Single Nucleotide Polymorphism (SNPs) Of TCF7L2 Gene with Susceptibility to Type 2 Diabetes in Vokkaliga Community of Mysuru

Diabetes was one of the first diseases described which meant "too big urine emptying." The first cases described are believed to be of type 1 diabetes. Indian doctors recognized the disease at about the same time and named it as madhumeha or "honey urine," nothing but the urine will attract ants. Diabetes mellitus is a globally highly prevalent chronic disease. This happens when the insulin released by the pancreas is not enough or when the body cannot use the insulin produced effectively¹. Prevention and treatment involve keeping a healthy diet, regular physical exercise, normal body weight and avoiding tobacco use. In people with the disease it is important to control blood pressure and to maintain proper foot care. Insulin injections shall be used to treat T1DM. T2DM may be treated with insulin-free or with medication. Approximately 366 million people were diagnosed with DM worldwide in 2011 and this is expected to increase to 552 million by 2030 as per the WHO². DM is categorized according to the pathogenic mechanism leading to hyperglycemia, as opposed to earlier parameters such as age of onset or form of therapy. Type 1 and Type 2 are classified as two broad categories of DM. As pathogenic processes proceed, both forms of diabetes are followed by a period of irregular ho-meostasis of glucose. Type 1 DM is the product of insulin deficiency in its entirety or near totality. Type 2 DM is a heterogeneous group of disorders with varying degrees of insulin resistance, impaired insulin secretion and increased production of glucose³. As per the study conducted by Anthropological Survey of India, Southern Regional Centre, Mysuru, it shows that the Gangadikara Vokkaligas community of Karnataka is having high prevalence to T2D. Gangadikara Vokkaliga also referred to as Gowda, is a community with its origin in India. They are landlords, village headmen and known to be dominant and powerful caste which is also a major dominant agricultural community⁴. Gangadikara Vokkaliga of Mysore has a very high prevalence of diabetes (27.5 %) compared to coastal Karnataka population (16 percent) and Chennai population (15.5 percent). All male and female diabetes are found to have more body weight compared to non-diabetes, although they are subject to restrictions on medicine and diet. However, compared to male diabetes, female diabetes is obese⁵. Single Nucleotide Polymorphism (SNP) is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present in some appreciable degree within a population. SNPs of the following genes are commonly associated with type 2 diabetes. Transcription factors 7 like 2 also known as TCF7L2 or TCF4 is a protein acting as a transcription factor in humans which is encoded by the TCF7L2 gene⁶.The TCF7L2 gene is located on the chromosome 10q25.2-q25.3 which contains 19 exons, and has autosomal dominate inheritance^7,8. KCNJ11 is a protein coding gene. Kir6.2 is a major subunit of the ATP-sensitive K+ channel, and inward-rectifier potassium ion channel. The gene encoding the channel is called KCNJ11 and mutations in this gene are associated with congenital hyperinsulinism⁹.Adiponectin also referred to as GBP-28, apM1, AdipoQ and Acrp30 is a protein hormone which is involved in regulating glucose levels as well as fatty acid breakdown. In humans, it is encoded by the ADIPOQ gene and it is produced in the Adipose tissue¹⁰. DM in India probably approaches epidemic proportions. Worryingly, it is now known that diabetes is associated with a number of complications and happens within the nation at a comparatively younger age. The incidence of diabetes is expected to double globally from 171 million in 2000 to 366 million in 2030 with a highest rise in India, according to Wild et Al^11,12. The SNPs of genes such as TCF7L2, KCNJ11 and ADIPONECTIN which are susceptible to type 2 diabetes is carried out in the Gangadikara Vokkaliga community of Mysuru district. The main aim of the study is to assess the single nucleotide polymorphism of TCF7L2 gene is carried out which is susceptible to type 2 diabetes in Gangadikara Vokkaliga community of Mysuru district. The biochemical parameters will be correlated with the identified SNPs in the study population.

MATERIALS AND METHODOLOGY

Study Design and Population

A community-based case-control study was conducted over a period of 18 months (August 2018 to February 2020) to investigate the association of TCF7L2 gene polymorphism with Type 2 Diabetes Mellitus (T2DM) in the Vokkaliga community of Mysuru, Karnataka. Participants were recruited from community-dominated regions such as K.G. Koppal and Bannur. Ethical approval was obtained from the Institutional Ethical Committee, and informed written consent was collected from all participants.

Sample Size and Selection Criteria

Based on an expected diabetes prevalence of 27.5% in the target population, a confidence level of 95%, absolute precision of 10%, and power of 80%, the estimated sample size was calculated to be 80 in each group. Ultimately, a total of 130 participants were recruited, including 71 confirmed T2DM patients (cases) and 59 non-diabetic individuals (controls) aged 40–55 years.

• Inclusion Criteria:
  • Cases: Diagnosed T2DM patients with random blood sugar (RBS) >150 mg/dL.
  • Controls: Non-diabetic individuals with RBS <150 mg/dL.
• Exclusion Criteria:
  • Individuals with Type 1 diabetes, other systemic illnesses, or outside the age range.
  • Pregnant or lactating women and individuals from other communities.

Clinical and Anthropometric Data Collection

Participant data were collected using a pre-tested and validated questionnaire covering:

• Demographics
• Medical and family history
• Lifestyle and dietary habits
• Anthropometric measurements including height, weight, waist and hip circumference, and blood pressure
• Body Mass Index (BMI) was calculated using the formula:
  BMI = Weight (kg) / Height² (m²)

Sample Collection and Processing

Venous blood samples (4 ml) were collected under aseptic conditions:

• 2 ml in plain vacutainer for biochemical analysis
• 2 ml in EDTA vacutainer for DNA extraction

Samples were transported in portable cold boxes to the laboratory within 30 minutes. Serum was separated by centrifugation at 6000 rpm for 10 minutes.

Biochemical Analysis

• Random Blood Sugar (RBS) was measured using the GOD-POD enzymatic method.
• Glycated haemoglobin (HbA1c) levels were estimated using the HPLC method.

DNA Extraction and Quantification

Genomic DNA was extracted from whole blood using the salting-out method (TKM buffer). Purity and concentration were assessed using a Nanodrop spectrophotometer (DeNovix) by measuring absorbance ratios at A260/280 and A260/230. DNA integrity was confirmed via 1.5% agarose gel electrophoresis.

Primer Design and Validation

Specific primers targeting the TCF7L2 gene were designed and validated using NCBI Primer-BLAST and bioinformatics tools for specificity, melting temperature (Tm), and ΔG values. The expected PCR product size was 201 bp.

Polymerase Chain Reaction (PCR)

PCR amplification was optimised using gradient PCR. The reaction mix included:

• DNA template (~100 ng)
• Forward and reverse primers (10 µM)
• dNTPs, Taq DNA polymerase, buffer, and MgCl?

Thermal cycling conditions:

• Initial denaturation: 94°C for 5 min
• 35 cycles of: 94°C for 30 sec, annealing at optimised Tm (56–60°C) for 30 sec, 72°C for 45 sec
• Final extension: 72°C for 7 min

Amplified products were visualised using gel electrophoresis with ethidium bromide staining.

Restriction Fragment Length Polymorphism (RFLP)

Amplified TCF7L2 gene fragments were digested with the RsaI restriction enzyme under optimal conditions. Restriction products were analysed by agarose gel electrophoresis to detect polymorphic patterns.

• Positive controls (Lambda DNA) and No Template Controls (NTC) were included in each batch.
• DNA fragment sizes were compared against a 100 bp molecular marker.

High Resolution Melt (HRM) Analysis

Samples with ambiguous or unexpected banding patterns in RFLP were subjected to HRM analysis using real-time PCR. Amplification and melting curve profiles were used to identify sequence variations.

Sanger Sequencing

Selected samples (suspected positive cases and matched controls) were subjected to Sanger sequencing for confirmation of SNPs. Chromatograms were aligned with the reference TCF7L2 sequence using BLAST for mutation identification.

Statistical Analysis

Statistical analysis was performed using Multifactor Dimensionality Reduction (MDR) and standard descriptive and inferential techniques. Group comparisons (e.g. HbA1c, BMI) were evaluated using Student’s t-test or Mann-Whitney U test, where appropriate. A p-value <0.05 was considered statistically significant.

RESULTS

A total of 130 participants were included in the study, comprising 71 individuals with Type 2 Diabetes Mellitus (T2DM) and 59 age- and gender-matched non-diabetic controls from the Vokkaliga community of Mysuru. The mean age of participants was comparable across groups. Significant differences were observed in random blood sugar (RBS) and HbA1c levels between the diabetic and control groups (p < 0.001 and p = 0.0018, respectively). Body mass index (BMI) was higher in diabetic subjects, though not statistically significant (Table 1).

Table 1: Demographic variables for Cases and Control.

Genomic DNA extracted from all samples showed good purity and concentration as illustrates in Figure 1 and Table 2.

Figure 1: Blood Genomic DNA separated under 1.5% Agarose Gel Electrophoresis, and Image captured using Gel documentation unit (SYNGENE). A representative image for 1st 10 cases and control samples.

The TCF7L2 gene primer validation was done using several bioinformatics tools and was found to be acceptable as depicted in Table 3 and Figure 2.

Figure 2: Primer for TCF7L2 gene utilized in the study showed its specificity towards target sequence as validated by using NCBI primer designing too/ Primer Blast.

Figure 3 showing Polymerase Chain Reaction (PCR) successfully amplified a 201 bp fragment of the TCF7L2 gene in all samples.

Figure 3: Detection of Human-TCF7L2 gene using PCR at optimized PCR condition yielded specific pcr product with size of 201bp. The above representative image is of TCF7L2 gene pcr product derived from Cases (D1-D9) and control (C1-C9) which are specific in size as expected. Similarly, all samples from cases and controls yielded specific product which validated all samples for the presence of TCF7L2 gene. 50bp marker was loaded along with samples in each gel to mark the size of pcr product. Restriction Fragment Length Polymorphism (RFLP) analysis using RsaI enzyme predominantly showed undigested products in both groups, indicating the absence of polymorphism at the targeted site as depicted in Figure 4

Figure 4: Detection of Human-TCF7L2 gene polymorphism using RFLP at optimized restriction digestion condition yielded mainly undigested product (wild type) with specific product size of 201bp, similar with that of PCR product. Some of the samples from Cases group (Diabetics) as mentioned above yielded spurious fragment corresponding to the size of one of the DNA fragment of TCF7L2 i.e. 176bp. These samples were further screened through HRM to find if these samples possess different Tm value or melting temperature. Positive control PC and No Template Control NTC yielded expected result as depicted in the gel image. 100bp marker was loaded along with samples in each gel to mark the size of digested product. A few diabetic samples displayed atypical banding patterns suggestive of possible sequence variation, which were further analysed using High Resolution Melt (HRM) (Figure 5) and Sanger sequencing (Figure 6). However, HRM analysis revealed no significant difference in melting temperature profiles between cases and controls, (Table 4) and Sanger sequencing confirmed the absence of single nucleotide polymorphisms (SNPs) in the amplified region. Overall, no TCF7L2 gene polymorphism was detected among the T2DM cases studied.

Figure 5: DNA samples with spurious fragment after RFLP for TCF7L2 gene were subjected for HRM along with Beta Actin as an internal control. Melting peaks (Left side) apparently at the same position of melting temperature are of TCF7L2 gene from both suspected cases and respective controls. Melting peaks (Right side) belong to Beta actin-an internal control. NTC didn’t show any amplification as expected and hence no melting peak was generated.

Table 4: HRM result of suspected cases and controls.