Transcript capture and ultradeep long-read RNA sequencing (CAPLRseq) to diagnose HNPCC/Lynch syndrome

Abstract

Purpose: Whereas most human genes encode multiple mRNA isoforms with distinct function, clinical workflows for assessing this heterogeneity are not readily available. This is a substantial shortcoming, considering that up to 25% of disease-causing gene variants are suspected of disrupting mRNA splicing or mRNA abundance. Long-read sequencing can readily portray mRNA isoform diversity, but its sensitivity is relatively low due to insufficient transcriptome penetration.

Methods: We developed and applied capture-based target enrichment from patient RNA samples combined with Oxford Nanopore long-read sequencing for the analysis of 123 hereditary cancer transcripts (capture and ultradeep long-read RNA sequencing (CAPLRseq)).

Results: Validating CAPLRseq, we confirmed 17 cases of hereditary non-polyposis colorectal cancer/Lynch syndrome based on the demonstration of splicing defects and loss of allele expression of mismatch repair genes MLH1, PMS2, MSH2 and MSH6. Using CAPLRseq, we reclassified two variants of uncertain significance in MSH6 and PMS2 as either likely pathogenic or benign.

Conclusion: Our data show that CAPLRseq is an automatable and adaptable workflow for effective transcriptome-based identification of disease variants in a clinical diagnostic setting.

Keywords: Gastrointestinal Diseases; Gene Expression Profiling; Nanopore Sequencing; RNA-Seq.

Figure 1

Outline of capture and ultradeep long-read RNA sequencing. The different experimental steps and approximate times are shown for a representative mRNA (see text for details). ONT, Oxford Nanopore Technology.

Figure 2

Effect of single-nucleotide variants on MSH2 mRNA expression and splicing as determined by CAPLRseq. (A) RNA isolated from PBMCs of a patient carrying the indicated genomic variant was sequenced with the CAPLRseq protocol. Prior to RNA isolation, one of two parallel cultures received puromycin to inhibit nonsense-mediated mRNA decay (see the Methods section). Allele-specific expression was assessed based on the ratio of the T variant to the C allele (representative example shown, averages of three technical replicates with SD). (B) CAPLRseq analysis of a patient carrying the indicated splice site variant in MSH2. Percent skipping of exon 5 is indicated. Numbers are averages of two technical replicates (three for the reference sample) with SD. (C) MLH1 expression levels in a sample from patient 8, which showed an MLH1 promoter methylation by multiplexed ligation-dependent probe analysis. MLH1 mRNA levels were quantified as described in the Methods section relative to 37 reference RNA samples of subjects without a known variant in MLH1. Log2-fold change of MLH1 mRNA (red dot) is shown in a volcano plot (n=3). CAPLRseq, capture and ultradeep long-read RNA sequencing; PBMC, peripheral blood mononuclear cell; SD, standard deviation.

Figure 3

Effect of single-nucleotide and structural variants and on mRNA structure and expression as determined by CAPLRseq. (A) CAPLRseq analysis of a patient carrying the indicated intronic variant in MSH2. The extension to exon 15 (left panel) and the resulting frame shift (right panel) are shown. Numbers indicate the percentage of the variant mRNA detected with and without puromycin (averages of three technical replicates with SD). (B) CAPLRseq analysis of a patient carrying the indicated SINE-VNTR-Alu insertion in PMS2 intron 7 schematically shown in the top panel. The 71 nucleotide extensions to exon 8 and the mismatches to the reference sequence are shown at the bottom right. Numbers indicate the percentage of the variant mRNA detected with and without puromycin (averages of three technical replicates with SD). (C) CAPLRseq analysis of a patient carrying the indicated structural variant involving the MLH1 locus and the predicted MLH1–DCLK3 fusion transcripts arising from it. The bottom left panel shows the fusion transcripts rendered in the Integrative Genomics Viewer. The bottom right plot shows MLH1 expression levels relative to 37 reference RNA samples of subjects without a known variant in MLH1. Log2-fold change of MLH1 mRNA (red dot) is shown in a volcano plot (n=2). CAPLRseq, capture and ultradeep long-read RNA sequencing.

Figure 4

Variants of uncertain significance reclassified by CAPLRseq analysis (A) CAPLRseq analysis of a patient carrying the indicated intronic variant in PMS2. Percent skipping of exon 2 is indicated. Numbers are averages of three technical replicates with SD. Allelic expression was evaluated based on the ratio of reads for the indicated SNVs. (B) CAPLRseq analysis of a patient carrying the indicated intronic variant in MSH6. Splicing of intron 3 was assessed by visualisation in the Integrative Genomics Viewer. Images show representative data of three technical replicates. Allelic expression was evaluated based on the ratio of reads for the indicated SNVs. CAPLRseq, capture and ultradeep long-read RNA sequencing; SNV, single-nucleotide variant; gDNA, genomic DNA.