Evaluating the clinical utility of a long-read sequencing-based approach in genetic testing of fragile-X syndrome

Fragile X syndrome (FXS) arises from the FMR1 CGG expansion. Comprehensive genetic testing for FMR1 CGG expansions, AGG interruptions, and microdeletions is essential to provide genetic counseling for females carrying premutation alleles. However, conventional PCR-based FMR1 assays mainly focus on CGG repeats, and could detect AGG interruption only in males. The clinical utility of a long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was evaluated in 238 high-risk samples by comparing to conventional PCR assays. PCR assays identified five premuation and three full mutation categories alleles in all the samples, and CAFXS successfully called all the FMR1 CGG expansion. CAFXS identified 24-bp microdeletions upstream to the trinucleotide region with 30 CGG repeats, which was miscalled by the length-based PCR methods. CAFXS also identified a 187-bp deletion in about 1/7 of the sequencing reads in a male patient with mosaic full mutation alleles. CAFXS allowed for precise constructing the FMR1 CGG repeat and AGG interruption pattern in all the samples, and identified a novel and alternative CGA interruption in one normal female sample. CAFXS represents a more comprehensive and accurate approach for FXS genetic testing that potentially enables more informed genetic counseling compared to PCR-based methods.