A deep intronic recurrent CHEK2 variant c.1009-118_1009-87delinsC affects pre-mRNA splicing and contributes to hereditary breast cancer predisposition

Abstract

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.

Keywords: Breast cancer; Deep intronic CHEK2 variant; Genetic testing; NGS; RNA analysis.

Fig. 1

Characterization of the c.1009–118_1009–87delinsC CHEK2 variant. (A.) NGS-based DNA sequencing visualized in the Integrative Genomics Viewer (IGV). The dashed gray lines indicate the deletion borders, and the dashed blue arrow denotes the deletion of 32bp following the nucleotide c.1009-86 at the 3′-end of intron 9 with the insertion of cytosine. (B.) DNA Sanger sequencing of the variant and wild-type samples. (C-E.) RNA (cDNA) Sanger sequencing of wild-type sample (C.), variant sample without (D.) and with (E.) NMD inhibition (cycloheximide), showing the increase of aberrant splicing variant signal peaks after NMD inhibition. (F.) RNA panel NGS from a wild-type control (top) and from a carrier of the c.1009-118_1009-87delinsC variant after NMD inhibition (bottom). Note the difference between wild-type and variant RNA in number (coverage) of intronic retentions (red dashed-line boxes) resulting from the aberrant pre-mRNA splicing. Solid blue arrows indicate sequencing context of aberrant reads showing 86b retained from intron 9, interrupted by 32b deletion replaced by single cytosine insertion, followed by 24b from intron 9: r.1008_1009ins1009-142_1009-1del1009-118_1009-87insC. We hypothesize that the reassembled primary transcript enhances the pre-existing alternative acceptor splice site TT|ga in intron 9, which precedes the canonical acceptor splice site upstream of exon 10. Note: The IGV visualizes the CHEK2 sequence in a reverse complement according to the CHEK2 gene reverse orientation on the chromosome 22. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)