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.

Pseudohypoparathyroidism type Ib (PHP1B) is characterized predominantly by resistance to parathyroid hormone (PTH) leading to hypocalcemia and hyperphosphatemia. These laboratory abnormalities are caused by maternal loss-of-methylation (LOM) at GNAS exon A/B, which reduces in cis expression of the stimulatory G protein α-subunit (Gsα). Paternal Gsα expression in proximal renal tubules is silenced through unknown mechanisms, hence LOM at exon A/B reduces further Gsα protein in this kidney portion, leading to PTH resistance. In a previously reported PHP1B family, affected members showed variable LOM at exon A/B, yet no genetic defect was found by whole-genome sequencing despite linkage to GNAS. Using targeted long-read sequencing (T-LRS), we discovered an approximately 2800-bp maternally inherited retrotransposon insertion nearly 1200 bp downstream of exon XL not found in public databases or in 13,675 DNA samples analyzed by short-read whole-genome sequencing. T-LRS data furthermore confirmed normal methylation at exons XL, AS, and NESP and showed that LOM comprising exon A/B is broader than previously thought. The retrotransposon most likely causes the observed epigenetic defect by impairing function of a maternally derived NESP transcript, consistent with findings in mice lacking full-length NESP mRNA and in PHP1B patients with deletion of exon NESP and adjacent intronic sequences. In addition to demonstrating that T-LRS is an effective strategy for identifying a small disease-causing variant that abolishes or severely reduces exon A/B methylation, our data demonstrate that this sequencing technology has major advantages for simultaneously identifying structural defects and altered methylation.

Rapid genetic testing in the critical care setting enables targeted evaluations, directs therapies, and helps families and care providers make informed decisions about goals of care. We tested whether we could perform ultra-rapid assessment of genetic risk for a Mendelian condition, based on information from an affected sibling, in a newborn via whole-genome sequencing using the Oxford Nanopore platform. By optimization of the DNA extraction and library preparation steps paired with targeted analysis, we were able to demonstrate within three hours of birth that the newborn was neither affected nor a carrier for variants underlying acrodermatitis enteropathica. This proof-of-concept experiment demonstrates how prior knowledge of familial variants can be used to rapidly evaluate an individual at-risk for a genetic disease.

Heterozygous chromosome inversions suppress meiotic crossover (CO) formation within an inversion, potentially because they lead to gross chromosome rearrangements that produce inviable gametes. Interestingly, COs are also severely reduced in regions nearby but outside of inversion breakpoints even though COs in these regions do not result in rearrangements. Our mechanistic understanding of why COs are suppressed outside of inversion breakpoints is limited by a lack of data on the frequency of noncrossover gene conversions (NCOGCs) in these regions. To address this critical gap, we mapped the location and frequency of rare CO and NCOGC events that occurred outside of the dl-49 chrX inversion in D. melanogaster. We created full-sibling wildtype and inversion stocks and recovered COs and NCOGCs in the syntenic regions of both stocks, allowing us to directly compare rates and distributions of recombination events. We show that COs are completely suppressed within 500 kb of inversion breakpoints, are severely reduced within 2 Mb of an inversion breakpoint, and increase above wildtype levels 2-4 Mb from the breakpoint. We find that NCOGCs occur evenly throughout the chromosome and, importantly, occur at wildtype levels near inversion breakpoints. We propose a model in which COs are suppressed by inversion breakpoints in a distance-dependent manner through mechanisms that influence DNA double-strand break repair outcome but not double-strand break location or frequency. We suggest that subtle changes in the synaptonemal complex and chromosome pairing might lead to unstable interhomolog interactions during recombination that permits NCOGC formation but not CO formation.