Enter Note Done


Keywords: Ataxia; Polyglutamine diseases; Triplet and other repeats; Diagnostics; Clinical testing

M. Deshotel 1; M. Jama 1; G. Pont-Kingdon 1; P. Bayrak-Toydemir 1,2

1) Molecular Genetics, ARUP Laboratories, Salt Lake City, UT; 2) Department of Pathology, University of Utah, Salt Lake City, UT

Spinocerebellar ataxias (SCAs) belong to a family of neurological diseases that lead to progressive cerebellar atrophy and eventual loss of peripheral motor functions. Of the roughly 40 hereditary SCAs described to date, the subtypes SCA1 [MIM 164400], SCA2 [MIM 183090], Machado-Joseph disease/SCA3 [MIM 109150], SCA6 [MIM 183086], and SCA7 [MIM 164500] are the most common autosomal dominant ataxias worldwide and are caused by pathogenic expansion of exonic CAG repeats in SCA-associated genes. Symptoms of these five subtypes are clinically heterogeneous, and de novo mutations are known to arise, so molecular genetic testing is required for differentiation. Here, we describe the development of a test for the quantification of CAG repeats in the SCA-associated genes ATAXIN1 (SCA1), ATAXIN2 (SCA2), ATAXIN3 (SCA3), CACNA1A (SCA6), and ATAXIN7 (SCA7). Quantification of CAG repeats was performed using a chimeric triplet repeat-primed PCR method, which was originally developed at ARUP Laboratories for detection of repeat expansions in Fragile X, Huntington disease, and myotonic dystrophy-1. Forward primers were designed with HEX, NED, and FAM fluorescent dyes to facilitate multiplexing, and reverse primers were designed with a CAG5 repeat region and flanking region. The amplicons were separated by capillary electrophoresis, and the characteristic ladder of peaks counted to determine the number of CAG repeats in each allele. Repeats in all five SCA-associated genes can be quantified in just two multiplex PCR reactions, an improvement over previously published methods. Our method also allows detection of CAT interruptions in ATAXIN1 and CAA interruptions in ATAXIN2, both of which have clinical utility for patients. We have detected CAG expansions with as many as 60 (SCA1), 44 (SCA2), 72 (SCA3), 22 (SCA6), and 62 repeats (SCA7) in patient samples, but our method is expected to reveal even larger pathogenic expansions. To verify correct repeat allele sizing, deidentified normal patient samples with homozygous alleles were confirmed by Sanger sequencing. Nine samples from the Coriell Institute and three CAP proficiency testing samples were also tested to verify correct repeat allele sizing. These results indicate that the triplet repeat-primed PCR technique is a robust and flexible method that allows for accurate quantification of CAG repeats and CAA/CAT interruptions in SCA-associated genes.