Pesquisa de variantes patogênicas no gene gnal em pacientes com distonia isolada idiopática

• Ano de defesa: 2016
• Autor(a) principal: Alves, Camila Oliveira dos Santos [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Dystonia; Genetics; Gnal; Dyt25; Gaolf; Distonia; Genética
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3907222; http://repositorio.unifesp.br/handle/11600/46677

Introduction: Due to advancement of Next Generation Sequencing technologies, new genes have been identified as dystonia causes. GNAL gene was recently identified and characterized as a new genetic cause represented by the DYT-GNAL (DYT-25) locus. This gene encodes α subunit of the stimulatory G protein (Gαolf) which is highly expressed in striatum, and acts in the signal transduction mediated by D1 dopamine and A2A adenosine receptors. Prevalence of GNAL variants in dystonia was initially reported as 15% in individuals of European ethnicity, which would have made of this gene a common cause of dystonia. However, studies conducted in Asian and European populations reported frequencies below of 2.6%. Therefore, it is necessary to collect more data to determine the prevalence of pathogenic variants in GNAL in patients with dystonia in other populations. Objective: The aim of this study was to standardize the molecular analysis of the GNAL gene by Sanger sequencing technique and investigation of the prevalence of variants in this gene as the cause of dystonia in a group of Brazilian patients. Methods: Ninety-one patients with isolated idiopathic dystonia, negative for mutations in THAP1 and TOR1A genes, were investigated for variants in GNAL by Sanger sequencing. Non-synonymous variants identified in patients were also investigated in 234 chromosomes from healthy individuals and their frequencies were compared to the populational database ExAC (Exome Aggregation Consortium). For in silico analysis the Mutation taster, Polyphen-2, PROVEAN, SIFT and CADD algorithms were used. To verify the degree of conservation of the amino acid throughout evolution the alignment of protein sequences of different species was performed with the ClustalW software. To investigate the pathogenicity of a novel non- synonymous variant identified in a dystonia patient, a functional assay was performed by Bioluminescence Resonance Energy Transfer (BRET) assay. Results: One novel non-synonymous variant (p.F133L) was identified in a female patient with cervical and laryngeal dystonia since the third decade of life, with no family history. This variant was not found in 234 chromosomes of Brazilian healthy controls and was not described in 63.000 exomes of the public database ExAC. In silico Analysis by Mutation Taster, PROVEAN, Polyphen-2 and CADD algorithms demonstrated that this variant is deleterious, consistent with result of functional analyses of the mutated Gαolf protein obtained by BRET assay, which demonstrated that this variant leads to the increase of basal BRET signal and to the decrease of the response elicited by dopamine, demonstrating a considerable impairment of protein function in signal transduction. Conclusion: Pathogenic variants in GNAL are cause of dystonia among Brazilians, and are less frequent than variants in THAP1 and TOR1A (1.09%, 2.6% and 8.8%, respectively). In this group of patients we identified a case with typical phenotype, however, with no family history. The absence of family history does not exclude the genetic cause, as this could be due to a de novo mutation or a gene with incomplete penetrance. We identified a novel variant (p.F133L) and confirmed that it leads to partial loss of function of G?olf related to signal transduction of dopaminergic pathways, which may contribute to the pathophysiology of dystonia. The elucidation of these and of others physiological mechanisms will enable the development of more effective treatments for dystonia.