Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

Abstract

Background Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. Methods We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. Results PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1g>t Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). Conclusions We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

Naturally occurringBRCA2alternative mRNA splicing events in clinically relevant samples

“…The corresponding isoform, using the same acceptor site in a wild-type allele, would be the ▾5p alternate-splicing event described here. Although the ▾5p splicing event was not observed previously in RNA samples from LCLs of BRCA1/2 pathogenic variant carriers or controls5 or by contributing members for phase Ia of this study, the isoform-specific RT-PCR analysis revealed this splicing event in 5/10 LCLs, BT20, MCF10A and 184A1 (table 2). …”

“…In an unrelated study,5 an isoform containing 18 intronic nucleotides upstream exon 5 was reported in a subject carrying a c.426–12_c.426-8delGTTTT that results in the use of a cryptic acceptor site. The corresponding isoform, using the same acceptor site in a wild-type allele, would be the ▾5p alternate-splicing event described here.…”

“…These results are of importance to the design and interpretation of mRNA splicing assays, construct-based or of patient material, that are commonly used to assess whether VUS leads to aberrations that are phenotypically equivalent to a molecular null (ie, a gene deletion). Recent studies have shown that detection of alternate splice events can be highly variable between laboratories and is quite sensitive to variations in cell types, cell growth conditions, mRNA preparation and RT-PCR methodology 5 6 24. Recommendations have been made to help make methodologies and reporting of alternate-splicing isoforms more uniform, including inhibition of NMD, sequence confirmation, proper primer design, presentation of data from at least 10 control samples of the same cell type to reveal potentially rare alternate splice isoforms, quantifying the variant allele contribution to full-length mRNA isoforms and quantifying the level of ‘aberrant’ transcripts relative to the full-length transcript 24.…”

“…Two additional potential alternate-splicing events not seen in phase Ia were also tested: Δ9–11, which was reported in the ENCODE RNA-Seq data set from the California Institute of Technology http://www.ncbi.nlm.nih.gov/geo/info/ENCODE.html, and ▾5p, which was predicted from a spliceogenic variant reported in a previous study 5…”

“…To address this problem, the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) is exploring a series of approaches to characterise the potential pathogenicity of BRCA1 /2 VUS identified during clinical screening, including those that may affect splicing 2 4 5. These investigations can be complicated by the observation that both BRCA1 and BRCA2 genes are transcribed into multiple naturally occurring mRNA splice isoforms independent of any genetic variants, and that these isoforms can vary in expression level between different individuals.…”