Rapid Molecular Screen of Spinocerebellar Ataxia Types 1, 2, and 3 by Triplet-Primed PCR and Melting Curve Analysis

Abstract

The autosomal dominantly inherited spinocerebellar ataxias (SCAs) can be caused by dynamic mutations of short tandem repeats within various genes. Because of the significant clinical overlap among the various SCA types, molecular screening of multiple genetic loci by fluorescent PCR and capillary electrophoresis is necessary to identify the causative repeat expansion. We describe a simple, rapid, and inexpensive strategy to screen for CAG repeat expansion mutations at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Plasmid DNAs of known repeat sizes were used to generate threshold melt peak temperatures, which rapidly and effectively distinguish samples carrying an expanded allele from those carrying nonexpanded alleles. Melting curve analysis–positive samples were confirmed by capillary electrophoresis sizing of the triplet-primed PCR products. All three assays achieved 100% sensitivity, with 95% CIs of 67.86% to 100% (SCA1), 74.65% to 100% (SCA2), and 91.58% to 100% (SCA3). The SCA1 assay also achieved 100% specificity (95% CI, 97.52%–100%), whereas the SCA2 and SCA3 assays achieved specificity of 99.46% (95% CI, 96.56%–99.97%) and 99.32% (95% CI, 95.70%–99.96%), respectively. These screening assays provide robust and highly accurate detection of expanded alleles and are amenable to large-scale screening while minimizing the need for capillary electrophoresis sizing for every sample.