Beyond Repeats: Intragenic Variants in FMR1 and Their Contribution to Fragile X Syndrome Pathogenesis.

Abstract

Fragile X Syndrome (FXS), the most common inherited cause of intellectual disability, is typically caused by expansion of a CGG triplet repeat in the 5' untranslated region (5'-UTR) of the FMR1 gene. Growing evidence indicates that intragenic mutations in FMR1, including single-nucleotide variants (SNVs) and structural changes, can also alter FMR1 function without the classical pathogenic expansion of CGG repeats. This study re-analysed publicly available next-generation sequencing (NGS) data from BioProject PRJNA745542 using a reproducible Galaxy-based bioinformatics workflow to identify non-repeat intragenic FMR1 variants. Of 18 available datasets, 11 paired-end Illumina samples passing quality control (Phred > 30) were analysed for non-repeat intragenic FMR1 variants. Ninety-one unique variants were identified: 61 single-nucleotide variants (SNVs), 26 insertions/deletions (indels), three mixed (complex) variants, and one multi-nucleotide polymorphism (MNP), producing 1,107 predicted gene effects. SnpEff predicted sixteen variants to have high or moderate impact, predominantly within the KH1 and KH2 RNA-binding domains of FMRP. Most predicted effects (94.3%) were intronic or non-coding, suggesting a possible regulatory role in the expression or processing of FMR1 transcripts. These preliminary findings warrant validation in larger cohorts of subjects with verified disease status. High-impact KH-domain variants are predicted to disrupt hydrogen bonding required for FMRP RNA-binding and mRNA transport. This study extends the known mutation spectrum of FMR1 and supports the development of comprehensive NGS-based diagnostic assays that combine intragenic SNV and indel detection with quantitative CGG repeat analysis. Further studies using cellular or animal models are needed to validate the candidate variants identified and their potential role in Fragile X syndrome pathogenesis.