Poster Presentation 25th Lorne Cancer Conference 2013

Functional analysis of TERT locus polymorphisms which are associated with risk of breast and ovarian cancer (#367)

Michael D Stutz 1 2 , Hilda A Pickett 1 2 , Stig E Bojesen 3 , Sharon E Johnatty 4 , Jonathan Beesley 4 , Georgia Chenevix-Trench 4 , Roger R Reddel 1 2
  1. Children's Medical Research Institute, Westmead, NSW, Australia
  2. Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
  3. Copenhagen General Population Study and Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark
  4. Department of Genetics, Queensland Institute of Medical Research, Brisbane, Queensland, Australia

Several independent genome-wide association studies have implicated polymorphisms in the TERT-CLPTM1L locus at 5p15.33 in cancer risk. Fine-mapping of this region has identified single nucleotide polymorphisms (SNPs) in both intron 4 and the upstream promoter region of TERT, which encodes the reverse transcriptase subunit of telomerase.

The minor alleles at intron 4 SNP sites rs10069690 and rs2242652 are associated with an increased risk of oestrogen receptor-negative breast cancer (BC), BC in BRCA1 mutation carriers and serous invasive epithelial ovarian cancer (EOC). We inserted intron 4 into a full-length TERT cDNA construct to determine the role of these SNPs in alternative splicing. Constructs were transiently transfected into a BC cell line (MDA-MB-468) and an EOC cell line (A2780), and TERT transcripts analysed by reverse transcriptase PCR (RT-PCR). We identified a novel splice variant (termed INS1b), specifically associated with the minor allele at rs10069690. INS1b contains the first 480 bp of intron 4, which introduces a premature stop codon 16 amino acids into the intronic region. This generates a severely truncated protein lacking the catalytic domain, and which is detectable by Western analysis upon over-expression. We are currently investigating the cellular consequences of INS1b expression.

The minor alleles at promoter SNP sites rs2736107, rs2736108 and rs2736109 are associated with a decreased risk of oestrogen receptor-negative BC and BC in BRCA1 mutation carriers. We used luciferase reporter constructs containing 3.9 kb of TERT promoter sequence to assess the consequence of these SNPs for TERT promoter activity. Constructs were transiently transfected into BC cell lines (MDA-MB-468 and MCF7) and normal breast epithelial cells (Bre16). The construct harbouring all three minor alleles severely reduced luciferase signals close to baseline in all cell types. These alleles are highly correlated and associate with longer telomeres. This result illustrates an apparently paradoxical association between decreased TERT promoter activity and longer telomeres, and underscores the complex and poorly understood nature of TERT regulation.