Protocol for the Characterization of the CAG Tract and Flanking Polymorphisms in Machado-Joseph Disease: Impact on Diagnosis and Development of Gene-Based Therapies.

Abstract Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. A definitive diagnosis relies on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide CAG in coding exons. The assessment of single nucleotide polymorphisms (SNPs) associated with the CAG expansion in the context of SCAs is also relevant for the improvement of molecular diagnosis and for the generation of novel therapeutic strategies. Here we focused on Machado-Joseph disease (MJD)/SCA3, aiming to develop 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 SNPs 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 MJD/SCA3 and may prove its utility when applied to other polyglutamine SCAs.