Splicing and multifactorial analysis of intronic BRCA1 and BRCA2 sequence variants identifies clinically significant splicing aberrations up to 12 nucleotides from the intron/exon boundary

Abstract

Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twenty-six intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple-case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants.

Figure 1

Instances of aberrant splicing arising from BRCA1 intronic sequence variants detected by RT-PCR. VC+ and VC− represent RT-PCR on mRNA from cycloheximide variant carrier treated and untreated LCLs, respectively, and nonvariant carrying controls are represented by C− or C+ according cycloheximide treatment status. A: BRCA1 c.593+4A>G displays a minor Δexon9 product confirmed by subcloning and sequencing of PCR products. The Δexon9/10 naturally occurring isoform is represented by the 362-bp fragment. B: BRCA1 c.4484+2ins8 resulted in the comigration of two bands: a full-length product and a second containing an eight nucleotide insertion, characterized by directly sequencing the PCR product. C: BRCA1 c.4675+1G>A exhibits an exon 15 deletion. D: BRCA1 c.5194–12G >A results in a predicted insertion of 10 nucleotides into exon 20 undetectable by gel electrophoresis but subsequently identified by cloning and sequencing. E: BRCA1 c.5467+ 5G 4>C is associated with Δexon 23. [Color figures can be viewed in the online issue, which is available at www.wiley.com/humanmutation.]

Figure 2

Instances of aberrant splicing arising from BRCA2 intronic sequence variants detected by RT-PCR. VC+ and VC− represent RT-PCR on mRNAfrom cycloheximide variant carrier treated and untreated LCLs, respectively, and nonvariant carrying controls are represented by C− or C+ according cycloheximide treatment status. A: BRCA2 c.426–12_8del5 is associated with Δexon 5. The Δexon 7/39 bp of exon 6 common isoform is also observed in controls. B: BRCA2 c.516+1G>T results in two aberrant splice products; Δexon 5/6 and Δexon 6. The Δexon 7/39 bp of exon 6 common isoform is also observed in controls. C: BRCA2 c.7007+1G>C results in Δexon 12 and Δexon 12/13 aberrations. D: BRCA2 c.7618–1G >A creates a 44 nucleotide exon 16 deletion and Δexon 16/69 bp of exon 17. E: BRCA2 c.8953+1G>T creates a minor 31-bp nucleotide deletion of exon 22 and a major Δexon 22 splice product. F: BRCA2 c.9257–1G >C creates an in-frame 27 nucleotide deletion from exon 25 and Δexon 25. G: BRCA2 c.9501+3A>T is associated with Δexon 25. [Color figures can be viewed in the online issue, which is available atwww.wiley.com/humanmutation.]