Long range polymerase chain reaction-based diagnosis of friedreich ataxia in the clinical molecular diagnostics laboratory

Background: Friedreich ataxia, an autosomal recessive neurodegenerative disease, is one of the recently identified trinucleotide repeat disorders. Approximately 94% of affected individuals are homozygous for an intronic GAA repeat within the frataxin gene. The identification of this trinucleotide expansion as the causative mutation in the majority of affected individuals has resulted in the availability of molecular testing for Friedreich ataxia in the clinical molecular diagnostics laboratory. Methods and Results: A Friedreich ataxia protocol has been implemented using two long-range polymerase chain reaction-based assays primed with the GAA-F/GAA-R and Bam/2500F primer sets [1]. The amplified products are analyzed on precast minigels and visualized by Sybr Green staining. The GAA primer set, which produces a normal product of approximately 500 bp and an expanded product of greater than 800 bp, is used to approximate the size range of expanded repeats. Because the GAA primer set occasionally demonstrates preferential amplification of the normal allele in heterozygotes, the Bam/2500F primer set is used to verify results. Conclusions: This protocol has been used to analyze 42 specimens from patients about whom we had detailed clinical information. Concordant results obtained from the GAA and Bam/2500F primer sets, as well as correlations between clinical information and molecular results, indicate that, for the majority of patient specimens, this protocol allows rapid analysis and sizing of GAA repeats in normal and expanded alleles.