Protocol for the characterization of the Cytosine-Adenine-Guanine tract and flanking polymorphisms in Machado-Joseph disease: impact on Diagnosis and Development of Gene-Based Therapies

Sara M. Lopes, Rosário Faro, Miguel M. Lopes, Isabel Onofre, Nuno Mendonça, Joana Ribeiro, Cristina Januário, Rui Jorge Nobre*, Luís Pereira de Almeida

*Autor correspondente para este trabalho

Resultado de pesquisarevisão de pares

1 Citação (Scopus)

Resumo

Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. Definitive diagnoses rely on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons. Assessment of single nucleotide polymorphisms associated with the CAG expansion in the context of SCAs is also relevant for improving molecular diagnosis and for generating novel therapeutic strategies. The current study is focused on Machado-Joseph disease/SCA type 3, with the aim of developing a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three single nucleotide polymorphisms in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118, and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis, and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of Machado-Joseph disease/SCA type 3 and may prove useful when applied to other polyglutamine SCAs.
Idioma originalEnglish
Páginas (de-até)782-793
Número de páginas12
RevistaJournal of Molecular Diagnostics
Volume22
Número de emissão6
DOIs
Estado da publicaçãoPublicado - jun. 2020
Publicado externamenteSim

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