New technique aids discovery of cancer gene candidate

Stem cells
Stem cells grown in the laboratory

Dr Ben Davies and his colleagues in WTCHG’s transgenics core have developed a new method for comparing the functional effects of genetic variants in non-coding parts of the genome. The technique has helped an international team of collaborators to discover how a variant strongly associated with testicular cancer drives the development of the disease.

DNA that encodes protein sequences represents only around 2 per cent of the human genome. Much of the rest, far from being the redundant debris of evolution, plays a role in regulating the activity of genes. Investigating the functional consequences of variation in these regions remains a challenging task, central to unravelling the meaning behind the multitude of single-letter variants, known as SNPs, found to be associated with susceptibility to disease.

Dr Gareth Bond and his colleagues at the Ludwig Institute of Cancer Research, WTCHG’s neighbour on the University of Oxford’s Biomedical Research Campus, investigated approximately 900 SNPs associated with the risk of cancer. At the binding site of the key tumour suppressor protein, p53, in a non-coding region within the KITLG gene, they found a SNP strongly associated with the risk of developing testicular cancer. The product of this gene is known to play an essential role in the regulation of cell survival and proliferation.

As they report in Cell, p53 bound much more strongly to sequence containing the cancer-related SNP, increasing the activity of the KITLG gene and causing cells to proliferate. They suggest that this regulatory circuit may underlie the increased risk of testicular cancer in people carrying the SNP. Interestingly, the same circuit may reduce the risk of skin cancer by increasing the production of pigmented skin cells that protect against UV radiation.

Working with embryonic stem cell lines, Davies and his colleagues fused different versions of the variant KITLG DNA to a reporter gene, whose activity can easily be monitored. They positioned these constructs at identical sites within the genome using a new method known as cassette exchange, which made it easier to compare them. The piece of non-coding DNA from the KITLG region enhanced the reporter gene's activity. Importantly, the presence of the cancer-associated variant turned up the dial even further.

This study represents the first use of cassette exchange technology in stem cells to allow a more precise and accurate comparison of regulatory sequence variants. ‘We’re keen to exploit this approach in validating other key regulatory SNPs associated with disease that are emerging from the Centre’s genetic association and resequencing studies’, says Davies, who heads the Transgenics Core at the WTCHG.

Further reading:

Jorge Zeron-Medina, Xuting Wang, Emmanouela Repapi et al. A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection. Cell. 2013; 155:410-422.

‘Testicular cancer may be the price paid for sun protection’, Science online, 10 October 2013.