Balkan Journal of Medical Genetics

INTERLEUKIN-1β AND TUMOR NECROSIS FACTOR-α GENE POLYMORPHISMS IN SYSTEMIC SCLEROSIS
Hakami M.A1, Alotaibi B.S1, Alkhalil S.S1, Das S2, Nasreen N3, Jeraiby M.A4, Jawed A5, Lohani M5, Dar S.A5*
*Corresponding Author: *Corresponding Author: Sajad Ahmad Dar, Department of Nursing, College of Nursing and Health Sciences, Jazan University, Jazan – 45142, Saudi Arabia; Email: sdar@jazanu.edu.sa
page: 59

MATERIALS AND METHODS

Samples Patients clinically diagnosed with SSc, confirmed through established laboratory investigations and meeting the American College of Rheumatology (ACR) criteria [20], were consecutively enrolled at a tertiary care hospital in North India. The rarity of SSc, its genetic heterogeneity, and strict diagnostic criteria present significant challenges in participant recruitment for SNP studies. Additionally, specific disease subtypes, comorbidities, drug exclusions, geographic barriers, and ethical concerns further limit the eligible patient pool. Our study involved 23 SSc patients and 80 age- matched healthy volunteers of Asian ethnicity. Patients and healthy volunteers were unrelated, and the latter had no clinical history of skin disease, minimizing potential con- founders and ensuring clear group distinction. Peripheral venous blood (2ml) was collected aseptically from each patient and healthy volunteers into EDTA vacutainer tubes and used for DNA extraction. The study was approved by the Institutional Ethical Committee-Human Research, and written informed consent was obtained from each patient and healthy volunteer before enrollment in the study. Genomic DNA Extraction from Blood Genomic DNA was extracted from blood samples of 23 SSc patients and 80 healthy controls for cytokine genotyping of 22 SNPs in 13 cytokine genes using PCR with sequence-specific primers. DNA extraction was per- formed using the HiPurA TM blood genomic DNA extrac- tion kit (HiMedia Laboratories) as per the manufacturer’s protocol. Briefly, 200μl of blood was mixed with 20μl Proteinase K solution, vortexed, then treated with 20μl RNase A solution. After incubation, 200μl of lysis buf- fer (C1) was added, followed by a 10-minute incubation at 55°C. Ethanol (200μl) was added, and the lysate was transferred to a spin column for centrifugation. The column was washed with prewash and wash buffers, then eluted with 100μl elution buffer after a 5-minute incubation. DNA was stored at -20°C for PCR analysis. Cytokine Genotyping by PCR Cytokine genotyping was carried out from genomic DNA by PCR with sequence-specific primers using com- mercially available Cytokine Genotyping Kit (Invitrogen Corporation, USA). Twenty two SNPs (IL-1α –889 T/C; IL-1β –511 C/T and +3962 T/C; IL-1R pst1 1970 C/T; IL- 1RA mspa1 11100 T/C; IL-4Rα +1902 G/A; IL-12 –1188 C/A; IFN-γ +874 A/T; TGF-β1 codon 10 T/C and codon 25 G/C; TNF-α –308 G/A and –238 G/A; IL-2 –330 T/G and +166 G/T; IL-4 –1098 T/G, –590 T/C, and –33 T/C; IL-6 –174 G/C and nt565 G/A; IL-10 –1082 G/A, –819 C/T, and –592 C/A) in thirteen cytokine genes were as- sessed in all the patients and healthy volunteers using the kit according to the included instructions. For 48 reactions/wells for each sample, 140μl of PCR buffer was mixed with 3.3μl of Taq DNA polymerase, 329μl of water and 50μl of 75-125ng/μl concentrated DNA tem- plate. The reaction mixture (10μl) was dispensed into each well and the following thermal cycler profile was used for amplification. Step 1 was denaturation for 2 minutes at 94oC; Step 2 comprised 10 cycles of 94oC for 15 seconds and 65oC for 60 seconds with no separate extension step; Step 3 (20 cycles) consisted of 94oC for 15 seconds, 61oC for 50 seconds, and 72oC for 30 seconds. The profile was set on hold at 4oC. The PCR products were loaded onto a 2 percent aga- rose gel in a specific order for electrophoresis and run at 150 volts for 20-25 minutes for separating the DNA. After electrophoresis, the ethidium bromide stained gel was photographed and interpreted for specific amplifica- tion patterns using the worksheet provided with the kit. Presence of a control band in each lane was ascertained. Wells identifying the IL-2, IL-4, IL-6, and IL- 10 cyto- kines contained an 89 bp fragment of the β-globin gene as an internal control. Wells identifying the IL-1α, IL-1β, IL-1R, IL-1Rα, IL-4Rα, IL-12, IFN-γ, TGF-β, and TNF-α cytokines contained a 440 bp fragment of the human C- reactive protein gene as an internal control. Statistical Analysis Two-sided Fisher’s exact test was used to compare al- lele, genotype and haplotype frequencies between patients and controls. The threshold for significance was p<0.05, and the relative risks associated with rare alleles, genotypes and haplotypes were estimated as odds ratios (ORs) with 95% confidence intervals (CIs). The deviation from Hardy- Weinberg equilibrium (HWE) was determined using a goodness-of-fit Chi-square test to compare the observed genotype frequencies with the expected frequencies among the patients and healthy volunteers. The polymorphisms were excluded if they deviated from HWE. All statistical analyses were performed by SPSS 16.0 (SPSS Inc).

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