To assess the frequency allele and genotypic distribution of polymorphism rs165599 in catechol-O-methyltransferase in Kinh ethnic-patients with schizophrenia. Subjects and methods: A case-control study consisted of 227 schizophrenic patients and 92 healthy controls in the Kinh ethnic.
Journal of military pharmaco-medicine no8-2019 POLYMORPHIC CHARACTERISTICS OF RS165599 IN COMT GENE IN SCHIZOPHRENIA PATIENTS IN KINH POPULATION Dinh Viet Hung1; Dang Tien Truong2; Ngo Van Nhat Minh2 Cao Tien Duc1; Tran Hai Anh2 SUMMARY Objectives: To assess the frequency allele and genotypic distribution of polymorphism rs165599 in catechol-O-methyltransferase in Kinh ethnic-patients with schizophrenia Subjects and methods: A case-control study consisted of 227 schizophrenic patients and 92 healthy controls in the Kinh ethnic The rs165599 was genotyped by PCR-RFLP and statistical analysis was performed by Chi-Square test Results: The frequency of allele A and G in schizophrenic patients were 72.36% and 69.94%, respectively The frequency of allele A and G in healthy controls were 27.64% and 30.06%, respectively (p > 0.05) In terms of distribution of AA genotype, the rate was 78.75% (patients) and 21.25% (controls) In terms of distribution of AG and GG genotype in schizophrenic patients and healthy controls were respectively: AG (66.05%, 33.95%); GG (74.03%, 25.97%) (p > 0.05) Conclusion: There was no correlation among the frequency of allele, genotypic distribution of polymorphism rs165599 and schizophrenia in the Kinh ethnic * Keywords: Schizophrenia; Rs165599; COMT gene; PCR-RFLP; Allele frequency; Genotypic distribution INTRODUCTION Enzyme catechol-O-methyltransferase (COMT) is encoded by the COMT gene, involved in degradation of essential neurotransmitters such as dopamine, epinephrine, nor-epinephrine COMT gene has been studied the most in behavioral genetics related to metabolism of catecholamine, a neurotransmitter related to psychosis and treatment in psychiatry COMT has also been proven in many studies about interaction between risky environmental factors and mental disorders [1, 2] The common polymorphisms of COMT genes that have been researched are rs4680, rs7378645 and rs165599, in which, rs4680 is the most common [3] Another polymorphism (rs165599) is at the beginning of 3' of the COMT gene, next to exon The relationship between rs165599/COMT and clinical features in schizophrenia was published in several studies with different results [4, 5, 6, 7, 8, 9] 103 Military Hospital Vietnam Military Medical University Corresponding author: Dinh Viet Hung (bshunga6@gmail.com) Date received: 11/09/2019 Date accepted: 11/10/2019 148 Journal of military pharmaco-medicine no8-2019 In Vietnam, there have been no studies (synthesized by PHUSA Bio-Chemistry to assess the relationship between Co, polymorphism rs165599 and schizophrenia PCR amplification was carried out in The target of our study: To assess the ProFlex x 32 thermal cycler (ABI, USA) frequency of allele and genotypic distribution with an initial denaturation step at 94°C of polymorphism rs165599/COMT in Kinh for minutes, followed by 35 cycles of ethnic-patients with schizophrenia Ltd), Master mix QIAGEN 1X 94°C for 30 seconds, 55°C for 45 seconds and 72°C for 60 seconds, and a final SUBJECTS AND METHODS extension step at 72°C for 10 minutes with storage at 4°C The forward Subjects and reverse primer sequences were Subjects included 227 schizophrenic 5’-CGACTTAGTACATCCTTC-3’ and patients and 92 healthy controls in the 5’-GAGTAGAATCTTGGCTAG-3’, respectively Kinh ethnic All patients were hospitalized The 628 bp fragment products were and diagnosed at Department of Psychiatry, 103 Military Hospital, using criteria of the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) separated on 1% agarose gel PCR products were treated with enzyme MSPI (NEB, USA) according to the manufacturer's recommendations, summarized as follows: 10 µL mixture containing µL products, Methods 10 U enzymes, buffer The mixture was * Methods of genotyping of rs165599 incubated at 37°C for 10 minutes The SNP: CC/GG sequence of 628 bp fragment was Peripheral blood samples were collected cut and formed 403 bp and 225 bp from 319 subjects and anti-coagulated fragments, respectively The genotyping by EDTA Genomic DNA was extracted method of single nucleotide polymorphism using QIAGEN Blood Mini kit (QIAGEN, (SNP) rs165599 was more detail elsewhere Germany), following the manufacturer’s in our previous report [10] o protocol and then stored at -20 C The * Statistical analysis: fragment, containing the single nucleotide SNP allele and genotype frequencies polymorphism, was amplified using the were analyzed by Chi-Square test and polymerase chain reaction (PCR) method Hardy-Weinberg equilibrium (HWE) for The PCR reaction was carried out in the the SNP was assessed in both cases and total volume of 25 µL containing 20 ng controls The level of significance was set genomic DNA, 0.2 µM of each primer at p < 0.05 149 Journal of military pharmaco-medicine no8-2019 RESULTS Results of amplification of fragment with SNP rs165599 Figure 1: Artwork of the targeted fragment results on agose electrophoresis (1%) White identical lanes in figure showed that all PCR products were specific and sufficient for PCR-RFLP method and for next genotyping Results of genotyping of rs165599 SNP Performance of PCR-RFLP by restriction fragment length polymorphism cutting enzyme and electrophoresis for fragmented products on agarose 1% had separated bands at 225 bp, 403 bp and 628 bp, which appeared upon the genotype of SNP rs165599 (figure 2) Figure 2: Results of genotyping of SNP rs165599 by electrophoresis for fragmented products 150 Journal of military pharmaco-medicine no8-2019 Samples had three bands at 628, 403, and 225 bp was of AG genotype; samples had two bands as 403 bp and 225 bp of GG genotype; and samples with AA genotype had only one band at 628 bp This showed that by combining techniques of restriction fragment length polymorphism cutting enzyme and electrophoresis for fragmented products had determined clearly genotypes of SNP Genotypic distributions of rs165599 satisfied the Hardy-Weinberg equilibrium in both schizophrenia patient (χ2 = 0.73) and control (χ2 = 3.57) groups Table 1: Allele frequencies SNP of the rs165599 of the two groups Groups Allele frequency [n (%)] Allele number (%) A G Schizophrenia (n = 227) 454 (100) 233 (51.32) 221 (48.68) Control (n = 92) 184 (100) 89 (48.37) 95 (51.63) 322 316 638 Total (n) χ = 0.456; p = 0.499 The result showed that there was no difference in frequencies of both A and G allele of SNP rs165599 between two groups (p = 0.499) Table 2: Allele frequencies of the SNP rs165599 in males Groups Allele frequency [n (%)] Allele number (%) A G Schizophrenia 312 (100) 165 (52.88) 147 (47.12) Control 102 (100) 48 (47.06) 54 (52.94) 213 201 414 Total (n) χ = 1.044; p = 0.307 Table illustrated that there was no difference in frequencies of allele A and G of SNP rs165599 in males (p = 0.307) Table 3: Allele frequencies of the SNP rs165599 in females Groups Allele frequency [n (%)] Allele number (%) A G Schizophrenia 142 (100) 68 (47.89) 74 (52.11) Control 82 (100) 41 (50.00) 41 (50.00) 213 115 Total (n) 414 χ = 0.093; p = 0.760 There was no difference in frequencies of allele A and G of rs165599 SNP between patient and control groups in females (p = 0.760) 151 Journal of military pharmaco-medicine no8-2019 Table 4: Genotypic distribution of SNP rs165599 in the two groups Groups Genotype number (%) n (%) AA AG GG Schizophrenia 227 (100) 63 (22.75) 107 (47.14) 57 (25.11) Control 92 (100) 17 (18.48) 55 (59.78) 20 (21.74) 319 80 162 77 Total (n) χ = 5.355; p = 0.069 There was no difference in frequencies of distribution of AA, AG, and GG genotypes between patient and control groups (p = 0.069) Table 5: Genotypic distribution of SNP rs165599 in males of the two groups Groups Genotype number (%) n (%) AA AG GG Schizophrenia 156 (100) 46 (29.49) 73 (46.79) 37 (23.72) Control 51 (100) (17.65) 30 (58.82) 12 (23.53) 207 55 103 49 Total (n) χ = 3.849; p = 0.146 There was no difference in distribution of homozygous and heterozypous genotypes of the rs165599 polymorphism in males of the two groups (p = 0.207) Table 6: Genotypic distribution of SNP rs165599 in females of the two groups Sample Genotype frequency Groups AA AG GG Schizophrenia 71 (100) 17 (23,94) 34 (47,89) 20 (28,17) Control 41 (100) (19,51) 25 (60,98) (19,51) 112 25 59 28 Total χ = 1,910; p = 0,385 In females of the two groups also found no differences in genotypic distribution of SNP rs16559 (p = 0.385) DISCUSSION Our results of genotype and allele frequencies suggested that the COMT rs165599 SNP was not a significant association with schizophrenia in Kinh ethnic 152 Our findings were consistent with previous studies A study by Acar C et al was conducted on Terkey population, the results showed that there was no relation between the three prevalent SNP (rs4680, Journal of military pharmaco-medicine no8-2019 rs737865, and rs165599) and schizophrenia in Turkey population [8] Moreover, a meta-analysis study from 20 independent reports concluded the same results [9] However, our study was inconsistent with other studies on Israel and Iran populations [5, 6, 7] Our research also showed no statistically significant difference in genotype frequencies and allele frequencies among groups in male and female This result supported some studies but was not in agreement with others Study on Turkey population [8] gave a similar conclusion with our research, whereas study on Israel population suggested the contrary one and showed a more significant association in female [5] Besides, there was study supporting the relation between SNP rs165599 with schizophrenia in female, especially for G allele, but no statistically significant difference for male [9] In summary, there were no general agreements about the association between the COMT rs165599 SNP and schizophrenia in different population, our study showed no relation between rs165599 SNP and schizophrenia in Vietnamese population These results suggested influence of other factors such as ethnicity Therefore, the research about suspected polymorphisms in general and rs165599 in particular with schizophrenia are crucial, should be furthered CONCLUSION These findings showed that there was no correlation among the frequency of allele, genotypic distribution of polymorphism rs165599 and schizophrenia in a population of Kinh ethnicity in Vietnam REFERENCES Li T et al Preferential transmission of the high activity allele of COMT in schizophrenia Psychiatric Genetics 1996, (3), pp.131-133 Henquet C et al An experimental study of catechol-O-methyltransferase Val158Met moderation of delta-9-tetrahydrocannabinolinduced effects on psychosis and cognition Neuropsychopharmacology 2006, 31 (12), pp 2748-2757 Henquet C et al Cannabis use and expression of mania in the general population Journal of Affective Disorders 2006, 95 (1 - 3), pp.103-110 Truong T.D, Dam M.N et al Val158Met polymorphism in catechol-o-methyltransferase gene and schizophrenia in a Vietnamese population Jounal of 108 Clinical Medicine and Pharmacy 2015, 12, pp.143-147 Shifman S et al A highly significant association between a COMT haplotype and schizophrenia 2002, 71 (6), pp.1296-1302 Ahmadi L et al Genetic variations of DAOA (rs947267 and rs3918342) and COMT genes (rs165599 and rs4680) Schizophrenia and Bipolar I Disorder 2018, (6), p.429 Behbahani P et al Association study of single nucleotide polymorphism rs165599 of COMT gene, with schizophrenia and bipolar mood disorder in the south-west of Iran 2015, (2), p.67 Acar C et al Lack of association between catechol-o-methyltransferase and schizophrenia in a Turkish population 2015, 40 (3), pp.205-209 Gozukara Bag H.G.J.M.G, G Medicine Association between COMT gene rs165599 SNP and schizophrenia: A meta‐analysis of case‐control studies 2018, (5), pp.845-854 153 ... rs165599 and schizophrenia in a population of Kinh ethnicity in Vietnam REFERENCES Li T et al Preferential transmission of the high activity allele of COMT in schizophrenia Psychiatric Genetics 1996,... 10 µL mixture containing µL products, Methods 10 U enzymes, buffer The mixture was * Methods of genotyping of rs165599 incubated at 37°C for 10 minutes The SNP: CC/GG sequence of 628 bp fragment... genotypes of the rs165599 polymorphism in males of the two groups (p = 0.207) Table 6: Genotypic distribution of SNP rs165599 in females of the two groups Sample Genotype frequency Groups AA AG GG Schizophrenia