Robust and accurate detection and sizing of repeats within the DMPK gene using a novel TP-PCR test.

Myotonic dystrophy type 1 is a multisystem disorder caused by the expansion of a trinucleotide repeat in the DMPK gene. In this study we evaluated the performance of the FastDM1TM DMPK sizing kit in myotonic dystrophy type 1 testing. This commercially available triplet repeat-primed PCR based kit was validated using reference and clinical samples. Based on testing with 19 reference samples, the assay yielded repeat sizes within three repeats from the consensus reference length, demonstrating an accuracy of 100%. Additionally, the assay generated consistent repeat size information with a concentration range of template-DNA, and upon repetition and reproduction (CV 0.36% to 0.41%). Clinical performance was established with 235 archived prenatal and postnatal clinical samples, yielding results of 100% sensitivity (95% CI, 97.29% to 100%) and 100% specificity (95% CI, 96.19% to 100%) in classifying the samples into the respective genotype groups of 5-35 (normal), 36-50 (non-pathogenic pre-expansion), 51-150 (unstable intermediate-sized pathogenic) or >150 (unstable pathogenic) CTG repeats, respectively. Furthermore, the assay identified interrupted repeat expansions in all samples known to have interruptions, and also identified interruptions in a subset of the clinical samples.

Validation of a commercially available test that enables the quantification of the numbers of CGG trinucleotide repeat expansion in FMR1 gene.

In the present study, we evaluated a commercially available TP-PCR-based assay, the FastFraXTM FMR1 Sizing kit, as a test in quantifying the number of CGG repeats in the FMR1 gene. Based on testing with well characterized DNA samples from Coriell, the kit yielded size results within 3 repeats of those obtained by common consensus (n = 14), with the exception of one allele. Furthermore, based on data obtained using all Coriell samples with or without common consensus (n = 29), the Sizing kit was 97.5% in agreement with existing approaches. Additionally, the kit generated consistent size information in repeatability and reproducibility studies (CV 0.39% to 3.42%). Clinical performance was established with 198 archived clinical samples, yielding results of 100% sensitivity (95% CI, 91.03% to 100%) and 100% specificity (95% CI, 97.64% to 100%) in categorizing patient samples into the respective normal, intermediate, premutation and full mutation genotypes.