Systematic analysis of dark and camouflaged genes: disease-relevant genes hiding in plain sight

Ebbert, Mark T. W.; Jensen, Tanner D.; Jansen-West, Karen; Sens, Jonathon P.; Reddy, Joseph S.; Ridge, Perry G.; Kauwe, John S. K.; Belzil, Véronique V.; Pregent, Luc; Carrasquillo, Minerva M.; Keene, Dirk; Larson, Eric B.; Crane, Paul K.; Asmann, Yan W.; Ertekin-Taner, Nilüfer; Younkin, Steven G.; Ross, Owen A.; Rademakers, Rosa; Petrucelli, Leonard; Fryer, John Denis

doi:10.1101/514497

Cited by 2 publications

(2 citation statements)

References 113 publications

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“…Targets disproportionately affected by this limitation include families of gene and pseudogene homologs (Ebbert et al, 2019). Other genomic regions are intractable to analysis due to difficulties caused by the sequencing chemistry itself.…”

Section: Introductionmentioning

confidence: 99%

Long‐read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel–Gruber syndrome

et al. 2019

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The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule longread sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.

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Section: Introductionmentioning

confidence: 99%

Long‐read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel–Gruber syndrome

et al. 2019

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“…“Population-scale” sequencers, capable of performing cheap whole genome sequencing (WGS) have been used extensively by large research programs, but they are now also being deployed in diagnostic practice [1]. Despite this shift towards WGS, which simplifies laboratory workflows by eliminating the need for target enrichment, a number of clinically relevant genomic regions are known to be intractable to analysis by short-read-sequencing [2].…”

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confidence: 99%

Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications

Watson

Crinnion

Hewitt

et al. 2020

Laboratory Investigation

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The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient’s father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumour cells, and for precisely identifying transgene integration sites in mouse models.

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Systematic analysis of dark and camouflaged genes: disease-relevant genes hiding in plain sight

Cited by 2 publications

References 113 publications

Long‐read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel–Gruber syndrome

Long‐read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel–Gruber syndrome

Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications

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