Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.

Vanessa L Lattimore,John F Pearson,Logan C Walker,Margaret J Currie,Amanda B Spurdle,Bridget A Robinson

doi:10.3389/fonc.2018.00140

Abstract

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

Highlights

Using targeted RNA-seq, we detected 40 BRCA1 and 17 BRCA2 alternate isoforms in lymphoblastoid cell lines (LCLs) from non-variant carrier controls cultured with or without a nonsense-mediated decay (NMD) inhibitor (Tables S6–S7 in Supplementary Material). These include 25/63 of the BRCA1 isoforms identified by Colombo et al (12) and/or Davy et al (10) (Table S6 in Supplementary Material), in addition to 5/22 BRCA2 isoforms identified by Fackenthal et al (13) (Table S7 in Supplementary Material)
Of the previously reported naturally occurring isoforms not detected in BRCA1 and BRCA2 using targeted RNA-seq (12, 13), the majority (28/32 in BRCA1 and 13/17 in BRCA2) were due to target probe placement, while the remainder (11/32 BRCA1 and 4/17 BRCA2) were presumably not expressed at levels that were detectable in our cell lines (Tables S6 and S7 in Supplementary Material)
BRCA1 and BRCA2 mRNA splicing assays are often carried out in a diagnostic and research setting to assess the effects of variants of uncertain clinical significance

Summary

Introduction

At least 20% of hereditary breast and ovarian cancer cases contain germline pathogenic variants in breast cancer susceptibility genes BRCA1 (MIM #113705) or BRCA2 (MIM #600185) (1). Functioning as tumor suppressor genes, BRCA1 and BRCA2 repair single and double-stranded breaks in DNA, a process which can be compromised when variants that disrupt pre-mRNA splicing to create aberrant splice isoforms are present (2–4). These variants may directly disrupt splice sites or splicing regulatory regions, such as exonic splicing enhancers or exonic splicing silencers (5). Resulting splicing aberrations, such as major deletion/retention events and frame shifts, can lead to loss of function through the introduction of premature termination codons, leading to non-functional isoforms that are generally destroyed by nonsense-mediated decay (NMD), or via the production of truncated proteins (6). A better understanding of expression level changes that reflect normal variation in BRCA1/2 splicing patterns between individuals would improve our understanding of isoform regulation for identifying variability that is likely to be of clinical relevance

Methods

Results

Conclusion