PCR-RFLP Optimisation For OPRM1 Rs540825 and Rs510769 Gene Polymorphisms and Their Allele/Genotype Frequencies in Turkish Population

Abstract

Objective: Opioid dependence, which has environmental and genetic components, is an important public health problem. OPRM1 gene encodes mu opioid receptor (MOR) which is a primary target for opioids. Polymorphisms on the OPRM1 gene have been shown to alter the properties and physiology of MOR and also may have impact on opioid dependence. The association between OPRM1 rs540825 and rs510769 polymorphisms and substance dependence have been shown through various studies. The purpose of this study is to develop reliable, robust and easily applicable genotyping procedures for OPRM1 rs540825 and rs510769 polymorphisms as they possess rising importance in the context of addiction and therapy success. Methods: A novel and an improved method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to determine OPRM1 gene polymorphisms at positions rs510769 and rs540825, respectively. OPRM1 gene regions containing these two polymorphisms were amplified using PCR; then, RFLP method was performed using the restriction enzymes NlaIII and SspI for rs540825 and rs510769, respectively. A total of 70 healthy samples from the Turkish population were tested to evaluate these two developed PCR-RFLP methods. Results: Amplicons were 276 base pair (bp) and 563 bp in length for rs540825 and rs510769, respectively. The length of the restriction products of OPRM1 rs540825 were 276 bp for wild type AA genotype, 153 bp and 123 bp for polymorphic TT genotype. Digestion of PCR region including rs510769 polymorphism yielded 364 bp, 146 bp and 53 bp fragments for polymorphic TT genotype and 417 bp and 146 bp fragments for wildtype CC genotype. Allelic and genotypic frequencies of rs540825 in the Turkish population were calculated as 29 % for allele A and 71 % for allele T, 10 % for AA, 38.6 % for AT, and 51.4 % for TT. For rs510769 polymorphism, 60 % of the individuals had CC genotype, 24.3 % and 11.4 % of them had CT and TT, respectively. Allele frequencies of rs510769 polymorphism were 75 % for allele C and 25 % for allele T. Conclusion: Novel, reliable and easy-to-apply PCRRFLP technologies were developed to detect rs540825 and rs510769 polymorphism in the OPRM1 gene. Moreover, genotype and allele frequencies of them were determined in the Turkish population.