Guidelines for clinical interpretation of variant pathogenicity using RNA phenotypes

Hum Mutat. 2022 Aug;43(8):1056-1070. doi: 10.1002/humu.24416. Epub 2022 Jun 29.


Over the last 5 years, RNA sequencing (RNA-seq) has been established and is increasingly applied as an effective approach complementary to DNA sequencing in molecular diagnostics. Currently, three RNA phenotypes, aberrant expression, aberrant splicing, and allelic imbalance, are considered to provide information about pathogenic variants. By providing a high-throughput, transcriptome-wide functional readout on variants causing aberrant RNA phenotypes, RNA-seq has increased diagnostic rates by about 15% over whole-exome sequencing. This breakthrough encouraged the development of computational tools and pipelines aiming to streamline RNA-seq analysis for implementation in clinical diagnostics. Although a number of studies showed the added value of RNA-seq for the molecular diagnosis of individuals with Mendelian disorders, there is no formal consensus on assessing variant pathogenicity strength based on RNA phenotypes. Taking RNA-seq as a functional assay for genetic variants, we evaluated the value of statistical significance and effect size of RNA phenotypes as evidence for the strength of variant pathogenicity. This was determined by the analysis of 394 pathogenic variants, of which 198 were associated with aberrant RNA phenotypes and 723 benign variants. Overall, this study seeks to establish recommendations for integrating functional RNA-seq data into the the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines classification system.

Keywords: RNA sequencing; guidelines; rare disorders; variant interpretation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Genetic Variation*
  • Humans
  • Phenotype
  • RNA* / genetics
  • Sequence Analysis, DNA
  • Virulence


  • RNA