TY - JOUR T1 - Targeted long-read sequencing identifies missing pathogenic variants in unsolved Werner syndrome cases JF - Journal of Medical Genetics JO - J Med Genet DO - 10.1136/jmedgenet-2022-108485 SP - jmedgenet-2022-108485 AU - Danny E. Miller AU - Lin Lee AU - Miranda Galey AU - Renuka Kandhaya-Pillai AU - Marc Tischkowitz AU - Deepak Amalnath AU - Avadh Vithlani AU - Koutaro Yokote AU - Hisaya Kato AU - Yoshiro Maezawa AU - Aki Takada-Watanabe AU - Minoru Takemoto AU - George M. Martin AU - Evan E. Eichler AU - Fuki M. Hisama AU - Junko Oshima Y1 - 2022/05/09 UR - http://jmg.bmj.com/content/early/2022/05/08/jmedgenet-2022-108485.abstract N2 - 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.Data are available on reasonable request. Data that support the findings of this study are available on request from the corresponding authors. ER -