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Thèse de Doctorat
DOI
10.11606/T.5.2008.tde-24112008-113934
Document
Auteur
Nom complet
Milena Gurgel Teles Bezerra
Adresse Mail
Unité de l'USP
Domain de Connaissance
Date de Soutenance
Editeur
São Paulo, 2008
Directeur
Jury
Xavier, Ana Claudia Latrônico (Président)
Abucham Filho, Júlio Zaki
Bronstein, Marcello Delano
Damiani, Durval
Trarbach, Ericka Barbosa
Titre en portugais
Estudo do gene GPR54 nos distúrbios puberais centrais idiopáticos
Mots-clés en portugais
Hipogonadismo hipogonadotrófico isolado
Mutações ativadoras
Mutações inativadoras
Puberdade precoce dependente de gonadotrofinas
Receptor GPR54
Retardo constitucional do crescimento e desenvolvimento
Resumé en portugais
O complexo de sinalização kisspeptina-GPR54 é um regulador chave para ativação dos neurônios de GnRH e do eixo reprodutivo. Mutações inativadoras no GPR54 foram identificadas em pacientes com hipogonadismo hipogonadotrófico normósmico isolado (HHIn). A partir desse achado, hipotetizamos que mutações ativadoras no GPR54 resultariam na liberação prematura de GnRH e, conseqüentemente, no aparecimento de puberdade precoce, dependente de gonadotrofinas (PPDG). No presente estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos em pacientes com PPDG, assim como a presença de mutações inativadoras e/ou polimorfismos em pacientes HHIn ou retardo constitucional do crescimento e desenvolvimento puberal (RCCP). Cento e catorze pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 53 com PPDG, 33 com HHIn e 28 com RCCP. Cento e cinqüenta controles brasileiros que relatavam desenvolvimento puberal normal foram estudados. A região codificadora do GPR54 de todos os pacientes foi amplificada utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e seqüenciamento automático. No grupo de puberdade precoce, identificamos uma nova variante em heterozigose no exon 5 do GPR54, que se caracterizou pela troca do aminoácido arginina por prolina na posição 386 (R386P) do receptor. Esta substituição foi encontrada em uma menina adotada com PPDG e estava ausente nos controles normais. Estudos in vitro demonstraram que as quantidades de fosfatidil-inositol (IP) e o grau de fosforilação da quinase regulada por sinal extracelular (pERK) em condições basais não foram significativamente diferentes entre as células transfectadas com o receptor selvagem ou com o receptor contendo a mutação R386P, indicando que não havia ativação constitutiva do receptor. No entanto, estudos por tempos mais prolongados demonstraram que a quantidade de IP e o grau de pERK permaneceram significativamente mais altos nas células transfectadas com o receptor mutante quando comparadas ao selvagem, indicando ativação da sinalização intracelular, porém por um mecanismo não-constitutivo. No grupo de hipogonadismo, duas novas variantes foram identificadas em três pacientes. Uma mutação do tipo inserção/deleção (indel) em homozigoze no sítio aceptor de splicing no intron 2 (IVS2-4_-2delGCAinsACCGGCT) do GPR54 foi identificada em dois irmãos com HHIn. Uma troca em heterozigose, E252Q, foi identificada em um paciente com HHIn esporádico. As duas alterações estavam ausentes no grupo controle. Polimorfismos foram encontrados nos pacientes com RCCP. Em conclusão, descrevemos a primeira mutação ativadora do GPR54 associada ao fenótipo de PPDG. Descrevemos uma nova mutação inativadora em sítio de splicing em pacientes com HHIn, entretanto mutações inativadoras do GPR54 são uma causa rara de HHIn.
Titre en anglais
GPR54 gene analysis in patients with idiopathic central pubertal disorders
Mots-clés en anglais
Activating mutations
Constitutional delay of puberty
Gonadotropin-dependent precocious puberty
GPR54 receptor
Inactivating mutations
Normosmic isolated hypogonadotropic hypogonadism
Resumé en anglais
The kisspeptin-GPR54 signaling complex is a gatekeeper of pubertal activation of GnRH neurons and of the reproductive axis. Inactivating mutations in the GPR54 receptor were identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). Based on this observation, we hypothesized that gain-of-function mutations of the human GPR54 receptor might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In the present study, we investigated the presence of GPR54 activating mutations or polymorphisms in patients with GDPP and inactivating mutations or polymorphisms in patients with nIHH or constitucional delay of puberty (CDP). A hundred fourteen patients were selected; 53 with GDPP, 33 with nIHH and 28 with CDP. A hundred and fifty Brazilian controls who reported normal pubertal development were also studied. The entire coding region of GPR54 of all patients was amplified using specific intronic oligonucleotides followed by enzymatic purification and automated sequencing. We have identified a novel variant in heterozygous state in exon 5 of GPR54, R386P, in an adopted girl with GDPP. This substitution was absent in all controls. Basal inositol phosphate (IP) and phosphorilated extracellular signalregulated kinase (pERK) levels in cells transfected with WT or R386P GPR54 were not significantly different indicating that there was not a constitutive activation of the receptor. However, studies performed in more prolonged times demonstrated that the IP and the pERK levels were significantly higher in cells transfected with the mutant receptor when compared to the wild type, indicating that the signaling pathway was still activated although by a non-constitutive mechanism. In the nIHH cohort, we have identified two novel variants in three patients. The first variant was an insertion/deletion (indel) in homozygous state within the constitutive acceptor splice site of intron 2 of GPR54 (IVS2-4_-2delGCAinsACCGGCT) identified in two male siblings with nIHH. The second variant was the change E252Q in heterozygous state in a patient with sporadic nIHH. Both alterations were absent in the control population. We have found only polymorphisms in patients with CDP. In conclusion, we have described the first activating mutation in GPR54 associated with the GDPP phenotype. We have also described a novel splice site inactivating mutation in patients with nIHH however, inactivating mutations of GPR54 represent a rare cause of nIHH.
 
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Date de Publication
2008-12-15
 
AVERTISSEMENT: Le matériau se réfère à des documents provenant de cette thèse ou mémoire. Le contenu de ces documents est la responsabilité de l'auteur de la thèse ou mémoire.
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