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Doctoral Thesis
DOI
10.11606/T.5.2012.tde-03092012-090655
Document
Author
Full name
Cíntia Tusset
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2012
Supervisor
Committee
Xavier, Ana Claudia Latrônico (President)
Abucham Filho, Júlio Zaki
Guerra Júnior, Gil
Jorge, Alexander Augusto de Lima
Trarbach, Ericka Barbosa
Title in Portuguese
Pesquisa de mutações na neurocinina B e no seu receptor em pacientes com distúrbios puberais centrais idiopáticos
Keywords in Portuguese
Hipogonadismo
Hormônio liberador de gonadotrofinas
Mutação
Neurocinina B
Puberdade precoce
Receptor da neurocinina B
Retardo constitucional do crescimento e desenvolvimento
Abstract in Portuguese
Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmico
Title in English
Analysis of mutations in the neurokinin B and its receptor in patients with idiopathic central pubertal disorders
Keywords in English
Constitutional delay of growth and puberty
Gonadotropin- releasing hormone
Hypogonadism
Mutation
Neurokinin B
Neurokinin B receptor
Precocious puberty
Abstract in English
Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH
 
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Publishing Date
2012-09-05
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • Beneduzzi, D., et al. Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism [doi:10.1530/EJE-11-0199]. European Journal of Endocrinology [online], 2011, vol. 165, p. 145-150.
  • Montenegro, L.R., et al. Combined use of multiplex ligation-dependent probe amplification and automatic sequencing for identification of KAL1 defects in patients with Kallmann syndrome [doi:10.1016/j.fertnstert.2013.04.045]. Fertility and Sterility [online], 2013, vol. 100, p. 854-859.
  • Noel, S.D., et al. TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients [doi:10.1096/fj.13-240630]. The FASEB Journal [online], 2014, vol. 28, p. 1924-1937.
  • Tusset, C., et al. Mutational analysis of TAC3 and TACR3 genes in patients with idiopathic central pubertal disorders [doi:10.1590/s0004-27302012000900008]. Arquivos Brasileiros de Endocrinologia e Metabologia [online], 2012, vol. 56, p. 646-652.
  • Tusset, C., et al. Clinical and molecular analysis of TAC3 and TACR3 genes in brazilian Patients with isolated hypogonadotropic hypogonadism. In The Endocrine Society, Boston, 2011. Program & Abstracs., 2011. Abstract.
  • Tusset, C., et al. Influence of FSH receptor genotype on ovarian response to FSH in brazilian women undergoing assisted reproduction. In The Endocrine Society, Boston, 2011. Program & Abstracts., 2011. Abstract.
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