Application Of Pcr-Rflp Techinique In Genotyping For Rs165599 Polymorphism On Comt Gene - Dinh Viet Hung

Journal of military pharmaco-medicine n o 5-2019 164 APPLICATION OF PCR-RFLP TECHINIQUE IN GENOTYPING FOR RS165599 POLYMORPHISM ON COMT GENE Dinh Viet Hung1; Dang Tien Truong1; Cao Tien Duc2; Tran Hai Anh1 SUMMARY Objectives: To establish and complete a protocol for genotyping of single polymorphism rs165599 on catechol-O-methyltransferase (COMT) gene by PCR-RFLP. Methods: DNA samples of rs165599 known genotypes by sequencing; using techniques including PCR amplification, restriction fragment length polymorphism, and agarose gel electrophoresis to determine parameters for optimization of the protocol. Results: Determining the temperature at 56 o C and 33 cycles were optimal for the protocol to genotype rs165599/COMT gene. Conclusion: The genotyping protocol for rs165599 polymorphism was established, which would be able to apply in elucidating the association of this polymorphism in neuropsychological disorders. * Keywords: COMT gene; PCR-RFLP; rs165599. INTRODUCTION COMT gene (Catechol-O-methyltransferase) encodes COMT enzyme, which several enzymes involved in degeneration of catecholamine neurotransmitters (including dopamine, epinephrine, and norepinephrine). COMT exists in two different forms: soluble short form, found in cell cytoplasm (S-COMT), and a larger form binding to the cell membrane (MB-COMT). Lachman et al (1996) found a single nucleotide polymorphism (SNP) on the COMT gene that encodes enzymes to change the activity of the COMT enzyme three to four times [6]. COMT is the most studied gene in behavioral genetics since the first report of the American Association of Schizophrenia in 1996 [7]. This gene has been studied extensively due to many factors, including the area associated with schizophrenia on chromosome 22 containing a significant 22q11 deletion, relating to metabolism of catecholamine - neurotransmitters regarded as having an association with mental disorders and psychiatric treatment [3]. Since then, many independent case-control studies, family-based as well as genome-wide studies have found associations of polymorphism on COMT gene with mental disorders [2, 3, 8]. Studies on COMT gene has also showed interactions with environmental risk factors related to mental disorders such as marijuana [4, 5]. The most common polymorphisms of COMT gene are rs4680, rs7378645, and rs165599. Rs4680 is the most studied, but found no association with schizophrenia in a Vietnamese population [1]. 1. Vietnam Military Medical University 2. 103 Military Hospital Corresponding author: Tran Hai Anh (anhhtr@yahoo.com) Date received: 10/04/2019 Date accepted: 20/05/2019 Journal of military pharmaco-medicine n o 5-2019 165 Another polymorphism has attracted attention is rs165599, which is located at the beginning of 3' end of the COMT gene, next to exon 6. This study aims at: Building a PCR-RFLP (restriction fragment length polymorphism) protocol, identifying genotypes of polymorphic rs165599/COMT for studies that evaluate the association of this SNP in schizophrenia. MATERIALS AND METHODS 1. Materials. Three DNA samples with known genotypes for polymorphic rs165599 on COMT gene by sequencing method. These genotypes were AA, GG, and AG, respectively. Other molecular chemicals include: forward and reverse primers (5’-CGACTTAGTACATCCTTC-3’ and 5’-GAGTAGAATCTTGGCTAG-3’, respectively) were synthesized by Phusa Biochem; Hot-tag master mix (QIAGEN), Emzym MspI (Invitrogen), Agarose (Thermo), pure water (Invitrogen), TBE 1X. 2. Equipment. Equipment used in PCR: small shaker for mixing - MS3 digital (IKA, USA); centrifugal centrifuge - E-centrifuge (Wealtec, USA); PCR machine - Proflex (ABI, USA). Equipment used in electrophoresis: electronic scales - TE612 (Sartorius, Germany); microwave oven (Sharp, Japan); electrophoresis (Scie-plas, UK); gel camera (UK) and some basic molecular equipment. 3. Methods. * PCR amplification: Amplification of COMT gene segment containing rs165599 by PCR reaction. 20 µL PCR reaction includes 100 ng genomic DNA, 0.5 µM per primer, Master mix i-taq 1X, and sufficient water. The amplification reaction runs on the ProFlex PCR system thermal cycle as follows: 94°C for 2 mins; 33 cycles of 94°C for 20 s, 56°C for 10 s, 72°C for 40 s; and the last step was 72°C in 4 mins; stored at 4°C. PCR products with calculated size are theoretically 628 bp. * Enzyme treatment: PCR products were treated with MspI enzyme according to the manufacturer's recommendations, summarized as follows: 10 µL PCR products, 0.5 µL enzyme, 2.5 µL Buffer, and 12 µL water. The mixture was incubated at 37°C for 5 mins. Enzyme MSPI will cut the CC/GG sequence of section 628 bp, forming two segments with sizes of 403 bp and 225 bp, respectively. * Agarose gene electrophoresis: PCR products after being treated with MSPI enzyme, ran on 2% agarose electrophoresis, at 100 V, for 60 minutes. Next, took the gel and determined the results. Results of electrophoresis of samples with AA genes showed one band sized 628 bp; samples with AG genotype showed three bands sized 628 bp, 403 bp, and 225 bp, respectively; and samples with GG genotypes showed two bands sized 403 bp and 225 bp, respectively. Journal of military pharmaco-medicine n o 5-2019 166 RESULTS AND DISCUSSION 1. Optimization of PCR. Many factors affect the results of PCR reactions such as annealing temperature (Ta), annealing time, time of the extension, enzyme activity, dNTP concentration, Mg++ concentration... Optimizing the PCR reaction needs to optimize all those factors to achieve the best results. However, this work consumes much time, efforts, and chemicals. Therefore, the actual optimization of PCR reaction usually performs the selection of standard reaction components and optimizes the annealing time (Ta) first. In the present study, we used 2x master mix solution of INtRON to save time and cost optimization of factors related to the reaction component. Ta of the reaction depends on the melting temperature (Tm) of the primer and usually less than 5 - 10oC. We conducted optimization tests at Ta range of 55 - 65 oC (figure 1). Figure 1: Results of optimization of Ta at 55 oC, 60oC, and 65oC. (55, 60, 65 were product bands of PCR reaction at Ta as 55 oC, 60oC, and 65oC, respectively; (-): negative control (in 60oC); M: Marker 100 bp) The results in figure 1 showed that the reaction occurred at Ta of 60°C for the darkest signal band (no bands at the other temperatures), with no by-products. The Ta was close to the theoretical calculations, and was no contaminated products. It can be showed that the optimal Ta for primer pairs ranged about 55°C to 60°C, therefore, we continued testing the optimization reaction at three Ta of 55°C, 56°C, and 58°C (figure 2). Journal of military pharmaco-medicine n o 5-2019 167 Figure 2: Results of optimization of Ta at 55 oC, 56oC, and 58oC. (55, 56, 58 were product bands of reaction at at Ta as 55 oC, 56oC, and 58oC; (-): Negative control; M: Marker 100 bp) The results in figure 2 showed that the reaction occurred at a Ta of 56°C giving the strongest band, then the product at Ta of 55°C, without by-product. The Ta was closed to the theoretical calculation, no contaminated products. It can be concluded that the optimal Ta for the primer pairs was about 56 oC. After repeatedly adjusting various conditions for the PCR reaction, we concluded that the annealing temperature of the reaction was 56°C and the number of cycles was 33 were optimal. Other components were recommended by the manufacturer. Thus, we have successfully optimized the PCR of the COMT fragment containing polymorphic rs165599. 2. Results of genotyping determined by RFLP. PCR products were treated with MspI enzymes according to the manufacturer's recommendations. The results were shown in figure 3 below. M 58 55 56 _ Journal of military pharmaco-medicine n o 5-2019 168 Figure 3: Electrophoresis results after enzyme treatment. (AG, GG, AA were product bands with corresponding genotypes of rs165599, respectively; (-): Negative proof; M: Marker 100bp) The results of enzyme treatment in samples with corresponding genotypes showed that the products were in accordance with those of theoretical calculations. Sample with AG genotype had three bands as 628, 403, and 225 bp; sample with GG genotype had two bands as 403 bp and 225 bp; and the sample with AA genotype had only one 628 bp band. Thus, we had successfully established the protocol for identifying the genotypes of polymorphic rs165599 on COMT gene. This protocol is simple, inexpensive, not requiring complex and expensive equipment. Thus, this protocol is applicable in studies elucidating the association of this polymorphism with neuropsychiatric diseases with a large sample size and less expense. (-) AG GG AA M 628 403 225 Journal of military pharmaco-medicine n o 5-2019 169 CONCLUSION Successfully developed the protocol for genotyping polymorphic rs165599 on COMT gene, serving studies on the association of the polymorphism and schizophrenia. REFERENCES 1. Dang Tien Truong, Nguyen Duy Bac, Pham Minh Dam, Tran Hai Anh. No association between rs821616 of DISC1 gene and susceptibility to schizophrenia in a Vietnamese population. Journal of Military Pharmaco-medicine. 2017, 42 (7), pp.48-52. 2. Allen N.C, Bagade S, McQueen M.B et al. Systematic meta-analyses and field synopsis of genetic association studies in schizophrenia: The SzGene database. Nature Genetics. 2008, 40 (7), pp.827-834. 3. Gothelf D, Law A.J, Frisch A et al. 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