A Multiplexed Assay for Exon Recognition Reveals that an Unappreciated Fraction of Rare Genetic Variants Cause Large-Effect Splicing Disruptions

Mol Cell. 2019 Jan 3;73(1):183-194.e8. doi: 10.1016/j.molcel.2018.10.037. Epub 2018 Nov 29.


Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.

Keywords: exon recognition; massively parallel reporter assay; population variation; rare variation; splicing; variant classification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Separation
  • Computational Biology
  • Exons*
  • Flow Cytometry
  • Gene Expression Profiling / methods*
  • HEK293 Cells
  • HeLa Cells
  • Hep G2 Cells
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Introns
  • K562 Cells
  • Mutation*
  • Oligonucleotide Array Sequence Analysis
  • RNA Splicing*
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*