Volume 29, Issue 6 (9-2022)
RJMS 2022, 29(6): 73-88 |
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Research code: 86589
Ethics code: IR.IAU.RASHT.RES.1398.013
Clinical trials code: IR.IAU.RASHT.RES.1398.013
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Haji Motalebi E, Assadian narenji S, Goleyjani moghadam R. Association of (-1082 A/G) (rs1800896) Promoter Region of Human IL-10 Gene Polymorphisms with Psoriasis Vulgaris in The West of Mazandaran and East of Guilan Provinces Using ARMS-PCR Technique. RJMS 2022; 29 (6) :73-88
URL: http://rjms.iums.ac.ir/article-1-6964-en.html
URL: http://rjms.iums.ac.ir/article-1-6964-en.html
Islamic Azad UniversityAssistant Professor, Department of Biology, Faculty of Biological Sciences,Tonekabon Branch, Islamic Azad University, Tonekabon, Iran , Assadiansomayeh@hotmail.com
Abstract: (1223 Views)
Background & Aims: Psoriasis is a chronic inflammatory skin disease with an immunogenetic background that is prevalent among ethnic groups. The prevalence of this disease varies from region to region; and it is reported to affect 2-4% of the population in Western countries. While in Asian and some African countries, the lowest prevalence rate (between 0.3 to 1.2% in China) has been reported. On the other hand, in the Scandinavian and Caucasian populations, this rate reaches 11%. In Iran, its prevalence is between 1.3% to 2.5% (1-3, 5, 7). IL-10 is a cytokine produced primarily by monocytes and lymphocytes. The gene encoding IL-10 is located on the long arm of chromosome 1. The promoter region is highly polymorphic, with 3-point mutations including: single nucleotide polymorphisms (SNPs) at positions: –1082 (G/A), –819 (C/T) and –592 (C/A) (4, 12, 13, 14). The association between interleukin-10 gene rs1800896 polymorphism and susceptibility to psoriasis has been investigated in several studies, but with contradictory findings. Therefore, the aim of the present study was to investigate the possible links between –1082(G/A) polymorphism of the IL-10 gene and psoriasis in cases from the north of Iran. The results of this study can be used to find biomarkers that are used in personal medical science for the early diagnosis of diseases and are highly regarded by the scientific community today.
Methods: This case-control study was performed on 50 patients and 35 healthy individuals in the west of Mazandaran and east of Guilan province. Genomic DNA was extracted from the blood samples and genotyping was performed by means of the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) method. This method is based on the design of specific pairs of allele primers and the amplification of the desired allele. To amplify the region containing the mutation, two primer pairs can be used, designed for mutated alleles and natural alleles, respectively. All primers used were designed using Gene Runner and Oligo7 software based on the IL-10 gene sequence on chromosome 1. After selecting the primers, their sequences were compared with the sequences of the human genome in the Gene Bank database. In the primer design, in addition to the 3′ terminal mismatch, at the third end of 3′ primers, a base was changed to ensure that the primer did not bind to the opposite allele.The results were validated using the DNA sequencing method. To perform this reaction, a vial containing 25 µl of Master Mix, 2µl of each primer; and 5 µl of DNA was used and reached to 50µl by adding 16µl of distilled water. The mix was then placed in a thermocycler with a annealing binding temperature of 61.5 ° C to perform the polymerase chain reaction. After PCR, qualitative analysis was performed using 2.5% agarose gel; and the rest of the PCR products with Reverse primer were sent to taq-Copenhagen (Denmark) for PCR Sequencing. The submitted sequence analysis was performed using Chromas software and the known mutations in the samples were confirmed. Data were analyzed using the chi-squared test. A logistic regression test was used to eliminate the effect of possible interfering factors and odd ratios (OR) and their respective 95% confidence intervals were calculated.
Results: AA, AG, and GG genotypes and A and G alleles were examined in patients with psoriasis. The genotypic distribution of the patient and control groups was in Hardy-Weinberg equilibrium. PCR products were analyzed on 2.5% agarose gel using a DNA marker with a molecular weight of 100 bp. The results of statistical studies showed that the studied groups did not differ significantly in terms of gender and age. The frequency percentages of AA, AG and GG genotypes were 82%, 18% and 0% in the control group and 56%, 32% and 12% in the patient group, respectively. This difference is significant at the level of 0.05 (p = 0.014). Also, the frequency of A and G alleles in the control group were 91% and 9%, respectively, and in the patient group were 72% and 28%, respectively. Therefore, this difference is significant at the level of 0.05 (p = 0.002).
The results showed that there is a significant difference in the frequency of alleles between the two groups; so the G allele is detected more in people with psoriasis than healthy group and in terms of genotypic distribution, the GG genotype in the psoriasis group is more than the controls. Also, due to the fact that this significant difference may be related to interfering and in Fisher's exact test, these factors were not controlled, so logistic regression was used to remove these factors. The results showed that even after matching and eliminating the interfering factors, there were still significant differences between the genotypes. Therefore, a significant relationship is observed between rs 1800896 rs polymorphism of interleukin 10 gene and susceptibility to psoriasis.
Conclusion: We conclude that IL-10 gene polymorphism (rs 180096) is likely to increase the risk of susceptibility to psoriasis in western Mazandaran and eastern Gilan. According to various studies, it can be seen that one of the reasons for explaining the contradictory results could be that these studies have been conducted on different populations around the world. Another possible reason is that the regulation of IL-10 varies in different cell types (T cells, B cells, and macrophages) and with different stimuli. In addition, transcriptional levels and IL-10 protein levels may differ due to post-transcriptional regulation of IL-10 gene expression. Other causes of different results in the study of gene polymorphisms include different gene pools, molecular methods used; and the size of the study population. Using the right sample size for each type of disease phenotype can better reflect the true effect of the polymorphism on the characteristics of that disease (1, 13,15, 44). More research is needed focusing on different populations to reach a definitive overall conclusion about this relationship. Also; studies should be conducted in a larger group of patients as high as possible and on all polymorphisms of this gene in order to validate these findings.
Methods: This case-control study was performed on 50 patients and 35 healthy individuals in the west of Mazandaran and east of Guilan province. Genomic DNA was extracted from the blood samples and genotyping was performed by means of the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) method. This method is based on the design of specific pairs of allele primers and the amplification of the desired allele. To amplify the region containing the mutation, two primer pairs can be used, designed for mutated alleles and natural alleles, respectively. All primers used were designed using Gene Runner and Oligo7 software based on the IL-10 gene sequence on chromosome 1. After selecting the primers, their sequences were compared with the sequences of the human genome in the Gene Bank database. In the primer design, in addition to the 3′ terminal mismatch, at the third end of 3′ primers, a base was changed to ensure that the primer did not bind to the opposite allele.The results were validated using the DNA sequencing method. To perform this reaction, a vial containing 25 µl of Master Mix, 2µl of each primer; and 5 µl of DNA was used and reached to 50µl by adding 16µl of distilled water. The mix was then placed in a thermocycler with a annealing binding temperature of 61.5 ° C to perform the polymerase chain reaction. After PCR, qualitative analysis was performed using 2.5% agarose gel; and the rest of the PCR products with Reverse primer were sent to taq-Copenhagen (Denmark) for PCR Sequencing. The submitted sequence analysis was performed using Chromas software and the known mutations in the samples were confirmed. Data were analyzed using the chi-squared test. A logistic regression test was used to eliminate the effect of possible interfering factors and odd ratios (OR) and their respective 95% confidence intervals were calculated.
Results: AA, AG, and GG genotypes and A and G alleles were examined in patients with psoriasis. The genotypic distribution of the patient and control groups was in Hardy-Weinberg equilibrium. PCR products were analyzed on 2.5% agarose gel using a DNA marker with a molecular weight of 100 bp. The results of statistical studies showed that the studied groups did not differ significantly in terms of gender and age. The frequency percentages of AA, AG and GG genotypes were 82%, 18% and 0% in the control group and 56%, 32% and 12% in the patient group, respectively. This difference is significant at the level of 0.05 (p = 0.014). Also, the frequency of A and G alleles in the control group were 91% and 9%, respectively, and in the patient group were 72% and 28%, respectively. Therefore, this difference is significant at the level of 0.05 (p = 0.002).
The results showed that there is a significant difference in the frequency of alleles between the two groups; so the G allele is detected more in people with psoriasis than healthy group and in terms of genotypic distribution, the GG genotype in the psoriasis group is more than the controls. Also, due to the fact that this significant difference may be related to interfering and in Fisher's exact test, these factors were not controlled, so logistic regression was used to remove these factors. The results showed that even after matching and eliminating the interfering factors, there were still significant differences between the genotypes. Therefore, a significant relationship is observed between rs 1800896 rs polymorphism of interleukin 10 gene and susceptibility to psoriasis.
Conclusion: We conclude that IL-10 gene polymorphism (rs 180096) is likely to increase the risk of susceptibility to psoriasis in western Mazandaran and eastern Gilan. According to various studies, it can be seen that one of the reasons for explaining the contradictory results could be that these studies have been conducted on different populations around the world. Another possible reason is that the regulation of IL-10 varies in different cell types (T cells, B cells, and macrophages) and with different stimuli. In addition, transcriptional levels and IL-10 protein levels may differ due to post-transcriptional regulation of IL-10 gene expression. Other causes of different results in the study of gene polymorphisms include different gene pools, molecular methods used; and the size of the study population. Using the right sample size for each type of disease phenotype can better reflect the true effect of the polymorphism on the characteristics of that disease (1, 13,15, 44). More research is needed focusing on different populations to reach a definitive overall conclusion about this relationship. Also; studies should be conducted in a larger group of patients as high as possible and on all polymorphisms of this gene in order to validate these findings.
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