J., Vitek, A., White, P.C., Characterization of two genes encoding human steroid 11β-hydroxylase (P-450(11β)) (1989) Journal of Biological Chemistry, 264 (35), pp. 20961-20967Spoudeas, H.A., Slater, J.D., Rumsby, G., Honour, J.W., Brook, C.G., Deoxycorticosterone, 11β-hydroxylase and the adrenal cortex (1993) Clin Endocrinol, 39, pp. 245-251. , OxfHague, W., Honour, J., Malignant hypertension in congenital adrenal hyperplasia due to 11β-hydroxylase deficiency (1983) Clin Endocrinol, 18, pp. 505-510. , OxfKrawczak, M., Cooper, D.N., The human gene mutation database (1997) Trends Genet, 13, pp. 121-122Chabre, O., Portrat-Doyen, S., Vivier, J., Morel, Y., Defaye, G., Two novel mutations in splice donor sites of CYP11B1 in congenital adrenal hyperplasia due to 11β-hydroxylase deficiency (2000) Endocrine Res, 26, pp. 797-801Curnow, K.M., Slutsker, L., Vitek, J., Cole, T., Speiser, P.W., New, M.I., White, P.C., Pascoe, L., Mutations in the CYP11B1 gene causing congenital adrenal hyperplasia and hypertension cluster in exons 6, 7, and 8 (1993) Proc Natl Acad Sci USA, 90, pp. 4552-4556Skinner, C.A., Rumsby, G., Honour, J.W., Single strand conformation polymorphism (SSCP) analysis for the detection of mutations in the CYP11B1 gene (1996) Journal of Clinical Endocrinology and Metabolism, 81 (6), pp. 2389-2393. , DOI 10.1210/jc.81.6.2389De Carvalho, C.E., Castro, M., Moreira, A.C., De Mello, M.P., CYP11B1 mutation and polymorphisms in congenital adrenal hyperplasia due to 11β-hydroxylase deficiency (1999) J Endocr Genet, 1, pp. 79-86Moreira, A.C., Elias, L.L.K., Pituitary-adrenal responses to corticotropin- releasing hormone in different degrees of adrenal 21-hydroxylase deficiency (1992) J Clin Endocrinol Metab, 74, pp. 198-203Mermejo, L.M., Elias, L.L.K., Marui, S., Moreira, A.C., Mendonca, B.B., De Castro, M., Refining hormonal diagnosis of type II 3β-hydroxysteroid dehydrogenase deficiency in patients with premature pubarche and hirsutism based on HSD3B2 genotyping (2005) Journal of Clinical Endocrinology and Metabolism, 90 (3), pp. 1287-1293. , DOI 10.1210/jc.2004-1552De-Araujo, M., Sanches, M.R., Suzuki, L.A., Guerra Jr., G., Farah, S.B., De-Mello, M.P., Molecular analysis of CYP21 and C4 genes in Brazilian families with the classical form of steroid 21-hydroxylase deficiency (1996) Brazilian Journal of Medical and Biological Research, 29 (1), pp. 1-13Soardi, F.C., Lemos-Marini, S.H.V., Coeli, F.B., Maturana, V.G., Silva, M.D., Bernardi, R.D., Justo, G.Z., De Mello, M.P., Heterozygosis for CYP21A2 mutation considered as 21-hydroxylase deficiency in neonatal screening (2008) Arq Bras Endocrinol Metab, 52, pp. 1388-1392White, P.C., Slutsker, L., Haplotype analysis of CYP11B2 (1995) Endocr Res, 21, pp. 437-442Ravichandran, K.G., Boddupalli, S.S., Hasemann, C.A., Peterson, J.A., Deisenhofer, J., Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's (1993) Science, 261 (5122), pp. 731-736Roumen, L., Sanders, M.P.A., Pieterse, K., Hilbers, P.A.J., Plate, R., Custers, E., De Gooyer, M., Hermans, J.J.R., Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics (2007) Journal of Computer-Aided Molecular Design, 21 (8), pp. 455-471. , DOI 10.1007/s10822-007-9128-9Mount, S.M., A catalogue of splice junction sequences (1982) Nucleic Acids Res, 10, pp. 459-472Buratti, E., Chivers, M., Královicová, J., Romano, M., Baralle, M., Krainer, A.R., Vorechovsky, I., Aberrant 5′ splice sites in human disease genes: Mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization (2007) Nucleic Acids Res, 35, pp. 4250-426

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