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 analysisepositive 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%e100%), whereas the SCA2 and SCA3 assays achieved specificity of 99.46% (95% CI, 96.56%e99.97%) and 99.32% (95% CI, 95.70%e99.96%), 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.

Triplet-primed PCR and Melting Curve Analysis for Rapid Molecular Screening of Spinocerebellar Ataxia Types 1, 2, and 3

“…The SCA1, SCA2, and SCA3 TP-PCR employs locus-specific flanking and TP primers and a common Tail primer, which together generate multiple products, each differing by one CAG repeat ( Lian et al, 2021 ). The shorter TP-PCR products generated from an allele with smaller CAG repeat size produce melt peaks with lower T m , whereas the longer TP-PCR products generated from an allele with larger CAG repeat size yield melt peaks with higher T m .…”

“…This work was supported by internal departmental funds. This protocol was derived from the original research paper ( Lian et al, 2021 ).…”

A comprehensive review of iPS cell line-based disease modelling of the polyglutamine spinocerebellar ataxias 2 and 3: a focus on the research outcomes

“…To improve the cost-effectiveness parameter, a screening technique for SCA subtypes 1, 2, and 3 was developed by coupling the TP-PCR and Melting Curve Analysis (MCA) techniques, allowing for 100% assay sensitivity in the detection of the expanded repeats in the disease proteins Ataxin-1, Ataxin-2, and Ataxin-3 involved in SCAs 1, 2, and 3, respectively. Moreover, the specificity of these assays was near absolute, with the SCA2 and SCA3 assays being 99.46% and 99.32% specific, respectively 25 . The significance of these new techniques is paramount in the pre-symptomatic diagnosis of genetic diseases to facilitate better family planning options for at-risk individuals and to aid them in better preparation for future ailments 26 .…”