Introduction

Diabetes Mellitus is a group of metabolic disorders characterized by hyperglycaemic resulting from the defects of insulin secretion, insulin action or both ^[1]. Diabetes Mellitus may present with characteristic symptoms such as thirst, polyuria, blurring of vision, and weight loss. In its most severe form ketoacidosis or a non–ketotic hyperosmolar state develop that may lead to stupor, coma and in the absence of an effective treatment it may lead to death.

The disease diabetes has been broadly classified into two types:

Type 1 Diabetes Mellitus.

Type 2 Diabetes Mellitus.

Type 1 Diabetes Mellitus (T₁DM) also known as Insulin Dependent Diabetes Mellitus (IDDM) is caused by auto-immune destruction of the β-cells of the pancreas, rendering the pancreas unable to synthesize and secrete insulin. Type 2 Diabetes Mellitus (T₂DM), also known as Non-Insulin Dependent Diabetes Mellitus (NIDDM) is a heterogeneous syndrome resulting from the defect of both insulin secretion and insulin action.

Compared to Type 1 Diabetes, T₂DM is the most common form of diabetes, as it accounts for over 90% of all diabetic cases worldwide ^[3]. The disease usually occurs after the age of 35-40 years but may be diagnosed earlier, especially in the populations with high prevalence of the disease. T₂DM can remain undetected (asymptomatic), for several years and the diagnosis is often made from the associated complications or through an abnormal blood or urine glucose test. Type 2 diabetes mellitus is an extremely complex disease with a tremendous social and economical burden. Consequently, diabetes is rapidly emerging as a global health care problem that threatens to reach pandemic levels. By 2030, the number of people with diabetes worldwide is projected to increase from 171 million in 2000 to 366 million by 2030 ^[4]. This will be the most noticeable increase in the developing countries, where the number of people with diabetes is expected to increase from 84 million to 228 million. According to World Health Organization, Southeast Asia and the Western Pacific regions are at the forefront of the current diabetes epidemic with India and China facing the greatest challenges.

Patients that suffer from T2DM have a reduced quality of life and decreased life expectancy. The patients suffer from conditions like retinopathy, kidney malfunctioning, heart and blood vessel diseases and nerve damage in some cases.

The etiology of T₂DM is not fully understood, but presumably, T₂DM develops when a diabetogenic lifestyle (i.e. excessive caloric intake, inadequate caloric expenditure, obesity etc.) acts in conjugation with a susceptible genotype. Majority of patients who develop T₂ DM are obese ^[4]. A variety of environmental factors have been implicated in the clinical expression of T2DM. Some of these factors are the degree and type of obesity, sedentarily, malnutrition in fetal and perinatal periods, lifestyle, and different kinds of drugs (e.g, steroids, diuretics, anti-hypertensive agents). People with a family history of the diabetes are at higher risk of developing the disease because they share genetic background and likely share the similar environments.  Family studies had revealed that first degree relatives of individuals with T2DM are about three times more likely to develop the disease than individuals without a positive family history of the disease ^[6&7].

Risk Factors Associated with T2DM

First-degree family history of Diabetes Mellitus (i.e., parents or siblings).

Overweight or obese

Habitual physical inactivity

Race or ethnicity (Native American, Latino/Hispanic American, Asian American, African American, and Pacific Islanders)

Prediabetes (i.e., previously identified with impaired glucose tolerance or impaired fasting glucose)

Hypertension (≥140/90 mm Hg)

High-density lipoprotein (HDL) less than 35 mg/dL (0.91mmol/L) and/or a triglyceride level greater than 250 mg/dL (2.83 mmol/L)

History of gestational diabetes or delivery of a baby weighing greater than 9 pounds (4.09 kg)

History of vascular disease

History of polycystic ovary disease

Other conditions associated with insulin resistance

Various studies through literature have shown that Asian Indians have an increased susceptibility to diabetes and increased insulin resistance; they are a unique population for carrying out genetic studies. Asian Indians develop T₂DM at lower levels of BMI and have stronger heritability factors as compared to Europeans. Recent genetic studies on Asian Indians indicated that certain genes appear to predispose Indians to diabetes while other genes, which afford protection against diabetes and insulin resistance to Caucasians and do not appear to protect Indians. Moreover, a plenty of data has been generated for the genetic susceptibility of various populations to T₂DM but it is scanty for Indian populations and almost absent for Northwest Indian population groups particularly from Jammu region of J&K State.

In order to know the molecular genetic cause of the T₂DM patients belonging to the Jammu region of J&K State, one of the genes known to be strongly associated with T₂DM was selected in the present study. A total of 250 confirmed cases & controls samples belonging to four population groups (Hindu, Muslim, Sikh & Christians) of Jammu region were also screened for UCP2 -866G/A promoter polymorphism (rs659366).

Materials and Method

In molecular genetic analysis, the present investigation was focused to study the association of a candidate gene Uncoupling protein 2 (UCP2) with Type 2 Diabetes Mellitus (T2DM) in the population groups of the Jammu region. The cytogenetic location of UCP2 Uncoupling protein 2 (mitochondrial proton carrier) is 11q13. UCP2 is a regulator of insulin secretion and is highly expressed in the lymphoid system, macrophages, and pancreatic islets & participating in intermediary metabolism and in particular in fatty acid Metabolism.

Genomic DNA isolated from blood samples of 250 T2DM patients was subjected to genotyping by using PCR-RFLP based method. PCR was performed using standard PCR conditions Initial pre-cycling denaturation 94⁰C for 4 min, Denaturation for 30 cycles at 94⁰C for 1 min followed by annealing (specific to each primer), Extension 72ºC for 1 min,  and Final extension 72ºC for 4 min.

(Table 1 & 2 showing the details of the Genes selected Primers sequences, Annealing temperatures, Amplified gene product, Restriction Endonuclease and product size of the gene after restriction digestion).

Table 1: Table showing details of Primers used during the present study.

Table 2: Table showing the PCR conditions.

Genotype and allele frequencies of UCP2 G/A -866 polymorphism were calculated in the selected controls. Based on the band size, 250 Controls were scored either homozygous (GG) for wild allele.

heterozygous (GA) and homozygous (AA) for mutant allele. The data of the allele and genotype frequencies of UCP2 -866 G/A polymorphism studied in the selected Controls has been provided in Table 3.

Results

Genotype and Allele frequencies of UCP2 promoter-866 G/A polymorphism in T₂DM patients

Genotype and allele frequencies of UCP2 promoter G/A -866 polymorphism were calculated in the selected T₂DM patients. Based on the band size, these 250 T₂DM patients were scored either homozygous (GG) for wild allele, heterozygous (GA) and homozygous (AA) for mutant allele. The data of the allele and genotype frequencies of UCP2 -866 G/A polymorphism studied in the selected T2DM patients has been provided in Table 3.

The genotype frequencies, allele frequencies and heterozygosity value of Uncoupling proteins 2 (UCP2) -866G/A polymorphism in 250 patients representing the different population groups are as under:

Hindu Population

A total of 104 Hindu individuals were genotypes for UCP2. Out of 104 individuals, 27 were homozygous (GG) for wild type allele, 46 were heterozygous (GA) and 31 individuals were found homozygous (AA) for mutant allele. Out of the total 208 alleles (2×104=208), the frequency of the G allele for UCP2 866G/A Polymorphism was found to be 0.48 where as the frequency of A allele for UCP2 866G/A Polymorphism was found to be 0.52 (Table 3).

Muslim Population

A total of 51 Muslim individuals were genotyped for UCP2. Out of the 51, 29 individuals were found to be homozygous (GG) for wild type allele, 10 were found heterozygous (GA) and 12 were found homozygous (AA) for mutant allele. Out of the total 102 alleles (2×51=102), the frequency of the G allele for UCP2 866G/A Polymorphism was found to be 0.67 where as the frequency of A allele for UCP2 866G/A Polymorphism was found to be 0.33 (Table 3).

Sikh Population

A total of 78 Sikh individuals were genotyped for UCP2. Of the 78 individuals, 40 were found homozygous (GG) for wild type allele, 28 were found heterozygous (GA) and 10 were found homozygous (AA) for mutant allele. Out of the total 156 alleles (2×78=156), the frequency of the G allele for UCP2 866G/A Polymorphism was found to be 0.69 whereas the frequency of the A allele for UCP2 -866G/A Polymorphism was found to be 0.31. (Table 3).

Christian Population

A total of 17 Christian individuals were genotyped for UCP2. Out of the 17 individuals, 08 were found to be homozygous (GG) for wild allele, 05 were found heterozygous (GA) and 04 individuals were found homozygous (AA) for mutant allele.  Out of the total 34 alleles (2×17=34), the frequency of the G allele for UCP2 866G/A Polymorphism was found to be 0.62 whereas the frequency of the A allele for UCP2 -866G/A Polymorphism was found to be 0.38 (Table 3).

Table 3: Distribution of UCP2 Genotypes and its Allele frequencies of the T₂DM patients.

The allelic odds ratio (OR) as observed for UCP2 -866G/A polymorphism in the four population groups showed significant association with Muslim & Sikh population groups (Table 4).

Table 4: Allelic odds ratio (OR) of UCP2 in the four selected population groups.

The genotype frequencies, allele frequencies and heterozygosity value of UCP2 866G/A polymorphism in the 250 Controls representing the selected population groups are as under

Hindu Population

A total of 104 individuals selected as Control from Hindus were genotyped for UCP2. Out of 104 individuals, 29 were homozygous (GG) for wild allele, 45 were found heterozygous (GA) and 30 individuals were found homozygous (AA) for mutant allele.  Out of the total 208 alleles (2×104=208), the frequency of UCP2 -866G/A polymorphism for G allele was found to be 0.50 whereas the frequency of A allele for UCP2 -866G/A polymorphism was found to be 0.50. (Table 5).

Muslim Population

Out of the total 102 individuals as controls, the frequency of the G allele for UCP2 866G/A polymorphism was found to be 0.53 where as the frequency of A allele for UCP2 866G/A polymorphism was found to be 0.47 (Table 5).

Sikh Population

A total of 78 Sikh individuals selected as Control were genotyped for UCP2. Of the 78 individuals, 24 were found homozygous (GG) for wild allele, 31were found heterozygous (GA) and 23 individuals were found homozygous (AA) for mutant allele.  Out of the total 156 alleles (2×78=156), the frequency of the G allele for UCP2 866G/A polymorphism was found to be 0.51 whereas the frequency of T allele for UCP2 866G/A polymorphism was found to be 0.49 (Table 5).

Table 5: Distribution of UCP2 Genotypes and its Allele frequencies of selected Controls.

Christian Population

A total of 17 Christian individuals selected as Control were genotyped for UCP2. Out of the 17 individuals, 22 were found to be homozygous (GG) for wild allele, 16 were found heterozygous (GA) and 11 individuals were found homozygous (AA) for mutant allele.  Out of the total 34 alleles (2×17=34), the frequency of the G allele for UCP2 866G/A polymorphism was found to be 0.62 whereas the frequency of T allele for UCP2 866G/A polymorphism was found to be 0.38.

Discussion

T₂DM is of major concern to man. In the past 5 years, Genome wide association studies have identified and replicated over 40 single nucleotide polymorphisms (SNPs) that predispose to T₂DM ^[8&9]. Study of the literature on the genes responsible for the disease T₂DM shows that several genes are behind the origin of the disease, however, some of the candidate gene variations include variants of calpain-10 (CAPN10), Peroxisome Proliferator-Activated Receptor-γ2 (PPARγ2), Potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11), ATP Binding Cassette, subfamily C, member 8 (ABCC8), Hepatocyte Nuclear Factor-1A (HNF1A), Hepatocyte Nuclear Factor4A (HNF4A), Glucokinase (GCK), Plasma Cell Glycoprotein-1/encoding Ectonucleotide Pyrophosphate Phosphodiesterase 1(PC-1/ENPPI), Insulin Receptor Substrate-1 (IRS-1), Protein Tyrosine Phosphatase 1B (PTPNI).

Among all T₂DM susceptibility genes studied till date, only few were found to be convincingly associated with the disease. UCP1, UCP2, PPP1R3A, P12A variant in peroxisome proliferator activated receptor gamma (PPARγ) gene ^[10], E23K in potassium inwardly rectifying channel, subfamily J, member 11(KCNJ11) ^[11]. All these genes have modest effect on the development of the disease T₂DM. The TCF7L2 gene is the most important T₂DM susceptibility gene found to date. Since its discovery, the association has been replicated in a variety of studies in subjects of different ethnicities ^[12&13].

UCP2 -866 G/A Polymorphism

Uncoupling protein 2 is an attractive candidate gene for obesity and T₂DM. UCP2 is a member of the mitochondrial inner membrane carrier family that is highly expressed in adipose tissue and pancreatic islets. UCP2 mediates mitochondrial proton leak releasing energy stored within the proton motive force as heat that, ultimately, results in a decrease in ATP production. β-cells sense glucose through its oxidative metabolism and the resulting increase in the ATP/ADP ratio plays a central role in glucose-induced insulin secretion by causing closure of the membrane ATP-sensitive potassium channel, membrane depolarization, influx of calcium, and finally, insulin granule exocytosis ^[14&15]. Uncoupling glucose metabolism from ATP generation would be expected to impair β-cell ability to secrete insulin in response to glucose. Thus, an increased expression or activity of UCP2 in pancreatic β-cell may contribute to impair insulin secretion.

Studies on UCP2 -866 G/A polymorphism and its association with T₂DM studies have been performed by various researchers seeking for an association between UCP2 866G/A gene polymorphism with T₂DM ^{[16,17,18,19,20&21]} in the different population and ethnicities]. These studies have demonstrated association of the -866A allele with increased ^[22&23] and decreased risk of T₂DM ^[24] as well as no association at all ^[25,26&27].

During the present findings UCP2 -866 G/A gene polymorphism was found to be less common in the selected population groups. Suggesting thereby that in the present study the UCP2 -866 G/A gene polymorphism was insignificant as far as the disease T2DM is concerned.

Workers like ^[28,29&30] in their studies on different population groups of their respective regions studied the genotype (-866 GG, -866 GA & 866 AA) of UCP2 -866 G/A polymorphism and observed the frequency of the -866 GA genotype found to be less common as compared to remaining two genotypes (GG and AA). Present study did not record the high prevalence of -866 G/A genotype in the selected population groups. Present study supports the previous findings investigated by various workers however it is an addition to the existing literature.

Conflicts of Interests

There is no conflict of interest.

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