Subjects

The present study included a total of 318 clinical subjects, comprising 130 controls, 124 hypothyroid patients, and 64 hyperthyroid patients aged between 20 and 65 years. These subjects were selected from various regions of Haryana, Delhi, and Uttar Pradesh in India. The research proposal was approved by the Institutional Human Ethical Committee (IHEC) of Kurukshetra University (letter no. DZ/17/IHEC/445). Fasting blood samples were collected from patients who met the research criteria. Relevant demographic information, such as age, gender, history of thyroid and other diseases, smoking history, and medication use, was collected. Patients were also examined for the presence of goiter or lumps. Body mass index (BMI) was calculated by dividing the person's weight (in kg) by their height (in meters squared, m2).

Inclusion criteria

This study included patients aged 20-65 years with newly diagnosed hyperthyroidism or hypothyroidism, confirmed by laboratory tests, and no prior treatment. Healthy individuals of the same age range with no personal or family history of thyroid disease, normal thyroid function tests, and no medications or illnesses affecting thyroid function were included as controls. All participants provided written informed consent.

Exclusion criteria

To ensure the validity of the study results, certain exclusion criteria were applied. Patients with pre-existing conditions such as heart disease, hypertension, diabetes, or pregnancy were excluded from the study to avoid any confounding factors that could potentially interfere with the accurate interpretation of hypothyroidism and hyperthyroidism conditions.

Blood sample collection genomic DNA extraction

A fasting venous blood sample of approximately 5 ml was collected from all subjects [19]. The blood sample was then divided into two aliquots: 3 ml was transferred into a plain tube, and 2 ml was transferred into K2EDTA-coated vials [20]. After 30 min, the blood samples in the plain tube were centrifuged to extract the serum, which was then stored at − 20 °C for future analysis [21]. Meanwhile, the blood samples collected in K2EDTA-coated vials were stored at − 20 °C for genetic analysis. This study modified Miller's protocol for extracting DNA from whole blood [22]. The protocol involves collecting blood in EDTA-anticoagulated tubes, centrifuging to separate the buffy coat from plasma, treating the buffy coat with protease to release DNA, adding a salt solution to precipitate DNA, centrifuging to remove cellular debris, adding ethanol to precipitate DNA, centrifuging to pellet DNA, washing the DNA pellet with 70% ethanol, and drying the DNA pellet and dissolving it in water. The isolated DNA was then stored in separate aliquots at − 20 °C for six months. The purity of the DNA was assessed by measuring the absorbance at 260/280 nm, which showed a ratio range of 1.6–2.0. The DNA was also visualized using 1.5% agarose gel electrophoresis and a UV transilluminator.

Biochemical analysis of blood samples

Biomarkers and biochemical parameters, including renal function tests (RFTs), liver function tests (LFTs), total protein (TP), serum uric acid, serum total calcium, and albumin, were analyzed using enzymatic methods. The RFTs included creatinine and urea, which were measured using a creatinine assay kit (ERBA) and a urea assay kit (ERBA), respectively. The LFTs included aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which were measured using an AST assay kit (ERBA) and an ALT assay kit (ERBA), respectively. Thyroid hormone levels, including triiodothyronine (T3) and thyroxine (T4), were measured using a chemiluminescent immunoassay technique (ERBA). The thyroid-stimulating hormone (TSH) levels were measured using a TSH assay kit (ERBA).

PCR and RFLP of TSHR SNP-rs2268458

Genotyping of candidate SNP were performed using the PCR–RFLP technique. The following set of primers was used: forward primer- 5’-CTAACCAGCAGAGGGAGCAC-3’, Reverse prime -5’-CCACTGCTTAAAGCCCAGAT-3’ [23]. The expected size of the PCR product obtained was 162bp. A total volume of 50µl of PCR reaction mixture was prepared, which included 5.0µl of PCR buffer (10X), 5.0µl of MgCl2 (25mM), 1µl of dNTPs (10mM), 0.5µl of each forward and reverse primer (10pmol), 2µl of Taq DNA polymerase, 1µl of DNA template (50ng), and 35µl of nuclease-free water. The reaction mixture was vortexed in tubes before being placed in the thermocycler. The PCR cycle started with a 5-minute enzyme activation step at 95 °C, followed by 35 cycles of denaturation for 45 s at 94 °C, annealing for 45 s at 57 °C, and extension for 45 s at 72 °C. The final extension was carried out at 72 °C for 5 min, and then the reaction was held at 4 °C. The PCR-amplified product (162bp) was validated using 2% agarose gel electrophoresis with a UV-transilluminator (Figure 1).

Fig. 1

PCR product (162 bp) of TSHR gene fragment containing SNP rs2268458 (lanes 2–6) and 100 bp DNA ladder in lane 1

Restriction enzyme analysis

The 162bp amplified PCR product was digested with the specific restriction enzyme Alu1 (New England Biolabs) to detect the presence of the SNP. The digestion reaction contained 10 units of Alu1 and was incubated overnight at 37 °C. The resulting fragments were separated by 2.5% agarose gel electrophoresis. The mutated heterozygote allele (TC) was expected to produce three fragments of 162bp, 100bp, and 62bp, while the wild-type homozygote allele (TT) would produce a single 162bp fragment. The mutated homozygote allele (CC) would yield two fragments of 100bp and 62bp. All samples were analyzed by electrophoresis on a 3% agarose gel, and the genotype pattern of the gene was determined. The gel was visualized under UV light using a transilluminator (Figure 2). Restriction enzyme Alu1 was selected using the online molecular calculator NEB cutter, and the results were validated using restriction mapper.

Fig. 2

Restriction digestion products at SNP rs2268458. Lane1- 50 bp DNA ladder; lanes 5 and 7- homozygous wild type genotype; lane 4- heterozygous mutant genotype, and lanes 3 and 6- CC homozygous mutant genotype

Statistical analysis

The allele and genotype frequencies among all participants were determined using the gene counting method. Quantitative data were presented as mean ± standard deviation (SD), while qualitative data were summarized as frequency (percentage, %). The Chi-square test was utilized to compare statistical differences in categorical variables, such as gender, allele, and genotype distribution, between the control group and thyroid patients (hypothyroid and hyperthyroid). Logistic regression analysis was performed to calculate the odds ratio (OR) and 95% confidence interval (CI). The demographic and clinical characteristics of thyroid patients and control participants were compared using Student's t-test, which is suitable for continuous and normally distributed quantitative data. All statistical analyses were conducted using IBM SPSS Statistics software version 23.0. A p-value < 0.05 was considered statistically significant, indicating the significance of the analyzed data.