Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia

Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease, is associated with an unstable expansion of a GAA trinucleotide repeat in the first intron of the frataxin gene on chromosome 9q13. Unequivocal molecular characterization of the FRDA triplet expansion requires the use of different PCR protocols to amplify normal and mutated alleles combined with Southern blotting analysis to accurately size the expansion. Nevertheless, expansion detection by PCR may be somewhat problematic in heterozygous individuals. The purpose of this study was to evaluate triplet repeat primed PCR (TP PCR) as a screening method for FRDA diagnosis in the diagnostic laboratory. Fifty-four cases referred either to confirm the diagnosis of FRDA or to detect carrier status were re-evaluated by the TP PCR method. The TP PCR assay correctly identified the FRDA status in all 54 individuals tested including homozygous expansions (9 individuals), heterozygous expansions (20 individuals), and non-carriers (25 individuals). Results showed 100% concordance with those obtained by Southern blot analysis. TP PCR allowed us to identify the expanded alleles or to demonstrate their absence in DNA samples where conventional PCR procedures failed to give a reliable result. TP PCR represents an additional valuable tool for mutation detection in FRDA patients and carriers, but also can be used as screening test in a diagnostic laboratory.