Expanding the diagnostic toolkit using long-read RNA-sequencing
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
Molecular diagnosis of inherited rare disease plateaus at around 50% and with progress in the development of disease-modifying treatments for rare diseases diagnostic speed has become increasingly important. However, significant diagnostic challenges remain and include technical limitations of short-read sequencing, novel gene-disease relationships yet to be discovered and the high proportion of variants of uncertain significance (VUS).
Methods:
In this study we evaluated the use of targeted long-read sequencing as an additional tool for the rapid assessment of aberrant splicing in patient samples to raise diagnostic yield. We assessed this technology in the context of undiagnosed paediatric patients with suspected neurometabolic disorders, previously tested by WES or WGS, and following an additional multidisciplinary review that suggested a monogenic disease with <5 genes of particular interest (based on a combination of clinical phenotype, the presence of VUS or a single pathogenic variant in the context of a probable autosomal recessive disorder). Focusing on a set of 10 patients for whom there was high confidence in a small number of candidate genes, we used patient blood samples or fibroblasts to generate cDNA, PCR for genes of interest and generate full length reads using Oxford Nanopore Technologies single molecule sequencing.
Results:
Using this approach we identified probable pathogenic splicing events in two patient samples. We used not only existing transcriptomic annotations, but also publicly available short- and long-read sequencing data from control individuals to determine the rarity of the events detected. Furthermore, by enabling the prediction of both ORFs and protein structures, the availability of full-length reads greatly increased our ability to identify events expected to disrupt gene function rapidly.
Conclusion:
Taken together, our findings suggest that targeted long-read RNA-sequencing in patients who remain undiagnosed following extensive testing could be an important tool particularly when a treatable genetic diagnosis is being considered.
Molecular diagnosis of inherited rare disease plateaus at around 50% and with progress in the development of disease-modifying treatments for rare diseases diagnostic speed has become increasingly important. However, significant diagnostic challenges remain and include technical limitations of short-read sequencing, novel gene-disease relationships yet to be discovered and the high proportion of variants of uncertain significance (VUS).
Methods:
In this study we evaluated the use of targeted long-read sequencing as an additional tool for the rapid assessment of aberrant splicing in patient samples to raise diagnostic yield. We assessed this technology in the context of undiagnosed paediatric patients with suspected neurometabolic disorders, previously tested by WES or WGS, and following an additional multidisciplinary review that suggested a monogenic disease with <5 genes of particular interest (based on a combination of clinical phenotype, the presence of VUS or a single pathogenic variant in the context of a probable autosomal recessive disorder). Focusing on a set of 10 patients for whom there was high confidence in a small number of candidate genes, we used patient blood samples or fibroblasts to generate cDNA, PCR for genes of interest and generate full length reads using Oxford Nanopore Technologies single molecule sequencing.
Results:
Using this approach we identified probable pathogenic splicing events in two patient samples. We used not only existing transcriptomic annotations, but also publicly available short- and long-read sequencing data from control individuals to determine the rarity of the events detected. Furthermore, by enabling the prediction of both ORFs and protein structures, the availability of full-length reads greatly increased our ability to identify events expected to disrupt gene function rapidly.
Conclusion:
Taken together, our findings suggest that targeted long-read RNA-sequencing in patients who remain undiagnosed following extensive testing could be an important tool particularly when a treatable genetic diagnosis is being considered.