Simultaneous Screening of the FRAXA and FRAXE Loci for Rapid Detection of FMR1 CGG and/or AFF2 CCG Repeat Expansions by Triplet-Primed PCR

Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 50-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 50-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXA-and FRAXE-associated disorders. (J Mol Diagn 2021, 23: 941-951; https://doi.org/10.1016/ j.jmoldx.2021.04.015)

Automated summary

A novel PCR assay was developed for simultaneous screening of trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, demonstrating accurate identification of expansions at the FMR1 and AFF2 trinucleotide repeat loci. Sanger sequencing of the AFF2 CCG repeat revealed a single-nucleotide polymorphism variant enriched in the Malay population and present in all expanded AFF2 alleles studied. Overall, this study offers a simplified approach to screening for FRAXA and FRAXE-associated disorders.