WITHDRAWN: Development and validation in 500 female samples of a TP-PCR assay to identify AFF2 GCC expansions

Abstract

The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to determine the allele size of both FRAXA and FRAXE in which around 20% of female samples are uninformative. The aim of this study is to develop a timely triplet-primed amplification (TP-PCR) screening strategy to size the AFF2 GCC repeat and accurately assess homozygosity in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Unlike the presence of AGG interspersion(s) in FRAXA triplet repeats, no interspersion was observed among the samples tested. Interestingly, the presence of a T>C SNP (rs868949662), contiguous to the GCC repetitive track, allows triplet primer binding in two additional GCCs, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range, eight intermediate and four premutated, were recognized, which seems relevant considering the rarity of the AFF2 expansions. Overall, this study confirmed the absence of interspersions in both normal and expanded FRAXE alleles, providing a strict, reproducible and low-cost homozygosity screening strategy. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency introduces new areas of clinical research in this cohort in understanding these less explored AFF2 repeat sizes and newly associated phenotypes.