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POLYMORPHIC CHARACTERISTICS OF RS165599 IN COMT
GENE IN SCHIZOPHRENIA PATIENTS IN KINH POPULATION
Dinh Viet Hung
1
; Dang Tien Truong
2
; Ngo Van Nhat Minh
2
Cao Tien Duc
1
; Tran Hai Anh
2
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 6. The relationship between
rs165599/COMT and clinical features in
schizophrenia was published in several
studies with different results [4, 5, 6, 7, 8, 9].
1. 103 Military Hospital
2. Vietnam Military Medical University
Corresponding author: Dinh Viet Hung (bshunga6@gmail.com)
Date received: 11/09/2019
Date accepted: 11/10/2019
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In Vietnam, there have been no studies
to assess the relationship between
polymorphism rs165599 and schizophrenia.
The target of our study: To assess the
frequency of allele and genotypic distribution
of polymorphism rs165599/COMT in Kinh
ethnic-patients with schizophrenia.
SUBJECTS AND METHODS
1. Subjects.
Subjects included 227 schizophrenic
patients and 92 healthy controls in the
Kinh ethnic. All patients were hospitalized
and diagnosed at Department of Psychiatry,
103 Military Hospital, using criteria of
the Diagnostic and Statistical Manual of
Mental Disorders, fifth edition (DSM-5).
2. Methods.
* Methods of genotyping of rs165599
SNP:
Peripheral blood samples were collected
from 319 subjects and anti-coagulated
by EDTA. Genomic DNA was extracted
using QIAGEN Blood Mini kit (QIAGEN,
Germany), following the manufacturer’s
protocol and then stored at -20
o
C. The
fragment, containing the single nucleotide
polymorphism, was amplified using the
polymerase chain reaction (PCR) method.
The PCR reaction was carried out in the
total volume of 25 µL containing 20 ng
genomic DNA, 0.2 µM of each primer
(synthesized by PHUSA Bio-Chemistry
Co, Ltd), Master mix QIAGEN 1X.
PCR amplification was carried out in
ProFlex 3 x 32 thermal cycler (ABI, USA)
with an initial denaturation step at 94°C
for 3 minutes, followed by 35 cycles of
94°C for 30 seconds, 55°C for 45 seconds
and 72°C for 60 seconds, and a final
extension step at 72°C for 10 minutes
with storage at 4°C. The forward
and reverse primer sequences were
5’-CGACTTAGTACATCCTTC-3’ and
5’-GAGTAGAATCTTGGCTAG-3’, respectively.
The 628 bp fragment products were
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 7 µL products,
10 U enzymes, buffer. The mixture was
incubated at 37°C for 10 minutes. The
CC/GG sequence of 628 bp fragment was
cut and formed 403 bp and 225 bp
fragments, respectively. The genotyping
method of single nucleotide polymorphism
(SNP) rs165599 was more detail elsewhere
in our previous report [10].
* Statistical analysis:
SNP allele and genotype frequencies
were analyzed by Chi-Square test and
Hardy-Weinberg equilibrium (HWE) for
the SNP was assessed in both cases and
controls. The level of significance was set
at p < 0.05.
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RESULTS
1. 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 1 showed that all PCR products were specific and
sufficient for PCR-RFLP method and for next genotyping.
2. 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.
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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 (χ
2
= 0.73) and control (χ
2
= 3.57) groups.
Table 1: Allele frequencies SNP of the rs165599 of the two groups.
Allele frequency [n (%)]
Groups
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)
638 322 316
Total (n) χ
2
= 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.
Allele frequency [n (%)]
Groups
Allele number (%)
A G
Schizophrenia 312 (100) 165 (52.88) 147 (47.12)
Control 102 (100) 48 (47.06) 54 (52.94)
414 213 201
Total (n)
χ
2
= 1.044; p = 0.307
Table 2 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.
Allele frequency [n (%)]
Groups Allele number (%)
A G
Schizophrenia 142 (100) 68 (47.89) 74 (52.11)
Control 82 (100) 41 (50.00) 41 (50.00)
414 213 115
Total (n)
χ
2
= 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).
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Table 4: Genotypic distribution of SNP rs165599 in the two groups.
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.
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.
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.
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,
Genotype number (%)
Groups 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
2
= 5.355; p = 0.069
Genotype number (%)
Groups n (%)
AA AG GG
Schizophrenia 156 (100) 46 (29.49) 73 (46.79) 37 (23.72)
Control 51 (100) 9 (17.65) 30 (58.82) 12 (23.53)
207 55 103 49
Total (n)
χ
2
= 3.849; p = 0.146
Genotype frequency
Groups
Sample
AA AG GG
Schizophrenia 71 (100) 17 (23,94) 34 (47,89) 20 (28,17)
Control 41 (100) 8 (19,51) 25 (60,98) 8 (19,51)
112 25 59 28
Total
χ
2
= 1,910; p = 0,385