Validation of a robust PCR-based assay for quantifying fragile X CGG repeats

Sizing of FMR1 trinucleotide repeats in the clinical laboratory requires the use of capillary sequencer by PCR, or by a labor intensive measurement using Southern blot method. Our aim was to validate an accurate and robust PCR assay for quantification of CGG repeats.We performed an analytical and clinical validation of a new PCR-based method that utilizes a low-cost capillary electrophoresis instrument and the FragilEase™ reagent kit. First, analytical performance was demonstrated on 12 Coriell reference samples comprising normal through full mutations. Subsequently, a cohort of 112 archived clinical DNA samples, enriched for premutation and full mutations, was analyzed.All samples were amplified successfully. Quantification of repeat numbers was interpreted by the use of standards with known repeats. Twenty-five full-mutation samples were successfully amplified with the largest allele size measured at 1380 repeats. The repeat numbers from the new assay were concordant with those obtained with the reference method. The intra-assay (CV<2.5%) and inter-assay imprecision was within 1 CGG repeat.This new PCR-based method is reproducible and capable of identifying all Fragile X alleles. It is an accurate and robust method that facilitates Fragile X testing in a broader spectrum of clinical laboratories.