Connecting the dots

Dr Stacey Edwards is uncovering the mechanism responsible for inherited disorders that occur seemingly without a clear genetic basis.

Why is it that female members of some families with a history of breast cancer can test negative for mutations or deletions in BRCA1 and BRCA2, along with the other usual genetic suspects in inherited breast cancers? And why is it that extended multigeneration pedigree diagrams for such families have an excess of black circles - each representing an female affected by breast cancer - which is strongly suggestive of inherited risk, but which defies explanation by Mendel’s rules of inheritance?

Breast cancer researcher Dr Stacey Edwards has been doing a spot of genomic ghost-busting in an attempt to resolve this mystery of the so-called ‘missing heritability’, where the mutations and deletions only tell a part of the story. “Inherited mutations and deletions in known breast cancer genes accounts for only 30% of familial breast cancers,” she says.

In the classic model of familial cancers, a mutation in a protein-coding region of a cancer gene disrupts its expression, leading to cancer. Yet, according to Edwards, at least some of the unexplained cases of families with an abnormally high risk of breast cancer can be put down to missing heritability in the form of DNA variants in unidentified regulatory elements or transcription factors.

She has recently been investigating this hypothesis by pouring over the data that came out of a recent study conducted by the UK-based Breast Cancer Association Consortium (BCAC), which assembled a huge data bank of genomic data from 39 breast cancer case-control sets, representing a total of 49,068 cases of breast cancer and 48,772 unaffected controls, predominantly of European ancestry.

They performed a genome-wide association study (GWAS) on the data looking for common variants associated with an increased susceptibility to breast cancer. The GWAS confirmed new breast-cancer susceptibility loci on chromosomes 9, 10 and 11. The strongest association involved a variant named rs614367, at the 11q13 locus on the long arm of chromosome 11.

The increased risk of breast cancer involving rs614367 was specific  to breast cancers that expressed oestrogen receptors (ERs), and was strongest for ER-positive breast cancers that also had an excess of  progesterone receptors.

Spooky action

Edwards was invited by the consortium to investigate the link between rs614367 and ER-positive cancers. Her collaborators fine-mapped the 11q13 region, again by GWAS, to determine if rs614367 was indeed the causal variant. What they found was that two quite different variants were responsible for breast cancer risk.

Edwards has proposed a model that explains the non-Mendelian pattern of inheritance in breast cancers associated with variation at the 11q13 as a consequence of a long-distance interaction between two DNA elements on chromosome 11. She has shown that the cell-cycle gene Cyclin D1 (CCND1) on chromosome 11 interacts with an enhancer that lies at a substantial remove from the gene, 125 kilobases away. Four wild-type alleles of the enhancer element, featuring different SNPs, can occupy the locus.

“Genetically, any one of the four could have a causal role, but our study showed that only two of these variants alter the function of the enhancer element,” says Edwards. “When the minor alleles of these two SNPs are present at the 11q13 locus, it stops the activation of CCND1.”

The explanation for this ‘spooky action at a distance’ (to appropriate Albert Einstein) lies in the fact that chromosomal DNA is nonlinear; it is packaged as chromatin, which are long, slender threads of DNA coiled around tiny beads of histone proteins. Multiple interactions of coiling and supercoiling compact some three metres of DNA, carrying the 3.08 billion base pairs of the human genome, to fit into the tiny volume of a cell nucleus - with room to spare.

Using a technique called chromosome conformation capture, Edwards showed that the 3D packaging arrangement positions CCND1 close enough to its enhancer sequence to ‘talk’ across the narrow gap. In molecular genetics terms, although they lie far apart in cis (on the same DNA strand), they lie physically close enough to interact in trans (between DNA strands).

According to Edwards, the minor allele carrier combination is the most potent off-switch for the enhancer, and a throw of the meiotic dice determines its presence or absence in family members; only females are at risk because males do not produce enough oestrogen to fuel the growth of oestrogen-dependent breast cancers.

Explanatory power

Edwards’ study of the interaction between CCND1 and its remote enhancer was one of the first of its type, and showed the value of GWAS in combination with follow-up functional studies for illuminating new mechanisms of cancer, particularly those that might account for the missing heritability.

“The chromosome conformation capture provides evidence that the CCND1 gene and its enhancer are interacting at long range,” she says. “I then went on to perform a series of reporter assays where I positioned the CCND1 promoter close to different alleles of the enhancer sequence.”

The technique involves adding the promoter and enhancer DNA sequences into a construct containing the luciferase gene and measuring the resulting luminescence when the nearby enhancer activates the gene. The brighter the glow, the more potent the enhancer. It turned out that some alleles acted as repressors, silencers or insulators, but the experiments confirmed for Edwards that the combined minor alleles act as a potent repressor of CCND1 expression.

She and her colleagues believe the inheritance of combinations of low-risk alleles with Cyclin D1 may explain some of the non-Mendelian, low-penetrance pattern of breast cancer in families where women are at increased risk of oestrogen-dependent breast cancers. Edwards expects the finding to be “extraordinarily valuable” for diagnostic and prognostic purposes in women whose families show a pattern of increased risk of certain oestrogen-dependent breast cancers.

She also works on the BRCA1 gene, another major player in breast cancer. “Coding mutations in BRCA1 and BRCA2 collectively account for only 5-10% of familial breast cancers,” she says. “BRCA1 mutations have only been identified in a proportion of patients with tumours suggestive of a BRCA1 defect, many of which display reduced levels of BRCA1 expression. I’m planning to investigate whether BRCA1 may have a strong enhancer that is required for normal expression. A mutation in the enhancer could explain loss of BRCA1 expression in some cases where the gene itself is unaffected by mutation.

“Little more than a decade ago, we didn’t have a human genome sequence or SNP chips to identify individual variation. Nobody appreciated how genetically unique we are, and why one sibling - even in identical twins - develops cancer while another doesn’t.

“We have already identified lots of genes involved in breast cancer, which is a very heterogeneous disease, and we already have some very good drugs in the clinic. The focus has been on single genes, but the future will be more about particular combinations of genes - or alleles - that confer an increased risk of breast cancer. Increasingly, the genes are going to tell us which drugs to use.”

She says the novel mechanism she has identified in breast cancer may have analogues in other diseases, such as heart disease, asthma or even Alzheimer’s disease - researchers in these fields may find a fruitful line of inquiry involving genomic action at a distance. It might appear spooky at first, but once the phenomenon is explained in concrete biochemical terms, it can become a potent tool for understanding the heritability, or otherwise, of many diseases, and may even help to improve diagnostics and treatment.

Dr Stacey Edwards completed her PhD in molecular biology and biochemistry at the University of Queensland in 2002. She received a C. J. Martin Fellowship for a postdoctoral position at the Breakthrough Breast Cancer Research Centre in London. On returning to the School of Chemistry and Molecular Biosciences at the University of Queensland, she was awarded a prestigious National Breast Cancer Foundation (NBCF) Fellowship to identify new mutation targets in breast cancer susceptibility genes. The NBCF is a sponsor of this year’s Lorne Cancer Conference.