Development and validation of a singleโtube multiplex PCRfor rapid screening of Fragile X and Fragile XE syndromesof FMR1 and FMR2 genes

Abstract

Fragile X (FRAXA) syndrome and fragile XE (FRAXE) syndrome are caused by the expansion of a trinucleotide repeat in the FMR1 and FMR2 genes, respectively. Currently, there are several methods available for fragile X syndrome screening in a large population, however these methods require relatively expensive equipment and have limitations in some laboratory settings. This study developed a multiplex PCR of triplet repeats in FMR1 and FMR2 genes using standard PCR instruments. The new multiplex PCR method was tested in known samples with variable repeat sizes of FMR1 and FMR2 genes to validate the technique and was then applied to prospective index male samples. All the multiplex PCR results matched well results from standard methods. We propose a single‐tube multiplex PCR technique, which is very reliable for rapid screening FMR1 and FMR2 normal and expanded alleles in males for a large cohort study, and can be used in limited-resource settings.