Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis as a Rapid Screening Tool for Identifying Actionable FMR1 Genotypes

To the Editor:Full mutation (FM)1 and premutation (PM) expansions of the fragile X mental retardation 1 ( FMR1 ) CGG repeat are the underlying causes of 3 FMR1 -related disorders: fragile X syndrome (FXS), fragile X–associated primary ovarian insufficiency (FXPOI), and fragile X–associated tremor/ataxia syndrome (FXTAS) (1). PM and FM females are also at risk of conceiving FXS-affected offspring.Molecular diagnosis of FMR1 -related disorders involves both repeat length and methylation state determination. The advantages and barriers to newborn, early childhood, and carrier screening have been debated at length, and recommendations have been proposed (2), but high test costs present a practical barrier to widespread implementation. We recently described a single-step, closed-tube, and readily scalable strategy for rapid large-scale screening detection of FMR1 expansion mutations by melting curve analysis (MCA) of triplet-primed PCR products from unmodified genomic DNA (3). However, the assay does not discriminate between males and females or between PM and FM expansions, thus potentially identifying nonactionable conditions. We have developed a modified strategy that can differentiate between actionable and nonactionable FMR1 expansion genotypes.We optimized the assay on cell line genomic DNA (Coriell Cell Repositories) and previously characterized reference DNAs (4). DNA samples were pretreated with sodium bisulfite (EZ-DNA Methylation Gold kit, Zymo Research), and then subjected to methylation-specific triplet-primed PCR (msTP-PCR) of the modified antisense strand. Each 50-μL reaction contained 0.2 mmol/L dNTP (deoxynucleotide triphosphate), 2.5 U HotStarTaq DNA polymerase in 1× supplied …