A repeat-primed PCR assay for pentanucleotide repeat alleles in spinocerebellar ataxia type 37

Spinocerebellar ataxia 37 (SCA37) is caused by an (ATTTC)_n insertion in a polymorphic ATTTT repeat in the non-coding region of DAB1. The non-pathogenic alleles have a configuration [(ATTTT)_7-400], whereas pathogenic alleles have a complex structure of [(ATTTT)_60-79(ATTTC)_31-75(ATTTT)_58-90]. Molecular diagnosis of SCA37 is laborious because about 7% of the pentanucleotide repeat alleles in DAB1 are larger than 30 units and, thus, fail to amplify with standard PCR conditions, resulting in apparently homoallelism or in complete lack of PCR amplification in several cases. The molecular test currently available requires long-range PCR and sequencing analysis for the detection and characterization of these large alleles. We developed a simple assay capable of rapidly detecting the presence or absence of large pentanucleotide repeat sizes. This assay is based on repeat-primed PCR followed by high-throughput capillary electrophoresis. Combining the standard PCR with RP-PCR allows completion of the diagnosis in more than 80% of individuals, minimizing the number of samples that require long-range PCR followed by Sanger sequencing analysis. This assay meets many of the requirements for pre-screening of large cohorts of affected individuals.