Vitamin D Receptor (VDR) Gene Polymorphism and Type 2 Diabeticmellitus (T2DM)-A Case Control Study in Ethnic Population of Kashmir Valley_Juniper Publishers
Authored by
Sabhiya Majid
Abstract
Background: An estimated one billion people
across various ethnic and age groups have vitamin D deficiency (VDD).
The high prevalence of VDD is predominantly imperative public health
issue, because hypovitaminosis D is a sovereign risk factor for total
mortality in the general population. Afar bone integrity and calcium
homeostasis, it is intricate in uncountable physiological and
pathological processes. The part of vitamin D in the pathogenesis and
prevention of Type 2 Diabetes Mellitus (T2DM) has sparked universal
interest.
Aims and objectives: The present study was
conducted to examine genetic polymorphism of Vitamin D receptor (VDR)
gene in patients with type 2 diabetes mellitus (T2DM) and control
subjects and valuation of 25(OH)2D3levels for the admittance of the
association between vitamin D levels and T2DM.
Material and Methods: The case-control study
was conducted in hundred clinically confirmed T2DM patients and hundred
healthy control subjects. All subjects underwent detailed clinical and
biochemical assays. Genomic DNA was extracted from peripheral blood by
kit method as per manufacture's protocol (Biotools). VDR gene
polymorphism was determined by polymerase chain reaction and restriction
fragment length polymorphism (PCR-RFLP) using restriction enzymes
Fok-Iand Bsm_I.
Result: A strong association was found between
Fok-I polymorphism and T2DM indicating that this gene polymorphism is
possibly a risk factor for T2DM (Ffvs FF p=0.0005; FFvsff p=0.0133;
ff+Ffvs FF p=0.001). However, the Bsm-I polymorphism had no significant
relation with T2DM (Bbvs BB: p=0.129; bb vsBB: p=0.23; bb+Bbvs BB:
p=0.10). 25 (OH) D3 levels were low in T2DM patients than in controls
being 19.26±0. 95ng/ml and 25.49±1.02ng/ml, respectively (p<0.001)
and were found to be inversely associated with HbA 1% in the diabetic
patients (p=0.008).
Conclusion: Vitamin D receptor (VDR) gene and its functional polymorphism seems to play an important role in pathogenesis of type 2 diabetes.
Keywords: Type2 Diabetes; Vitamin D receptor; Genetic polymorphisms; Restriction fragment length polymorphismsIntroduction
Vitamin D, the sunshine vitamin has been recently
implicated in a plethora of medical illnesses. Diminution in incidence
of rickets after fortification of foods with vitamin D led physicians to
believe that vitamin D related health disorders had come to an end. But
regrettably, rickets appears to be a mere drop in the vast ocean of
disorders resulting from vitamin D deficiency (VDD) [1].
Apart from its conventionally understood role in bone health and
calcium homeostasis, vitamin D is believed to have an effect on body's
endocrine system, immune system, cardiovascular system,
neuropsychological functioning, neuromuscular performance and is also
believed to act as a potent antioxidant protecting against free radical
damage, as well as being an inducer of cellular differentiation,
protecting against carcinogenesis [2,3].
Accumulating research suggests that circulating concentrations of
vitamin D may be inversely related to the prevalence of diabetes, the
concentrations of glucose and to insulin resistance [4-7].
Worldwide some 382 million people are estimated to have diabetes. About
80% live in low- and middle-income countries. If these trends continue,
by 2035, some 592 million people, or one adult in 10, will have
diabetes. This equates to approximately three new cases every 10 seconds
or almost 10 million per year. The largest increases will take place in
the regions where developing economies are predominant [8].
Diabetes is a multi factorial disorder; identification of modifiable
risk factors is of colossal importance to curtail this pandemic. Higher
plasma vitamin D has been shown to be related with a lower risk for the
development of diabetes mellitus in high risk patients [9].
Also some other studies have shown that vitamin D may play a functional
role on glucose tolerance through its effects on insulin secretion and
insulin sensitivity [10]. The presence of a correlation between VDR
polymorphisms and T2DM-associated metabolic parameters, including
fasting glucose, glucose intolerance, insulin sensitivity, insulin
secretion, and calcitriol levels, has been reported by observational
studies. Among such VDR polymorphisms, Fok1, Taq1, Bsm-I, EcoRV, and
Apal are suspected to alter the activity of the VDR protein [11].
Therefore, it was hypothesized that VDD may be prevalent in T2DM
patients and that vitamin D may be related to glucose control in this
group of patients. Further we aimed to investigate the prevalence of two
functional single nucleotide polymorphisms (Fok-I & Bsm-I) of the VDR gene and correlate this with the occurrence of T2DM.
Material and Methods
The case-control study was conducted on age and sex
matched hundred healthy control volunteers and hundred cases of
clinically confirmed T2DM (American Diabetes Association- 2010
criteria). All the cases were recruited from OPD and IPD of Department
of Medicine Government Medical College Srinagar (GMC Srinagar) and
associated Shri Maharaja Hari Singh (SMHS) hospital and were of Kashmiri
ethnic origin (J and K, India).
Pursuant to physical examination a record of clinical
history including that of hypertension, obesity, hypercholesterolemia,
stroke or transient ischemic attacks, smoking and other relevant details
was maintained. Questionnaire was used to assess demographic details
including dietary pattern, extent of exposure to sun, use of sunscreen.
BMI and complete demographic profile was recorded.
Routine and advanced biochemical investigations were
cARGHied out in Diagnostic wing of Biochemistry laboratory, Govt.
Medical College (GMC) Srinagar and associated SMHS hospital. Molecular
assays were cARGHied out in the Molecular research laboratory of
Department of Biochemistry GMC Srinagar.
Exclusion criteria
Chronic illnesses that potentially alters vitamin D
metabolism, use of medications that affect bone metabolism (vitamin D
supplements), pregnant or breast feeding women.
Biochemical analysis of blood samples
Sample collection: Overnight fasting venous
blood samples were collected from the subjects in EDTA containing tubes
using standardized protocol and equipment, separated into two samples
one whole blood for DNA and the measurement of glycated hemoglobin
(HbA1c). The other plasma specimen was used for measuring 25(OH) 2D3
levels. Other basic biochemical blood tests were measured by standard
chemical and enzymatic commercial methods in the Biochemistry department
and hospital laboratories. Laboratory measurements included estimation
of fasting blood sugar, Lipid profile. Lipid profile levels were
estimated by enzymatic kit method (Abbott) using analyzer (Architect
C4000), Glucose levels were estimated by Glucose oxidase kit method
(Abbott) using analyzer (Architect C4000). Serum HbA1c% was estimated by
enzymatic method/kit method (Abbott laboratories Illinois, USA) as per
manufacturers protocol, analyzed on Automatic Analyzer (Abbott C 4000).
25(OH)D was analyzed by Chemilumenescence method/kit method (Siemens,
USA) as per manufacturer's protocol, using Siemens ADVIA Centaur
analyzer
Ethical justification: Informed and written
consent [in language they best understood] was taken before collecting
data and blood sample. Only those individuals, who volunteered to
participate in the study, were selected and the data was kept
confidential. The study did not impose any financial burden on the study
subjects and the institute, therefore the study was ethically
justified.
Molecular Assay
Genomic DNA was extracted from EDTA blood sample
using Genomic DNA Purification Kit (Bio tools USA). Reaction mixtures of
25μl were used in PCR for the VDR gene (Fok-I and Bsm-I) polymorphism
and DNA samples were amplified. (Eppendorf Thermal Cycler). Followed by
Restriction Fragment Length Polymorphism. Gels were visualized under UV
spectrophotometer and photographed with gel doc (Genetix).
Fok-I polymorphism and RFLP: PCR cycle
conditions were initial denaturation at 94 °C for 5 min, followed by 35
cycles at 94 °C for 1 min, 61 °C for 1 min and 72 °C and one final cycle
of extension at 72 °C for 5 min. 25μl PCR reaction mixture consisted of
1μl of 0.5μg genomic DNA, 1μl of forward and reverse primers (10mM)
(eurofins Genomics) 12.5μl of PCR master mix (Thermo Scientific USA) and
9.5μl DD H2O. PCR products were verified using 1% agarose gel
containing ethidium bromide. PCR products (265bp) were digested with
Fok-I (Thermo Scientific USA) restriction enzyme at 65 °C for 16hrs
incubation and 8μl of the digested reaction mixture was then loaded into
6% agarose gel (Figure 1).
Bsm-I polymorphism and RFLP: PCR cycle
conditions were Initial denaturation at 94 °C for 10 min, followed by 35
cycles at 94 °C for 1 min, 60 °C for 1 min and 72 °C and one final
cycle of extension at 72 °C for 5 min. 25μl PCR reaction mixture
consisted of 1μl of 0.5μg genomic DNA, 1μl of forward and reverse
primers (10mM) primers 12.5μl (eurofins Genomics) of PCR master mix
(Blackbio Biotech India) and 9.5μl DD H2O. PCR products were verified
using 1% agarose gel containing ethidium bromide. PCR products (825bp)
were digested with Bsm-I (Thermo Scientific USA) restriction enzyme (50
units) at 65 °C for 16hrs and 8μl of the digested reaction mixture was
then loaded into 6% agarose gel (Figure 2).
Data management and statistical analysis
During data collection completed questionnaires were
checked regularly to rectify any discrepancy, logical errors or missing
information. All statistical analyses were performed with Statistical
Package for Social Services (SPSS vs 21 for Mac. IBM Inc.
Chicago).Descriptive data were expressed by mean± standard deviation
(95% CI) or frequency (95% CI). Comparisons between two groups were
performed using independent t-tests. Chi-square analysis was applied to
examine the variations of allele, genotype and genotype frequencies in
groups. Chi-square analysis was also used to test Hardy-Weinberg
equilibrium for the genotypes in all groups of subjects. Odds ratio and
their 95% confidence interval (CI) were performed for the risk alleles
from logistic regression analysis. A value of p<0.05 was considered
statistically significant.
Results
Hundred T2DM subjects and hundred normal healthy
controls were included in this study. Mean age was 51.20+8.79 and
45.02+11.01 years in patients and controls respectively. Gender
distribution of patients showed that there is high incidence of diabetes
in females.Significantly higher systolic blood pressure, BMI, and
waist-hip ratio, as well as higher serum concentrations of cholesterol,
low-density lipoprotein (LDL) and glucose levels were observed in T2DM
patients. Further Vitamin D levels in diabetes mellitus type 2 patients
were found to be inversely asso-ciated with HbA1c levels (p=0.008, r2=
0.058,linear regres-sion analysis) (Figure 3).
Vitamin D levels were found to be lower in T2DM
patients than in Controls being 19.26+0.95ng/ml and 25.49+1.02ng/ ml,
respectively (p<0.001). Out of 100 Cases, 25(OH)D levels were found
<30ng/ml, 48 showed <20ng/ml and remaining 09 showed <10ng/ml.
HbA1c% was higher in T2DM patients than in the control being 7.2+0.18 %
and 5.1+0.05% respectively (p<0.001).
Distribution of VDR (Fok-I and Bsm-I) gene polymorphism
Comparison of frequency distribution of different genotypes and alleles of VDR (Fok-I &Bsm-I) polymorphism was done in T2DM and control subjects. The genotype frequencies of the VDR
Fok-I and Bsm-I, were in agreement with Hardy-Weinberg equilibrium.
T2DM subjects have a higher genotypic and allelic frequency of Fok-I VDR
polymorphism(C>T) with a p-value of <0.001 and 0.0125
respectively. Frequency distribution of different genotypes and alleles
were as follows Ffvs FF: 0R=0.32, 95% CI: 0.1704-0.6154, P=0.0005; FF
vsff: 0R=0.37, 95% CI: 0.1715-0.8253, P=0.0133; ff+Ffvs FF: 0R=0.34, 95%
CI: 0.19110.6076, p=0.001). f(T) allele was found to be a risk factor
for T2DM (Figure 1).
Regarding the VDR Bsm-I polymorphisms, there
was no significant differences in genotypic and allelic frequency
between cases and controls (P>0.05).the frequency distribution of
different genotypes and alleles were not statistically significant Bb vs
BB: 0R=0.63, 95% CI: 0.35-1.14, P=0.129; bb vs BB: 0R=0.46, 95% CI:
0.12-1.67, P=0.23; bb+Bbvs BB: 0R=0.62, 95% CI: 0.35-1.09, P=0.10) (Figure 2).
On comparison of various metabolic variables in Cases with relation to VDR
gene polymorphism the Fok-I polymorphism (C>T) was found to be
significantly associated with increased levels of LDL cholesterol,
triglycerides cholesterol total cholesterol, systolic blood pressure and
plasma blood sugar. The analysis of the results of our study revealed
that ff genotype and f allele of the Fok-I are strongly linked with the
metabolic parameters associated with the T2DM. However, no such
association was found between distribution of Bsm-I genotype and allele
frequency and the various metabolic variables.
Discussion
Kashmir is at a higher altitude of 1,574-5,42 5 feet
above the sea level at latitudes of 32° 27'N-33° 50' N and longitude 73°
45'-75° 35' E in the Northern mountainous regions of India and due to
different topographical, geographical and climatic conditions, it does
not receive plenty of sunshine. Vitamin D can modulate insulin secretion
and also possesses pleiotropic effects on the pathogenesis of diabetes
mellitus. It is reported that vitamin D deficiency inhibits pancreatic
secretion and turnover of insulin (but not of other pancreatic
hormones), leading to impaired glucose tolerance [12].
Vitamin D is the most important regulator of calcium homeostasis in the
body by increasing absorption of calcium from food and reducing urinary
calcium loss, apart from calcitropic actions, vitamin D and its
metabolites appear to have important effects (through
paracrine/autocrine functions and gene modulation) on insulin synthesis,
secretion and actions as well as components of inflammation. The main
purpose of this case control study was to estimate the vitamin D levels
in diabetic patients and compare it with the controls, and to study any
possible association of VDR gene polymorphism with T2DM. It is possible
that genetic variants of the VDR gene may contribute to the development
of T2DM.Lower 25(OH) D3 levels were observed in diabetes mellitus type 2
patients than in a control group, an inverse relationship was observed
between glycosylated hemoglobin levels and 25(OH)D3 levels in the
patient group. To approve of our results, other studies on vitamin D
status and type 2 diabetes found the same results [13-15].
This prevalence of vitamin D deficiency in T2DM may be explained by the
fact that majority of the study subjects had indoor jobs, with mild
physical activity and not receiving ample sunshine, all these factors
contribute to hypovitaminosis D [16].
In the present study majority of the cases were females 62%, the reason
may be due to cultural and religious reasons because most Kashmiri
women wear clothes that cover their bodies completely which block
sunlight, thus preventing the synthesis of vitamin D, and this may be
responsible for vitamin D deficiency and T2DM.
In our study we analyzed a significant increase of
lipid profile parameters (TC, TG, LDL-C) with significant decreased
levels of HDL-C in diabetic patients when compared to control group.
Additionally, in the present study Vitamin D levels were negatively
correlated with TC. Our results were in harmony with Mackawy AMH [17].
It has been proposed that genetic variations in the VDR gene contribute
to the pathogenesis of type2 diabetes mellitus by altering calcium
metabolism, modification of adipocyte function and insulin secretion as
well as by modification of cytokine expression [18].
In current years the link between VDR polymorphisms, involving mainly
Fokl and Bsml, and different metabolic diseases, such as metabolic
syndrome, obesity and inflammatory activity, diabetes and vitamin D
deficiency, has been investigated [19].
Although the individual contribution of these polymorphisms in the
pathogenesis of T2D still needs to be confirmed, the analysis of this
association in our population produced some interesting results. Our
study demonstrated that VDR gene polymorphisms were associated with
susceptibility to T2DM which can be explained by differences in VDR FokI
genotype distributions between T2DM and control subjects. The results
of VDR polymorphisms in our study revealed that the genotype Ff and ff
is linked with increased risk of T2DM associated metabolic parameters
including LDL cholesterol, triglyceride cholesterol, total cholesterol,
systolic blood pressure and plasma blood sugar. In agreement with our
study many observational studies have reported the presence of
correlation between VDR polymorphism and T2DM associated metabolic
parameters including fasting glucose, glucose intolerance, insulin
sensitivity, insulin secretion [20,21].
In concordance with our study other meta-analysis concluded that Fok-I
could be the risk factor for T2DM especially in Asian population [22].
The biological consequences of this Fok-I polymorphism could be
explained by that the C to T transition not only destroys the Fok-I site
but also abolishes the first translation initiation of this gene, the
first translation initiation codon is shifted down steam by the amino
acids [23].
However there is no clear information in the literature regarding the
physiological role of VDR polymorphism and varying concentrations of
these T2DM associated clinical parameters including LDL cholesterol,
triglyceride cholesterol, total cholesterol, systolic blood pressure and
plasma blood sugar. On the antagonistic, there are educations
establishing no link between T2DM patients and controls in the allele
and genotype frequencies in VDR FokI gene polymorphism [24-26].
In conclusion, our data supports that VDR FokI polymorphism is
associated with T2DM, and the genotypes Ff and ff of this variant show a
strong link with various metabolic variables associated with T2DM.
The distribution of VDR Bsm-I genotype showed no
statistical difference between the control and T2DM patient. In approve
with our results, meta-analysis did not find any association of VDR
Bsm-I polymorphism with an increased T2DM risk in overall and subgroup
analysis [27].
Bsm-I, restriction site is localized to intron and is unlikely to cause
disease. In type 2 DM a link between Bsm-I and the onset of thedisease
has been found in Hungarians [28] and Germans [29]
but not in French [30], Bangladeshi [31] or Polish [32] populations.
Similar results have been found regarding the other most common
polymorphisms [30,32].
This discrepancy in findings from different studies
is attributed in fact to the differences in population
genetics.Examining population-based genetic association between VDR
variants and type 2 diabetes could of extreme importance for a specific
population that has a high prevalence of both type 2 diabetes and
Vitamin D deficiency.
Conclusion
Our findings indicate that a greater understanding is
needed with regards to the role of vitamin D in the metabolism of
glucose, insulin production, and regulation of α-cell function. Further
investigation is needed to have a better understanding of the role of
Vitamin D, VDR polymorphisms. We recommend larger scale studies for
detecting vitamin D deficiency in our population especially in patients
with type-2 diabetes mellitus and suggest planning aggressive strategies
to supplement our population with vitamin D. Awareness of the high
prevalence of vitamin D insufficiency among those with T2D may prompt
awareness among physicians and dietitians with intent to prevent vitamin
D insufficiency.
Acknowledgement
The authors would like to acknowledge the support
provided by the Technical Staff, Department of Biochemistry, GMC and
Associated Hospitals, Srinagar
To Know More About Advanced Research in Gastroenterology &
Hepatology Journal
click on:
https://juniperpublishers.com/argh/index.php
https://juniperpublishers.com/argh/index.php
Comments
Post a Comment