Abstract
Background: Polymorphism in metallothionein (MT) 1A gene (rs8052394) has been found to be associated with diabetes mellitus and its
complications. We conducted the present study to find prevalence of polymorphism in MT1A gene (rs8052394) in Nepalese type 2 diabetes
mellitus patients.
Method: A study was carried out among type 2 diabetes mellitus patients (n = 82: 44 males and 38 females, 54.7 ± 11.7 years) selected from
Samjhana Laboratory Clinic, Lalitpur, in Kathmandu, Nepal to investigate single nucleotide polymorphism in MT1 A gene (rs8052394) in Nepalese
patients with type 2 diabetes mellitus. Demographic parameters were noted and blood samples were collected, and polymerase chain reaction
restriction fragment length polymorphism (PCR-RFLP) analysis was performed to detect rs8052394 polymorphism.
Results: The mean age of study patients was 54.7 ± 11.7 years. Mean fasting blood glucose and HbA1c were 168.7 ± 42.4 mg/dL and 7.4 ±
2.1% respectively. AA, AG and GG genotype of MTA1 gene rs8052394 was observed in 60.9% (n=50), 35.3% (n=29) and 3.6% (n=3) patients
respectively. Allele frequency of A and G allele was 0.786 and 0.213 respectively.
Conclusion: Our findings suggest that polymorphism in metallothionein 1A gene (rs8052394) is common in Nepalese Type 2 diabetes
patients.
Keywords
Metallothionein 1A; Polymorphism; Type 2 diabetes mellitus
Introduction
Diabetes mellitus is being one of the greatest health threats for the
21st century [1]. It is rising rapidly throughout the world, and the latest
report from International Diabetes Federation (IDF) suggests that about
415 million adults have diabetes worldwide, and by the year 2040, 642
million will have diabetes [1,2]. Type 2 diabetes mellitus (T2DM) causes
important morbidity, disability and early mortality, and is associated with
a huge economic burden [3].
A number of risk factors like obesity, low physical activity, sedentary
behavior, ethnicity etc. have been implicated for the development of Type
2 diabetes [4]. A strong heritable component for T2DM with genetic
factors explaining around 25% of the variation in disease risk has been
observed. Genome wide association studies (GWAS) have identified
many single nucleotide polymorphisms (SNPs) consistently associated
with an increased risk for T2DM [5].
Polymorphism in metallothionein (MT) gene has been found to have
linked with T2DM. Three different SNPs (rs11640851, rs8052394 and
rs1610216) in MT genes were shown to be associated with diabetes and
its complications [6]. Polymorphisms of these genes have been associated
with reduced intracellular zinc ion availability. +1245 A/G MT1A
polymorphism (rs8052394) has also been implicated in T2DM onset [7].
Experimental data suggested that MT exerts cellular protection effects not
only against metal toxicity, but also against a variety of oxidative stimuli
including the protective effects in animal models of diabetes [8].
Diabetes and the disorders associated with it have been rising in Nepal
[9]. Besides other factors, genetic components may also have contribution
for the recent rapid rise in diabetes in Nepalese population. One of the
predominantly associated genetic factors for diabetes risk is polymorphism
in MT1A gene (rs8052394). Thus, we conducted the present study to
detect polymorphism in MT1A gene (rs8052394) in Nepalese population
with T2DM and to determine if rs8052394 has significant role for diabetes
epidemic in Nepal.
Methods
We conducted a study among 82 T2DM patients selected from
Samjhana Laboratory Clinic, Lalitpur, Nepal in 2013. Diabetes mellitus
was diagnosed on the basis of American Diabetes Association (ADA)
criteria [10]. Consents were taken from each patient and the study
protocol was approved by the review board of Nobel College, Kathmandu,
Nepal. Each patient age, sex, height and weight was noted, and blood
samples were analyzed for biochemical parameters (fasting blood glucose
and glycated hemoglobin A 1c (HbA1c)), and rs8052394 variation in
metallothionein gene was investigated. Blood glucose and HbA1c was
estimated by using enzymatic method and immuno chromatographic
principle respectively. Genomic DNA was extracted using protocol
of Lahiri et al. [11]. After extracting DNA, polymerase chain reaction
(PCR) amplification of MT1A gene was done using the forward
primer (5’-ACTAAGTGTCCTCTGGGGCTG 3’) and reverse primer
(5’-AATGGGTCACGGTTGTATGG 3’) by using XP thermal cycler.
After restriction digestion of each DNA sample obtained by PCR by PstI
(Fermentas), agarose gel electrophoresis (3% agarose gel in 1XTAE buffer)
of 15 µl of digested products (mixed with 3 µl of loading dye) along with
100 bp DNA ladder was done for 1 hour at 90 volts. The results were
viewed under UV transilluminator for number of DNA fragments (bands)
obtained. Individual bands resulting from enzyme digested product were
compared with molecular sized marker (100 bp ladder from Fermentas).
The data generated from the study were entered into MS excel and
analyzed using SPSS version 11.0. Continuous variables were expressed as
mean ± SD and categorical variables as percentage (number). Student t test
and chi square test were applied for continuous variables and categorical
variables at 95% confidence interval respectively.
Results
The study population comprised 44 diabetic males and 38 diabetic
females, with mean age of 54.7 ± 11.7 years. The mean blood glucose
and HbA1c was 168.7 ± 42.4 mg/dL and 7.4 ± 2.1% respectively. General
characteristics of study population are shown in table 1. Agarose gel
electrophoresis of the amplified MT gene after digestion with PstI showed:
3 bands of 118 bp, 165 bp and 283 bp in heterozygous patients with SNP
(rs8052394), 2 bands of 118 bp and 165 bp in homozygous patients
with SNP (rs8052394), and single band of 283 bp for patients with no
polymorphism (wild type). SNP was observed in 32 patients. AA, AG and
GG genotype of MTA1 gene rs8052394 was observed in 60.9% (n=50),
35.3% (n=29) and 3.6% (n=3) patients respectively. Allele frequency
of A and G allele was 0.786 and 0.213 respectively. Body mass index
(BMI), fasting blood glucose and HbA1c in patients with and without
polymorphism was 25.3 ± 2.6 Kg/m2 versus 24.7 ± 2.7 Kg/m2
; p=0.33,
169.7 ± 39.5 mg/dL versus 168.1 ± 44.5 mg/dL; p=0.867, and 7.4 ± 2%
versus 7.4 ± 2.2%; p=0.956 respectively.
Discussion
Type 2 diabetes develops as consequence of interplay of various genetic
and environmental factors [5]. Among different MT genes, the isoform
MT1A with SNP rs8052394 is found to have a significant relationship
with T2DM [7]. In the present study, we found that 39% Nepalese diabetic
patients have rs8052394 polymorphism. This polymorphism is the result
of a nucleotide change from A to G at position 55231329 of chromosome
16, and it results in the substitution of lysine by arginine at 51 position of
the MT1A protein. In the wild type, codon AAA codes for lysine but in
mutated type, codon AAA changes to AGA hence it codes for amino acid
arginine [12].
Table 1: General characteristics of study population
Data are presented as mean ± SD, percentage (number) and as frequency
(number). P value was calculated between diabetic males and females
at 95% confidence interval. BG, blood glucose; BMI, body mass index;
HbA1c, glycated hemoglobin A 1c.
Metallothioneins are cysteine rich metal binding proteins which,
by means of their antioxidant and zinc-buffering properties, might
prevent the development of diabetic cardiovascular complications [13].
Polymorphism in MT1A gene may lead to formation of nonfunctional
metallothionein, thus affecting diabetic patients. In the present study,
we observed three genotypes of MT1 gene polymorphism; AA, AG and
GG. AA, AG and GG genotypes were observed in 60.9% (n=50), 35.3%
(n=29) and 3.6% (n=3) patients respectively. Similarly, A and G allele
frequency was 0.786 and 0.213 respectively. In a study in Bulgaria, AA,
AG and GG genotype at MT1A (A+1245G) rs8052394 was observed in
63.4%, 32.7% and 4.0% of diabetic patients (101 patients) respectively.
Frequency of A and G allele was 0.798 and 0.204 respectively [6]. In a
study in china, a significant difference in the frequency distributions
of the allele A and G for SNP rs8052394 in the MT1A gene between
the T2DM (397 cases) and non-diabetic control (454 cases) groups
was found. Although no significant difference in the frequency
distributions of the genotype for MT1A gene rs8052394 between T2DM
and non-diabetic control groups was observed, but if combined GG and
GA together, there was a significant difference between the T2DM group
and the control group [12].
Diabetes mellitus is a chronic disease characterized by an
overproduction of reactive oxygen species (ROS), which perturbs zinc
metabolism and promotes the onset of CVD [13]. In the present study,
we however, did not assess status of CVD, kidney disease, and diabetes
complications which may have association with the polymorphism
in MT1A gene. In a study in Bulgaria, it was found that the single
nucleotide polymorphisms +1245 A/G MT1A and -209 A/G MT2A
are related with diabetic complications and coronary artery disease
(CAD) developing [6]. In a study in Greece, +1245 MT1A G+ genotype
significantly increased the risk of CVD in Greece (34.4% versus 23.2%;
odds ratio=1.88, 95% CI=1.14-3.08; p=0.013) but not in Italy [14]. A
study in China revealed that rs8052334 and rs10636 SNPs are significantly
associated with hyperlipidemia, and both SNPs rs11076161 and rs10636
are found to be significantly associated with the incidence of T2DM with
neuropathy [12].
Studies have reported increase in oxidative stress in diabetic patients
with rs8052394. In one study, +1245 MT1A AG+GG genotype showed
higher plasma advanced glycation end products (AGEs) and ROS
production in peripheral blood mononuclear cells (PBMCs) than AA
genotype at the baseline. Thus, +1245 MT1A A/G polymorphism has
a significant influence on baseline AGEs and ROS production [7]. In
China, a significant decrease in serum superoxide dismutase (SOD)
activity was observed in both GG carriers and GA carriers compared
with AA carriers [12].
In conclusion, 39% of Nepalese diabetic patients had polymorphism
in MT1A gene (rs8052394). The magnitude of polymorphism in MT1A
gene (rs8052394) is important to estimate the burden of diabetes and
its contribution to epidemic in Nepal. Further studies are required with
larger sample size to find the association of polymorphism in MT1A gene
with complications of diabetes.
Acknowledgements
We kindly acknowledge the patients who participated in the study.
Author’s contribution
SK, PT, and NDP designed the study. SK, PT, RS, PLD and NDP
performed laboratory analysis. SK and MKT performed statistical
analysis. SK wrote the manuscript and all authors reviewed manuscript.
All authors read and approved the final version of the manuscript.
Article Information
Article Type: Research Article
Citation: Khatiwada S, Tiwari P, Shrestha R, Das
PL, Tamang MK, et al. (2016) Polymorphism in
Metallothionein 1A Gene in Nepalese Patients with
Type 2 Diabetes Mellitus. J Dia Res Ther 2(2): doi
http://dx.doi.org/10.16966/2380-5544.116
Copyright: © 2016 Khatiwada S, et al. This is an
open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Publication history:
Received date: 04 Jan 2016
Accepted date: 05
Feb 2016
Published date: 10 Feb 2016