Unraveling missing variants through target capture-based long-read sequencing in autosomal recessive disorders

Area of Science:

• Genomics
• Rare disease diagnostics
• Molecular biology

Background:

• Short-read sequencing (SRS) gene panels are vital for rare disease diagnosis but struggle with autosomal recessive cases.
• Undiagnosed cases often involve variants in deep intronic regions or structural variants (SVs) missed by SRS.

Purpose of the Study:

• To enhance diagnostic yield for rare autosomal recessive disorders.
• To implement and evaluate a cost-effective target capture-based long-read sequencing (LRS) assay.

Main Methods:

• Retrospective analysis of 78 patients with unsolved autosomal recessive disorders after SRS.
• Application of a target capture-based LRS assay covering 56 genes.
• Functional validation using RT-PCR and minigene assays.

Main Results:

• LRS solved 25.6% (20/78) of previously undiagnosed cases.
• Identified 10 structural variants (SVs) and 3 deep intronic variants causing aberrant splicing.
• Haplotype phasing reclassified VUS as likely pathogenic in 7 cases.

Conclusions:

• Target capture-based LRS effectively detects diverse variants missed by SRS, including SVs and deep intronic variants.
• Integrating LRS into diagnostic workflows is a practical strategy to improve autosomal recessive disease diagnosis.
• The reported diagnostic yield is from a selected, enriched population and not directly generalizable to unselected cohorts.