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

Abstract

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.

Keywords: genetic variation; genomics; nanopore sequencing.

Figure 1

Targeted long-read sequencing (T-LRS) was used to identify missing variants. (A) T-LRS was used to target an approximately 3 Mbp region around WRN. Data shown for individual WV. Increased coverage (y-axis) represents the target region, decreased coverage represents background. The position of WRN is represented by the horizontal black bar around 31 Mbp (GRCh38). (B) T-LRS revealed a 339 kbp deletion in individual PD1010 that began within WRN. (C) Long-read sequencing of RT-PCR products from individual SIV1010 confirmed the absence of exon 20 in approximately half of the reads (arrow). Please see online supplemental figure 3 for an integrative genomics viewer screenshot of exons 19–21 that shows how reads are linked and that approximately half of reads skip of exon 20. (D) Long-read sequencing revealed RT-PCR products from individual SIV1010 did not carry the T allele present on haplotype 2 within exon 20, confirming that splicing was altered on haplotype 2. The previously known pathogenic variant (table 1) was predicted to be located on haplotype 1. No second pathogenic variant was identified in this individual.

Figure 2

Diagram of WRN and the location of variants identified in this study. Pathogenic variants found in this study are shown with respect to the 35 exons within WRN. Functional domains of WRN proteins are shown below the corresponding exons. Those functional domains are the exonuclease and helicase domains, RecQ helicase conserved region (RQC), helicase RNaseD C-terminal conserved region (HRDC) and the nuclear localisation signal (NLS).

Figure 3

Exon trapping of the EN1010 variant. (A) The pSPL3 vector contains splice donor (SD) and splice acceptor (SA) exons and functional introns, with transcription beginning following the SV40 promoter and ending at the poly(A) site. The wildtype (Wt) construct, pSPL3-EN1010-Wt, and the mutant (Mut) construct, pSPL3-EN1010-Mut, contain the 1.8 kbp fragment derived from EN1010, with the c.839+1309T>G variant at the asterisk (*). (B) Agarose gel electrophoresis of RT-PCR products shows the larger amplicon in the Mut compared with Wt due to the 171 bp cryptic insertion.