Genetic influences on methylation of the mu-opioid receptor gene in black South African nyaope users

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Background: Nyaope is a highly addictive heroin derivative that elicits its effects through interaction with the µ-opioid receptor. Genetic and epigenetic mechanisms, such as mutations and (cytosine-phospho-guanosine) CpG methylation, alter the function of the mu-opioid receptor gene (OPRM1) and protein. The single nucleotide polymorphisms (SNP) rs1799971 (118 A>G) and intronic rs3778150 (T>C) in the OPRM1 have previously been associated with heroin use. The SNP 118 A>G can influence methylation levels of OPRM1 and alter mRNA and protein expression. This study compared the degree of OPRM1 methylation and frequency of SNPs 118 A>G and SNP rs3778150 in peripheral blood samples of black South African male nyaope users (n=200) to age-and-gender-matched screened controls (n=55) in a South African population. Method: DNA was extracted from whole blood samples, bisulfite converted, amplified by methylation-specific PCR and sequenced to determine individual and total methylation levels across a total of 29 CpGs within the OPRM1 promoter region (chr6: 154331689- 154332224bp). The sequencing data was analysed by each of the two methods, methylation assignment bias and partial methylation bias methods. Results: We found that the total methylation levels were lower in nyaope-users than in controls when using the methylation assignment bias method. Only CpG 29 sites showed substantial methylation in the control group in both approaches. In the partial methylation bias method, our main findings showed no differences in the total methylation levels between the two groups while most CpGs displayed significantly higher levels of partial methylation in nyaope-users while controls exhibited significantly higher unmethylation sites at some CpGs. The AG genotype frequency for SNP 118 A>G, was higher in controls than in nyaope-users while other genotypes (AA & GG) for SNP rs1799971 and (TT, CT, CC) SNP rs3778150 were broadly similar in both groups. While the TT genotype SNP rs3778150 occurred more frequently in the control group, there was no haplotype relationship between SNP rs1799971 and rs3778150. Neither was there any correlation observed between nyaope consumption and the presence of SNP rs1799971 and/or rs3778150 in the study populations. Conclusion: This study provided evidence that methylation may not be associated with nyaope addiction and that SNPs rs1799971 and rs3778150 did not alter the methylation status of the OPRM1 promoter or contribute to nyaope addiction in a black South African male population. The findings of this study are a novel contribution to the body of literature as, to the best of our knowledge, this is the first study to investigate OPRM1 gene methylation patterns and the frequency of these specific SNPs in a South African population of nyaope users.
A research report submitted in partial fulfilment of the requirement for the degree of Master of Science in Medicine to the Faculty of Health Sciences, University of the Witwatersrand, School of Physiology, Johannesburg, 2023