A Comprehensive Triple-Repeat Primed PCR and a Long-Range PCR Agarose-Based Assay for Improved Genotyping of Guanine-Adenine-Adenine Repeats in Friedreich Ataxia.

ABSTRACT

Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide [guanine-adenine-adenine (GAA)] repeat in intron 1 of the FXN gene. It is common in the White population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease. A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles <200 GAA repeats (±1 to 5 repeats) and an agarose gel-based, long-range PCR (LR-PCR) assay to genotype expanded alleles >200 GAA repeats (±50 repeats) is described. Of the 1236 DNA samples tested using TR-PCR, 31 were identified to have expanded alleles >200 repeats and were reflexed to the LR-PCR procedure for confirmation and quantification. The TR-PCR assay described herein is a diagnostic genotyping assay that reduces the need for further testing. The LR-PCR component is a confirmatory test for true homozygous and heterozygous samples with normal and expanded alleles, as indicated by the TR-PCR assay. The use of this two-tier method offers a comprehensive evaluation to detect and genotype the smallest and largest number of GAA repeats, improving the classification of FXN alleles as normal, mutable normal, borderline, and expanded alleles.