PCR-RFLP optimisation for OPRM1 rs540825 and rs510769  gene polymorphisms and their allele/genotype frequencies  in Turkish population

KOTİLOĞLU, Selin ÖZKAN

doi:10.5505/TurkHijyen.2023.43433

PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population

Selin ÖZKAN KOTİLOĞLU (Kırşehir Ahi Evran Üniversitesi, Fen-Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik Bölümü, Kırşehir, Türkiye)

Türk Hijyen ve Deneysel Biyoloji Dergisi

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Yıl: 2023 Cilt: 80 Sayı: 3 Sayfa Aralığı: 355 - 364 Metin Dili: İngilizce DOI: 10.5505/TurkHijyen.2023.43433 İndeks Tarihi: 28-09-2023

PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population

Öz:

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 wild-type 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 PCR-RFLP 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.

Anahtar Kelime: OPRM1 gene single nucleotide polymorphism PCR-RFLP optimisation genotyping

OPRM1 rs540825 ve rs510769 gen polimorfizmleri için PCR-RFLP yöntemi optimizasyonu ve Türk popülasyonundaki alel/genotip frekansları

Öz:

Amaç: Opioid bağımlılığı, çevresel ve genetik bileşenleri olan önemli bir halk sağlığı problemidir. Opioidlerin başlıca hedefi olan mu opioid reseptörü (MOR) OPRM1 geni tarafından kodlanmaktadır. OPRM1 geninde bulunan polimorfizmlerin MOR’un fizyolojisini ve özelliklerini değiştirdiği gösterilmiştir ve bunun opioid bağımlılığında bir etkisi olabileceği düşünülmektedir. OPRM1 polimorfizmlerinden rs540825 ve rs510769’un madde bağımlılığı ile ilişkisi yapılan çalışmalarda gösterilmiştir. Bu çalışmanın amacı bağımlılıkta ve bağımlılık tedavisinde önemi gittikçe artan OPRM1 rs540825 ve rs510769 polimorfizmleri için güvenilir, güçlü ve her laboratuvarda uygulanabilecek bir genotiplendirme yöntemi geliştirmektir. Yöntem: OPRM1 gen polimorfizmlerinden rs510769 için yeni ve rs540825 içinse iyileştirilmiş polimeraz zincir reaksiyonu-restriksiyon parçacık uzunluk polimorfizmi (PCR-RFLP) tekniğine dayanan yöntemler geliştirilmiştir. OPRM1 geninde bu iki polimorfizmin bulunduğu bölgeler PCR ile çoğaltılmış ve sonrasında da rs540825 için NlaIII enzimi ve rs510769 için SspI enzimi kullanılarak RFLP yöntemi uygulanmıştır. Sağlıklı bireylerden oluşan 70 kişilik bir Türk popülasyonu örneklemi üzerinde geliştirilen yöntemler test edilmiştir. Bulgular: OPRM1 rs540825 ve rs510769 polimorfizmleri için PCR ürünleri sırasıyla 276 baz çifti (bç) ve 563 bç uzunluğundadır. OPRM1 rs540825 için kesim ürünleri yabanıl AA genotipi için 276 bç iken polimorfik TT genotipi için 153 bç ve 123 bç boyutundadır. OPRM1 rs510769 polimorfizmini içeren PCR ürününün kesimi sonucunda polimorfik TT genotipi için 364 bç, 146 bç ve 53 bç’lik ürünler elde edilirken, yabanıl CC genotipi için 417 bç ve 146 bç boyutunda kesim ürünleri elde edilmiştir. OPRM1 rs540825 polimorfizminin alel ve genotip frekansları A aleli için % 29 ve T aleli için % 71 olarak, AA genotipi için % 10, AT genotipi için % 38,6 ve TT genotipi için de % 51,4 olarak hesaplanmıştır. OPRM1 rs510769 polimorfizmi için, bireylerin %60’ının CC genotipine, sırasıyla % 24,3 ve % 11,4’ ünün CT ve TT genotiplerine sahip olduğu gösterilmiştir. OPRM1 rs510769 polimorfizminin alel frekansları C aleli için % 75 ve T aleli için % 25 olarak belirlenmiştir. Sonuç: Yeni, güvenilir ve uygulaması kolay PCR-RFLP yöntemleri OPRM1 geninde bulunan rs540825 ve rs510769 polimorfizmlerinin genotiplendirilmesi için geliştirilmiştir. Ayrıca bu polimorfizmlerin alel ve genotip frekansları Türk popülasyonunda belirlenmiştir.

Anahtar Kelime: OPRM1 geni tek nükleotit polimorfizmi PCR-RFLP optimizasyon genotiplendirme

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

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APA	KOTİLOĞLU S (2023). PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. , 355 - 364. 10.5505/TurkHijyen.2023.43433
Chicago	KOTİLOĞLU Selin ÖZKAN PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. (2023): 355 - 364. 10.5505/TurkHijyen.2023.43433
MLA	KOTİLOĞLU Selin ÖZKAN PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. , 2023, ss.355 - 364. 10.5505/TurkHijyen.2023.43433
AMA	KOTİLOĞLU S PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. . 2023; 355 - 364. 10.5505/TurkHijyen.2023.43433
Vancouver	KOTİLOĞLU S PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. . 2023; 355 - 364. 10.5505/TurkHijyen.2023.43433
IEEE	KOTİLOĞLU S "PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population." , ss.355 - 364, 2023. 10.5505/TurkHijyen.2023.43433
ISNAD	KOTİLOĞLU, Selin ÖZKAN. "PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population". (2023), 355-364. https://doi.org/10.5505/TurkHijyen.2023.43433

APA	KOTİLOĞLU S (2023). PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. Türk Hijyen ve Deneysel Biyoloji Dergisi, 80(3), 355 - 364. 10.5505/TurkHijyen.2023.43433
Chicago	KOTİLOĞLU Selin ÖZKAN PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. Türk Hijyen ve Deneysel Biyoloji Dergisi 80, no.3 (2023): 355 - 364. 10.5505/TurkHijyen.2023.43433
MLA	KOTİLOĞLU Selin ÖZKAN PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. Türk Hijyen ve Deneysel Biyoloji Dergisi, vol.80, no.3, 2023, ss.355 - 364. 10.5505/TurkHijyen.2023.43433
AMA	KOTİLOĞLU S PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. Türk Hijyen ve Deneysel Biyoloji Dergisi. 2023; 80(3): 355 - 364. 10.5505/TurkHijyen.2023.43433
Vancouver	KOTİLOĞLU S PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population. Türk Hijyen ve Deneysel Biyoloji Dergisi. 2023; 80(3): 355 - 364. 10.5505/TurkHijyen.2023.43433
IEEE	KOTİLOĞLU S "PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population." Türk Hijyen ve Deneysel Biyoloji Dergisi, 80, ss.355 - 364, 2023. 10.5505/TurkHijyen.2023.43433
ISNAD	KOTİLOĞLU, Selin ÖZKAN. "PCR-RFLP optimisation for OPRM1 rs540825 and rs510769 gene polymorphisms and their allele/genotype frequencies in Turkish population". Türk Hijyen ve Deneysel Biyoloji Dergisi 80/3 (2023), 355-364. https://doi.org/10.5505/TurkHijyen.2023.43433