High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes

Methylated FMR1 full-mutation expansions cause fragile X syndrome. FMR1 premutation carriers are susceptible to other late-onset conditions, and women with premutation are at risk of transmitting a fully expanded FMR1 allele to offspring. Identification of individuals with actionable FMR1 genotypes (full-mutation males and females, and premutation females at risk for primary ovarian insufficiency and/or having fragile X–affected offspring) can enable timely access to intervention services and genetic counseling. This study presents a rapid, first-tier test based on melting curve analysis of methylation-specific triplet-primed PCR amplicons (msTP-PCR MCA) for concurrent detection of FMR1 CGG-repeat expansions and their methylation status. The msTP-PCR MCA assay was optimized on 20 fragile X reference samples, and its performance was evaluated on 111 peripheral blood-derived DNA samples from patients who have undergone prior molecular testing with PCR and/or Southern blot analysis. The msTP-PCR MCA assay detected all samples with a methylated FMR1 CGG-repeat expansion, and had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 92.06%, 91.1%, and 100%, respectively. The msTP-PCR MCA assay identified premutation/full-mutation mosaicism down to 1%, detected skewed inactivation in females with FMR1 expansions, and enabled selective identification of all individuals with an actionable FMR1 genotype. The msTP-PCR MCA assay may aid in fragile X screening of at-risk populations and newborns and voluntary carrier screening of women of reproductive age.