We have used a functional approach to identify candidate genes from an in vitro model of ovarian cancer suppression, and evaluated whether common germline genetic variation for 63 SNPs in these genes is associated with low-penetrance susceptibility to the disease. Of the nine genes identified, we found statistically significant evidence of association with disease risk at the 5% level for three SNPs (in genes AXIN1, CASP5 and RUVBL1) when all ovarian cancer histological subtypes were analysed, and for an additional three SNPs (in genes RUVLB1, RBBP8 and STAG3) when the analysis was restricted to the serous subtype.
The strongest associations identified were for two SNPs in RUVLB1
. However, these associations may still be false positives; more stringent significance levels are required to ensure that an identified association is a true positive. It has been proposed that the significance level of candidate gene studies should be p
Therefore, we further investigated the associations for rs13063604 and rs7650365 in RUVBL1
as part of an international multicentre case control study by the OCAC. This analysis failed to replicate independently the association for either of these SNPs, although the combined analysis of all genotyping remained marginally significant for rs13063604. This variant is located in Intron 9 of the gene and does not appear to be functionally significant per se
; however, it tags nine other SNPs in the gene, two of which are located in the 3′untranslated region. Using the bioinformatics tool Pupasuite (http://pupasuite.bioinfo.cipf.es/
), none of these SNPs were predicted to be potentially functionally significant. There is evidence to suggest that RUVBL1
plays an important role in the development of ovarian cancer and other cancer types; RUVBL1
expression is elevated in ovarian, breast, colon, bladder and lung cancers [Oncomine(Compendia Bioscience, Ann Arbor, MI)].
The data for rs13063604 and rs7650365 in RUVBL1
were stronger in serous ovarian cancer cases than in all histological subtypes combined. If different germline genetic variants confer susceptibility to different disease subtypes, then disease heterogeneity may be one of the reasons for a lack of success in identifying genetic susceptibility alleles for ovarian cancer. There is increasing evidence from high-penetrance gene studies that the underlying genetic basis of ovarian cancer can contribute to disease heterogeneity. For example, BRCA1
appear to confer susceptibility mainly to the development of the serous subtype.30
Some studies have also suggested that disease heterogeneity can be influenced by common alleles of low penetrance. Studies by the OCAC that are sufficiently large to enable stratification by subtype have identified genetic variants that may be associated with specific subtypes of the disease.22,31
From the data presented, we cannot rule out the possibility that risk associations exist for other germline genetic variants in these genes (AIFM2, AKTIP, AXIN2, CASP5, FILIP1L, RBBP8, RGC32, RUVBL1 and STAG3). The combined sample size from the three studies provides 98% power at the 5% significance level to detect a co-dominant allele with a frequency of 0.3 that confers a relative risk of 1.2, and 95% power to detect a dominant allele with a frequency of 0.1 that confers a relative risk of 1.3. Of the 303 common variants, 288 were tagged with r2 ≥ 0.8 and 290 with r2 ≥ 0.5; however, some unidentified common as yet SNPs may not have been tagged efficiently, and we did not evaluate rarer variants with MAFs <0.05. Risk associations for these genes may also exist for some of the rarer histological subtypes of ovarian cancer, but we did not have sufficient statistical power to detect such associations.
During the course of this study, a GWAS for ovarian cancer was completed and published.22
This GWAS study analysed a partly overlapping set of patients as used in this study, with cases from UKOPS and SEARCH as well as other UK studies. With access to the data from this GWAS (which analysed 1,819 cases and 2,343 controls for 507,094 genotyped SNPs and ~2 million imputed SNPs) we were able to reevaluate all tSNPs identified in these nine genes including the five SNPs that were not analysed; we found no evidence of association with disease risk in the GWAS data for all histologies combined (Supporting Information Table 6). Three SNPs were significant in the serous only analysis; two in CASP5
rs518604 and rs523104 and one in FILIP1L
rs518604 is the only SNP significant in both studies, and it is currently being investigated further in additional cases and controls.
One of the features of our study is that candidate genes were selected on the basis of having a possible functional role in an in vitro
model of ovarian cancer suppression. To our knowledge, none of the nine candidate genes investigated have been previously assessed for their association with ovarian cancer risk. For some of the genes, we identified, there are previously reported evidence of a role in ovarian cancer development, in addition to that described for RUVBL1
: For example, we have previously shown that germline variants in the BRCA1
interacting gene RBBP8
may be associated with survival after a diagnosis of ovarian cancer,16
and another study has shown downregulation of FILIP1L
in primary ovarian tumours suggesting it may be an ovarian cancer tumour suppressor gene [Oncomine (Compendia Bioscience, Ann Arbor, MI)].
Although numerous common genetic variants have now been shown to be associated with the risks of several cancer types, rarely has any functional rationale been established for their risk association. Therefore, we investigated whether SNPs within all nine genes were somatically altered in primary ovarian cancers. The SNP rs1637001 in the STAG3
gene shows significant nonrandom allelic imbalance by LOH analysis in tumours. This was shown by array CGH to be the likely result of amplification of the minor G allele; amplification rather than deletion of an allele has previously been suggested as a potential mechanism to explain detected LOH at a SNP.32
The same SNP in this gene also showed evidence of association with disease risk in serous ovarian cancer cases, although this was not confirmed in the imputed data from the GWAS. The potential synergy between a putative risk association for a germline genetic variant and the preferential somatic amplification of one of the alleles during tumour development is intriguing, and further investigation is worthwhile.
Although there are several examples in the published literature of allele-specific imbalance of polymorphic markers in primary tumours (reviewed in Refs. 33-36
), synergy between genetic risk alleles and somatic alterations has not been reported before. For example, in one study, analysis of the DAL1
gene in breast cancer found that 94% of tumours showing LOH retained the C allele of the C2166T SNP; in another study, 73% of lung tumours with LOH involving the P34
gene retained the G allele of the A106G SNP; and a third study reported 83% of breast tumours with loss of the pro allele of Arg72Pro in the P53
gene. What is unclear for the rs1637001 variant located in the 3′UTR of STAG3
is whether or not the preferential allelic amplification targets rs1637001 or STAG3
specifically. The amplified region detected by aCGH in tumours extended across several genes including STAG3
; neither is it clear that the amplification is functionally relevant to ovarian cancer development. More detailed in vitro
cell biology studies will be needed to address this. STAG3
is a component of the meiosis specific cohesion complex.37,38
It is not expressed in embryonic stem cells that form follicle like ovarian structures, and this is thought to contribute to the inability of those cells to progress through meiosis.39
The gene is activated in lymphoma cells after mutant p53 has been induced by irradiation.40
Other studies have implicated STAG3
mutations with chromosomal instability in colorectal cancer,41
and the gene is associated with chromosome segregation and downregulation in testicular cancer.42
In conclusion, we have used a functional model of ovarian cancer suppression to identify and test candidate susceptibility genes for ovarian cancer. We have identified functional evidence suggesting allele-specific imbalance for somatic genetic alterations in primary ovarian tumours for a SNP in the STAG3 gene. These studies highlight the importance of international consortia like the OCAC to validate putative genetic risk associations; but they also emphasise some of the limitations and challenges that face the scientific community in trying to elucidate the functional rationale underlying the numerous genetic associations that have been identified for multiple complex disease traits.