Association Between RASSF1A Promoter Methylation and Testicular Germ Cell Tumor: A Meta-analysis and a Cohort Study

Abstract

Background: The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk.

Materials and methods: Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O⁶-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood.

Results: Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy.

Conclusion: The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.

Keywords: BEP chemotherapy; DNA methylation; MGMT; RASSF1; testicular cancer.

Figure 1. Location of the pyrosequencing assay within the RASSF1A (A) and MGMT (B) genes used for analysis of CpG methylation status. Position of the analyzed CpG sites is shown with absolute genomic coordinates according to the human genome GRCh37/hg19 assembly. Positions of the pyrosequencing assays on the chromosome 3 (A) and chromosome 10 (B) are indicated by the yellow bar. Translation start is indicated with the arrow above the ATG start codon (A).

Figure 2. Forest plot of the logarithm of odds ratio (OR) for the association between the RASSF1A promoter methylation and testicular cancer. Selected publications are listed on the Y-axis with the summary (random effects model) at the bottom. Error bars represent 95% confidence intervals.

Figure 3. Funnel plot of the logarithm of the odds ratio with its corresponding standard error (Y-axis), where each black dot represents one of the six studies included in the meta-analysis, shows no apparent asymmetry would have indicated bias across studies. The absence of bias was formally confirmed using Egger’s test (p=0.679).

Figure 4. Methylation levels of the individual CpG sites within the RASSF1A (A) and MGMT (B) gene promoters. Methylation level is determined by pyrosequencing and is expressed here as a percentage. The distance between the CpG sites corresponds to their relative position within the analyzed DNA fragment. Points (triangle, circle and square) represent the mean methylation level for each group. Lines are drawn as a visual aid for illustrating the spatial organization of methylation levels along the analyzed region. A) The CpG sites 2, 3, 4, 5 and 7 in the analyzed fragment within the RASSF1A promoter show statistically significant difference between patients (before chemotherapy) and healthy controls. Methylation at the site CpG5 shows statistically significant difference (p=0.0115) between the patients before and after BEP chemotherapy (indicated by vertical dashed line). Shaded area emphasizes CpG methylation difference between healthy controls and testicular cancer patients. B) No significant difference was found in the MGMT promoter methylation.