Targeted long-read sequencing identifies missing pathogenic variants in unsolved Werner syndrome cases.

Area of Science:

• Genetics
• Molecular Biology
• Rare Diseases

Background:

• Werner syndrome (WS) is a rare autosomal recessive progeroid disorder.
• Pathogenic variants in the WRN gene cause WS.
• Most classical WS cases have biallelic pathogenic WRN variants, but some remain unsolved.

Purpose of the Study:

• To identify the missing second pathogenic WRN variant in unsolved Werner syndrome cases.
• To evaluate the utility of targeted long-read sequencing (T-LRS) for detecting complex genetic variants.

Main Methods:

• Targeted long-read sequencing (T-LRS) on an Oxford Nanopore platform was employed.
• T-LRS was used to search for second pathogenic variants in the WRN gene in nine unsolved WS cases.
• Identified variants were confirmed using RT-PCR or exon trapping.

Main Results:

• A second pathogenic WRN variant was identified in eight out of nine unsolved WS cases.
• T-LRS detected intronic splice variants (five cases), a large deletion (one case), and missense variants (two cases).
• Phasing of long reads confirmed variants were on different haplotypes; one case with exon skipping remained unresolved by T-LRS DNA sequencing.

Conclusions:

• Targeted long-read sequencing (T-LRS) is a highly effective method for identifying missing pathogenic variants in Werner syndrome.
• T-LRS is particularly adept at detecting intronic splice variants.
• Challenges in variant interpretation may arise from computational prediction algorithm limitations.