Comprehensive Analysis of Fragile X Syndrome: Full Characterization of the FMR1 Locus by Long-Read Sequencing

Background Fragile X syndrome (FXS) is the most frequent cause of inherited X-linked intellectual disability. Conventional FXS genetic testing methods mainly focus on FMR1 CGG expansions and fail to identify AGG interruptions, rare intragenic variants, and large gene deletions. Methods A long-range PCR and long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was developed and evaluated in Coriell and clinical samples by comparing to Southern blot analysis and triplet repeat-primed PCR (TP-PCR). Results CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2-4-fold analytically more sensitive than TP-PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified 2 rare intragenic variants including the c.879A > C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP-PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in 2 samples. Conclusions Long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS.