Mapping RNA splicing variations in clinically accessible and nonaccessible tissues to facilitate Mendelian disease diagnosis using RNA-seq

Abstract

Purpose: RNA-seq is a promising approach to improve diagnoses by detecting pathogenic aberrations in RNA splicing that are missed by DNA sequencing. RNA-seq is typically performed on clinically accessible tissues (CATs) from blood and skin. RNA tissue specificity makes it difficult to identify aberrations in relevant but nonaccessible tissues (non-CATs). We determined how RNA-seq from CATs represent splicing in and across genes and non-CATs.

Methods: We quantified RNA splicing in 801 RNA-seq samples from 56 different adult and fetal tissues from Genotype-Tissue Expression Project (GTEx) and ArrayExpress. We identified genes and splicing events in each non-CAT and determined when RNA-seq in each CAT would inadequately represent them. We developed an online resource, MAJIQ-CAT, for exploring our analysis for specific genes and tissues.

Results: In non-CATs, 40.2% of genes have splicing that is inadequately represented by at least one CAT; 6.3% of genes have splicing inadequately represented by all CATs. A majority (52.1%) of inadequately represented genes are lowly expressed in CATs (transcripts per million (TPM) < 1), but 5.8% are inadequately represented despite being well expressed (TPM > 10).

Conclusion: Many splicing events in non-CATs are inadequately evaluated using RNA-seq from CATs. MAJIQ-CAT allows users to explore which accessible tissues, if any, best represent splicing in genes and tissues of interest.

Keywords: RNA-seq; alternative splicing; clinical genetics; diagnostic markers; medical genetics.

Figure 1:. Identification of splicing events inadequately represented by clinically-accessible tissues (CATs).

(a) Different pre-mRNA splicing decisions can have significant, potentially pathogenic, functional consequences. (b) Splicing events in inaccessible tissues (non-CATs) can only be adequately represented by accessible tissues as a proxy if the gene is both expressed and similarly spliced. (c) We used MAJIQ on RNA-seq samples from 56 different tissues to define and identify inadequately represented splicing events between inaccessible and accessible tissues.

Figure 2:. Mapping transcriptome variations identified in CATs vs non-CATs.

(a) Of an average of 9,966 genes with consistently spliced events per non-CAT, 3,925 (40.2%) were inadequately represented in at least one CAT, with 609 (6.3%) being inadequately represented by all CATs. (b) The percentages of genes with consistently spliced events that were inadequately represented over the 53 non-CATs were lowest in fibroblasts and highest in whole blood. (c) The percentage of junctions/retained introns that were consistently used in at least one non-CAT that were inadequately represented by each CAT was lowest in fibroblasts and highest in whole blood.

Figure 3:. Expression and pathogenicity of inadequately represented genes.

(a) The majority of inadequately represented genes are lowly expressed (TPM < 1) in CATs, but an average of 217 genes (5.8%) are well expressed (TPM > 10) in at least one inadequately representing CAT. (b) An average of 29.2% of inadequately represented genes are annotated as disease-causing.

Figure 4:. MAJIQ-CAT enables clinicians and scientists to explore inadequate representation of splicing by CATs in specific genes and tissues of interest.

(a) MAJIQ-CAT allows users to choose from predefined or custom gene sets and tissues (left) to quantify and understand the user-specific relevant limitations of RNA-seq in different accessible tissues (right). (b) Users can further explore individual genes for tissue-specific differences in gene expression and splicing. Shown here is a closer look at the gene MEF2C, with a violin plot of its expression in CATs and selected non-CATs (left) and violin plots of PSI for one of its inadequately represented splicing events (right). See main text for more details.