Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Genet Med. 2020 Jun;22(6):1005-1014. doi: 10.1038/s41436-020-0766-9. Epub 2020 Mar 3.

Abstract

Purpose: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories.

Methods: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software.

Results: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity.

Conclusion: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

Keywords: RNA splicing; RNA-seq; genetic diagnosis; genomic medicine; variant interpretation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Computational Biology
  • Exons
  • Humans
  • Mutation
  • RNA Splicing*
  • RNA* / genetics

Substances

  • RNA