Enhanced accuracy and sensitivity in detecting FMR1 CGG repeats: a multicenter evaluation of a novel PCR-capillary electrophoresis assay.

BACKGROUND

Fragile X syndrome (FXS) is primarily caused by the expansion of CGG repeats in the 5' untranslated region of the FMR1 gene. Accurate detection of expanded FMR1 alleles is essential for timely diagnosis and management. Triplet-repeat primed PCR is the most widely used method for detecting FXS; however, it has limitations in detecting low DNA input (< 10 ng/μL) and low-level mosaicism (< 5%). This study aimed to develop an improved method for detecting FMR1 CGG repeat expansions, outperforming existing methods in efficiency, reliability and sensitivity.

METHODS

We developed a novel four-primer PCR with capillary electrophoresis assay (FP-PCR/CE) and validated its performance in identifying and sizing FMR1 alleles using DNA standards and multi-center clinical samples (N = 1690). Comparative analyses were performed against the AmplideX FMR1 PCR/CE assay and Southern blot to assess the accuracy, sensitivity, and clinical reliability of this assay.

RESULTS

The FP-PCR/CE assay demonstrated 100% concordance with DNA standards for CGG repeat sizing and mosaicism detection. It detected DNA input ≥ 2.5 ng/μL and mosaic alleles at a mass fraction as low as 1%. In clinical validation, FP-PCR/CE achieved 100% concordance in FMR1 allele characterization with both the AmplideX assay and Southern blot, while exhibiting higher sensitivity for detecting mosaicism. Additionally, the assay identified AGG interruptions within FMR1 alleles. The FP-PCR/CE assay also reduced testing time to under 7 h and lowered the cost to < $80 per test.

CONCLUSIONS

The FP-PCR/CE assay is a rapid, accurate, and cost-effective method for FMR1 CGG repeat analysis, offering improved sensitivity for mosaicism detection. Its scalability and reliability support its potential for broader use in FXS carrier screening, clinical diagnosis and research.