A Kallikrein 15 (KLK15) single nucleotide polymorphism located close to a novel exon shows evidence of association with poor ovarian cancer survival

Abstract

Background: KLK15 over-expression is reported to be a significant predictor of reduced progression-free survival and overall survival in ovarian cancer. Our aim was to analyse the KLK15 gene for putative functional single nucleotide polymorphisms (SNPs) and assess the association of these and KLK15 HapMap tag SNPs with ovarian cancer survival.

Results: In silico analysis was performed to identify KLK15 regulatory elements and to classify potentially functional SNPs in these regions. After SNP validation and identification by DNA sequencing of ovarian cancer cell lines and aggressive ovarian cancer patients, 9 SNPs were shortlisted and genotyped using the Sequenom iPLEX Mass Array platform in a cohort of Australian ovarian cancer patients (N = 319). In the Australian dataset we observed significantly worse survival for the KLK15 rs266851 SNP in a dominant model (Hazard Ratio (HR) 1.42, 95% CI 1.02-1.96). This association was observed in the same direction in two independent datasets, with a combined HR for the three studies of 1.16 (1.00-1.34). This SNP lies 15 bp downstream of a novel exon and is predicted to be involved in mRNA splicing. The mutant allele is also predicted to abrogate an HSF-2 binding site.

Conclusions: We provide evidence of association for the SNP rs266851 with ovarian cancer survival. Our results provide the impetus for downstream functional assays and additional independent validation studies to assess the role of KLK15 regulatory SNPs and KLK15 isoforms with alternative intracellular functional roles in ovarian cancer survival.

Figure 1

Diagrammatic representation of human KLK15 mRNA transcripts and confirmation of the exon B variant by RT-PCR. (A) Various isoforms (1-6) of KLK15 derived from the NCBI database are shown with the two new isoforms identified from the EST database containing novel exons, A and B, highlighted in red boxes. The unshaded boxes represent the noncoding exons, shaded black boxes for the isoforms represent the coding exons and the connecting lines the introns. Numbers inside boxes or above connecting lines represent exon or intron lengths in base pairs respectively. H, denotes histidine; S, denotes serine; and D, denotes aspartate [amino acids of the catalytic triad of the (putative) encoded proteins]. The arrowhead points to the common start codon and astericks (*) to the stop codon positions. (B) PCR was conducted on the cDNA from five different cell lines HOSE17.1 (1), PEO1 (2), OVCA432 (3), SKOV3 (4), LNCaP (5) and no cDNA (6) using the Forward primer in exon B and reverse primer in exon 2. Two products were obtained corresponding to with (144 bp) and without (70 bp) exon 1.

Figure 2

The putative KLK15 promoter region: (A) The 11 kb region upstream of exon 1 on chromosome 19 (56020307 to 56037591 bp, NCBI build 36.3) was downloaded for in silico analysis (A) schematic of the 5' region of the KLK15 gene with significant motifs noted: 2 putative Androgen Response Elements (AREs) were found by both JASPAR and Cister programs (red arrows); 4 putative Estrogen Response Elements (EREs) were found by both ERE finder and Cister programs (blue arrows); 2 putative cis-element clusters which indicate putative promoter regions found by Cister (green arrows); 2 putative promoter regions that overlap with CpG islands found by WWWPromoter Scan, Promoter Inspector and CpG Island Finder (yellow arrows) (B) AREs (boxes), EREs (triangle) and Nuclear Hormone Receptor Binding Sites (NHRBs) (circles) as predicted by individual softwares. The ARE "cluster" consists of 16 AREs. The colored bars (blue, red, green, orange, yellow) in the sequence indicate the 5 regions that were chosen for sequencing genomic DNA. The single nucleotide polymorphism (SNP)-modeling results are shown by color coding the functional SNPs validated in bold. Orange and red text coloring indicate a gain or loss of ERE motifs respectively, while green and blue indicate ARE gain or loss. The two brown SNPs indicate a loss of one ERE and a loss of two AREs. An additional four validated SNPs viz rs2659052, rs11880663, rs2659051, rs2659055 have been added to NCBI databases (not modeled here) from the time of our analysis.