The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity

Abstract

Although the spliceogenic nature of the BRCA2 c.68‐7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real‐time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case‐control analysis in 83,636 individuals. Co‐occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5‐fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68‐7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10−115. There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86–1.24) nor for a deleterious effect of the variant when co‐occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68‐7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

“…Moreover, the reproducibility of MGBR2_2–9 splicing outcomes was supported by previous studies based on patient RNA or minigene data, according to which fourteen variants matched preceding results: c.67+3A>G, c.68‐7T>A, c.316+3del, c.316+5G>C, c.426‐12_426‐8del, c.467A>G, c.516+ 1G>T, c.517G>T, c.572A>G, c.617C>G, c.627C>A/T, c.631G>A, c.631+3A>G and c.681+4A>G . Conversely, in a recent publication, variant c.68‐7T>A induced 20% of transcript Δ3 in lymphoblastoid cell lines of carrier patients versus 2.8% of Δ3 and 7.2% of Δ(6q 39 ,7) found in our minigene study in MCF‐7 cells . These variations may be due to technical differences in RNA isolation methods, retro‐transcriptases, DNA polymerases, primers and product length, thermocycling protocol as well as cell‐type specific variations between the patient sample and the minigene assay.…”

Incorporation of semi‐quantitative analysis of splicing alterations for the clinical interpretation of variants inBRCA1andBRCA2genes

“…Authors found that although BRCA1 c.(594–2A>C; 641A>G) alleles did not contribute to full‐length transcript expression and produced a major frameshift event lacking exon 10, an estimated proportion of 30% of Δ9,10 isoform was sufficient to confer tumor suppressor capacity in variant carriers (la Hoya et al, ). Another example illustrating the complexity of splicing interpretation is BRCA2 c.68–7T>A variant, for which authors concluded that BRCA2 alleles producing up to approximately 20% exon 3 skipping were not associated with cancer risk (Colombo et al, ). Here, we describe three variants producing “leaky” splicing effects (see BRCA1 c.135–18 T>G, BRCA2 c.9501+3A>T and BRCA2 c.7447A>G variants in Figures a, and e,f, respectively), and the methodology used in this study is limited for the clinical interpretation of this type of variants.…”

“…A recent study reported that variants affecting splicing, namely spliceogenic, are the third most common type occurring in BRCA1 and BRCA2 genes, accounting for 10.1% and 7.6% of the total, respectively (Rebbeck et al, ). Although multiple spliceogenic variants producing abnormal transcripts have been identified in HBOC families, the clinical relevance of the splicing outcomes can be sometimes difficult to determine (de Caputo et al, ; Colombo et al, ; la Hoya et al, ; Montalban et al, ). Furthermore, numerous BRCA1/2 alternative splicing events have been described in several tissues and cell lines, adding complexity to the interpretation of splicing outcomes generated from variant alleles (Colombo et al, ; Fackenthal et al, ).…”

Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay

“…These assays are accessible to most laboratories, can be performed in patient samples, do not require the use of expression vectors, and can perform qualitative characterization of splicing variants; however, these assays only provide semi-quantitative data and lack the throughput required to analyze a large amount of variants ( 30 , 31 ). Alternatively, there are quantitative approaches, including real-time and digital PCR, that provide robust and reliable quantitative data ( 29 , 32 ), but cannot perform qualitative analysis (i.e., these assays are unable to reveal the precise transcript sequence). Here we describe a novel RNA MPS method, CloneSeq, and demonstrated that this technique is capable of performing reliable high-throughput quantitative and qualitative analysis of splicing variants, a necessary feature to obtain evidence for the large number of alterations predicted to affect splicing in HBOC genes.…”