A Splice Site Variant of CDK12 and Breast Cancer in Three Eurasian Populations

Abstract

CDK12 is a member of the cyclin-dependent kinase family that acts as regulator of DNA damage response gene expression. A c.1047-2A>G splice site variant of the CDK12 gene was recently reported to strongly associate with hereditary breast and ovarian cancer in patients of Tatar ethnic origin. To gain more insight into the potential risk and the population spread of the c.1047-2A>G variant, we have genotyped three breast cancer case-control series of Tatar, Bashkir and Kazakh ethnicity. We identified c.1047-2A>G in 6/155 cases and 12/362 controls of Tatar ancestry, 0/96 cases and 9/189 controls of Bashkir ancestry, and 1/131 cases and 0/154 controls of Kazakh ancestry (Mantel-Haenszel odds ratio 0.72, 95% CI 0.30-1.70, p = 0.45). Consistent with the absence of a large effect, bioinformatic analyses predicted that c.1047-2A>G modulates alternative splicing of a NAGNAG sequence rather than constituting a loss-of-function allele, and RT-PCR analyses of c.1047-2A>G heterozygous lymphocytes verified the usage of the predicted alternative acceptor site. Our study confirms a high prevalence of CDK12*c.1047-2A>G in the Tatar and Bashkir population but excludes a role as a clinically actionable high-risk breast cancer mutation.

Keywords: DNA double-strand break repair; breast carcinoma; chromosome breakage syndrome; founder mutation; genetic susceptibility.

Figure 1

Screening and splice site analysis of CDK12 c.1047-2A>G. (A) Identification of CDK12 c.1047-2A>G by means of MspI restriction fragment length polymorphism analysis on a 2% agarose gel. Mutation-specific cleavage produces a 154/150 bp band as exemplified in lane 2, with lanes 1 and 3 showing samples with wildtype genotypes. (B) Comparative assessment of Maximum Entropy 3′-splice site scores in the wildtype and the mutant context. Splice site scores were obtained from the MaxEntScore site (http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html), accessed on Jan 15, 2019. The wildtype sequence harbors two adjacent acceptor splice sites of similar scores (upper two sequences), and the mutant sequence still can make use of the downstream site (bottom two sequences). (C) Sanger sequencing of RT-PCR products at the border between CDK12 exons 1 and 2. Upper panel: wildtype control, Lower panel: CDK12 c.1047-2A>G heterozygous carrier. Exon borders are indicated by a blue bar, and the main isoform is marked in bold while the minor isoform is indicated in italics. Note that the triplet of the alternative site (TAG, blue) is included in a majority of transcripts represented by the wildtype control sequencing reads, but only in a minority of transcripts from the CDK12 c.1047-2A>G heterozygous carrier.