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

Abstract

BackgroundWerner 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.MethodsTargeted 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.ResultsWe 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.ConclusionT-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.

Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic

“…Although additional read depth would be required to confidently assess the parent of origin of the duplicated allele, we have demonstrated that methylated reads can be phased such that parent-of-origin could be determined in samples with sufficient depth. Parent-of-origin for this variant has potential prognostic implications because individuals with 15q11.2-q13 duplications of the paternal allele typically develop less severe autism spectrum disorder than individuals with duplications of the maternal allele [ 25 ]. In the case we presented, we relied on a single nearby SNV to phase reads.…”

NanoRanger enables rapid single base-pair resolution of genomic disorders

“…This may be particularly true in the case of genomic disorders, a term first coined by Lupski in 1998 to describe diseases that result from rearrangements of the human genome rather than from DNA sequence base changes 10 . There are indeed a few studies demonstrating the superiority of LRS over SRS in this class of disorders 11,12 . However, whole genome LRS is still prohibitively expensive for routine genetic diagnosis and remains largely inaccessible due to complex workflow and stringent sample requirement for ultrahigh molecular weight DNA.…”

Long‐read sequencing for molecular diagnostics in constitutional genetic disorders

“…Research studies over the last several years have highlighted the utility of long reads in diagnosing genetic disorders where the standard of care assays fail to achieve a molecular diagnosis (Amarasinghe et al, 2020;Ardui et al, 2018;Borras et al, 2017;Melas et al, 2022;D. E. Miller et al, 2021D. E. Miller et al, , 2022.…”

“…Research studies over the last several years have highlighted the utility of long reads in diagnosing genetic disorders where the standard of care assays fail to achieve a molecular diagnosis (Amarasinghe et al, 2020; Ardui et al, 2018; Borras et al, 2017; Melas et al, 2022; D. E. Miller et al, 2021, 2022). These studies fall into the two broad categories: (1) identifying pathogenic variants in regions difficult to sequence with short‐reads: repeat expansions, homopolymers, protein‐coding genes with pseudogenes, long insertions (>50 base pairs), and complex structural rearrangements, and (2) resolving unknown or uncertain phase in autosomal recessive conditions where short‐read technology requires additional sequencing of parental samples.…”

“…LRS revealed the deletion and the single nucleotide variant in the same experiment with phase resolution in the same experiment. More recently, Miller et al used Oxford Nanopore sequencing in a cohort of nine patients with a clinical diagnosis of Werner Syndrome (WRN; MIM# 277700) (D. E. Miller et al, 2022). Werner syndrome is an autosomal recessive disorder caused by loss of function variants in the gene WRN (MIM# 604611).…”