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...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 per-formance in identifying and sizing FMR1 alleles using DNA standards and multi-center clinical samples(N=1690).Com-parative 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 mosai-cism 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 car-rier screening,clinical diagnosis and research.展开更多
基金supported by the Key R&D Program of Zhejiang(No.2023C03003)National Natural Science Foundation of China(No.82373971)National Key R&D Program of China(No.2023YFC2706100).
文摘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 per-formance in identifying and sizing FMR1 alleles using DNA standards and multi-center clinical samples(N=1690).Com-parative 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 mosai-cism 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 car-rier screening,clinical diagnosis and research.
