SUMMARY
Prostate cancer (PCa) is recognized as one of the most common malignancies that affects the male population globally. The identification of PCa susceptibility genes is of great importance. Breast Cancer gene 1 (BRCA1) is an important tumor suppressor gene that plays a central role in the maintenance of genomic integrity by promoting the repair of double-strand breaks of DNA. Because of its indirect involvement, the impact of Ataxia Telangiectasia Mutated (ATM) in repairing DNA has led to its classification among other genes linked to compromised DNA repair. The aim of this study was to use exome sequencing to discover variants linked to PCa susceptibility. Furthermore, this investigation aimed to examine the silencing effect of promoter methylation on the gene expression of BRCA1 and ATM genes in patients with PCa in the Erbil governorate, Kurdistan region, Iraq.
The collection of Formalin Fixed Paraffin Embedded (FFPE) samples took place in Erbil City, Iraq, specifically at Rizgary Hospital, PAR Hospital, and Al-Mufti’s private laboratory. In the process of analysis, a total of 86 samples were examined, with 4 samples being chosen randomly for the execution of exome sequencing. In terms of gene expression, the study encompassed 30 paired cancer and non-cancerous neighboring tissues. The identification of mutations was achieved through exome sequencing. The assessment of BRCA1 and ATM expression in PCa patients was conducted using quantitative real-time PCR (qPCR). In the promoter methylation study, 40 FFPE tissue samples were collected from age-matched individuals, comprising 30 pathologically confirmed PCa cases and 10 prostatic tissues taken from individuals who, during diagnosis, were found to be negative for PCa. Data about demographic and clinical information, such as pathological stage, age, and PSA level, were gathered from the medical records. The impact of the promoter methylation was forecasted using the DNA bisulfite conversion technique and Methyl-Specific PCR (MSP) with specific primers for the BRCA1 and ATM promoter region. Sanger sequencing was used to validate the positive results of MSP through sequencing the promoter region of BRCA1 and ATM genes.
In the exome sequencing study, ten variants were detected in six genes: BRCA1, MSH6, TP53, BRCA2, NRAS, and PMS2. The analysis revealed a significant down-regulation of the BRCA1 (p<0.0001) and ATM (p<0.0001) gene expression in tumor samples as compared to non-cancerous neighboring tissues. Among the 30 patients examined, 76.6% (23 cases) and 60% (18 cases) were found to have BRCA1 and ATM promoter methylation respectively, and none of the non-cancerous tissues appeared to have DNA methylation. BRCA1 promoter methylation was positively associated with the advanced stage of disease (p=0.007) and Gleason score (p=0.01). There was a positive association observed between ATM promoter methylation and the advanced stage of disease (p=0.004) as well as Gleason score (p=0.01). The analysis revealed a significant down-regulation of the BRCA1 and ATM gene expression in methylated tumor samples as compared to non-methylated tissue samples, suggesting the role of epigenetic silencing. The results of Sanger sequencing showed multiple methylated CpG sites in the promoter region of BRCA1 and ATM genes of PCa tissue samples.
To the best of our knowledge, this is the first study investigating the variation of repair genes, methylation status and levels of BRCA1 and ATM mRNA transcripts among PCa patients in the Kurdistan region. Our findings reveal the presence of various genetic alterations within several genes. The findings of this study suggest that promoter hypermethylation of the BRCA1 and ATM genes could serve as a viable biomarker for PCa, marking a significant discovery. Assessing BRCA1/ATM gene expression in PCa patients using qPCR can provide valuable information for risk assessment in terms of overall survival and PCa-specific mortality.