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

Background: Werner syndrome (WS) is an autosomal recessive progeroid syndrome caused by variants in WRN . The International Registry of Werner Syndrome has identified biallelic pathogenic variants in 179/188 cases of classical WS. In the remaining nine cases, only one heterozygous pathogenic variant has been identified.
Methods: Targeted long-read sequencing (T-LRS) on an Oxford Nanopore platform was used to search for a second pathogenic variant in WRN . Previously, T-LRS was successfully used to identify missing variants and analyse complex rearrangements.
Results: We identified a second pathogenic variant in eight of nine unsolved WS cases. In five cases, T-LRS identified intronic splice variants that were confirmed by either RT-PCR or exon trapping to affect splicing; in one case, T-LRS identified a 339 kbp deletion, and in two cases, pathogenic missense variants. Phasing of long reads predicted all newly identified variants were on a different haplotype than the previously known variant. Finally, in one case, RT-PCR previously identified skipping of exon 20; however, T-LRS did not detect a pathogenic DNA sequence variant.
Conclusion: T-LRS is an effective method for identifying missing pathogenic variants. Although limitations with computational prediction algorithms can hinder the interpretation of variants, T-LRS is particularly effective in identifying intronic variants.
Competing Interests: Competing interests: DEM has received travel support from Oxford Nanopore Technologies (ONT) to speak on their behalf. DEM is a paid consultant for and holds stock options in MyOme. DEM and EEE are engaged in a research agreement with ONT. EEE is a scientific advisory board (SAB) member of Variant Bio, Inc.
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