Establishment of a New-Generation National Reference Material System for Fragile X Syndrome Using Targeted Long-Read...

Fragile X syndrome (FXS) is the most common monogenic cause of inherited intellectual disability and is primarily caused by CGG repeat expansion in the FMR1 gene. Conventional diagnostic methods have limited precision for sizing long repeat sequences and cannot resolve AGG interruptions, which are critical for comprehensive risk assessment. Existing national FXS reference materials are based on conventional methods and provide limited molecular information.

We developed a targeted long-read sequencing assay for comprehensive FMR1 characterization, termed tLRS-FMR1, and applied it to a panel of 22 national FXS reference materials.

The tLRS-FMR1 assay demonstrated 100% concordance with standard methods while overcoming key limitations of conventional approaches. It enabled precise quantification of CGG repeat numbers, including full mutations (>200 repeats) that were only qualitatively reported by traditional techniques and provided comprehensive mapping of AGG interruption patterns. The assay showed high reproducibility, with 100% genotyping concordance across intra- and inter-assay replicates and achieved a detection limit of 3 ng/μL.

This study successfully developed tLRS-FMR1 and established a new-generation national FXS reference material system with expanded molecular information and improved precision, providing a foundation for advancing the standardization and accuracy of FXS molecular diagnosis.