Polymorphism in IL-6(-174 G/C) Gene in Scabietic Patients

Background : Scabies ( Sarcoptes scabiei var. hominis ) is one of the parasitic diseases that is often ignored. It results in problems that trigger an allergic and inflammatory immunological response. Cytokines are crucial to the development of disease and its pathophysiology. This investigation looked at how scabies risk and a variation in the gene IL-6 affected IL-6 production. Materials and Methods : The goal of the current investigation was to determine the level of IL-6 by ELISA and its linked with IL-6(-174 G/C) polymorphisms in 60 scabietic patients and 30 healthy individuals. Results : In contrast, the IL-6(-174 G/C) CC genotype and C allele carrier frequencies were highly significant (P0.05) in the patients who had their first infestation. The results showed a significant increase (P0.05) in the IL-6(-174 G/C) GG genotype and G allele in the re-infestation group of patients compared to the healthy group. According to IL-6 genetic variations, low serum levels were connected with the CC genotype. Conclusion : IL-6(-174 G/C) Scabies susceptibility and polymorphism were related, providing evidence for the hereditary basis of the illness.


Introduction
S. scabiei, the scabies mite, continues to spread around the world, affecting an estimated 300 million people.This infection typically causes significant pruritus that must be treated with prescription scabicidal therapy since it worsens at night]1[.The S. scabiei that causes scabies in humans and sarcoptic mange in many other animals, on the other hand, are physiological variations of the same species.They don't completely have a host specificity, but they typically only last a short time on other hosts]2[.Pleiotropic cytokine IL-6 has important functions in both acute and chronic inflammation as well as having a variety of impacts on both immune and non-immune cell types.This cytokine is released by T-cells and macrophages to promote an immunological response ] 3[ .IL-6 plays harmful roles in a number of disorders, including inflammatory, autoimmune, and cancerous diseases]4[ .On chromosome 7p21, the human IL-6 gene spans 5 kb, has five exons, four introns, and a proximal promoter region.The IL-6 gene promoter region has more than 50 single nucleotide polymorphisms (SNPs), specifically IL-6(174 G/C)]5 and 6[ .The purpose of this study was to use the ARMS-PCR technology to examine the relationship between the promoter polymorphism of interleukin-6 (174 G/C) among some Iraqi scabietic patients.

Patients and control:
The current study comprised 30 healthy persons (15 males and 15 females) as well as 60 people who have Sarcoptes scabiei variant.hominis infection (35 females and 25 males).The patients were divided into two groups: first infestation and re-infestation.

Blood samples :
Five milliliters of blood were drawn from each patient hospitalized to the AL-Diwaniyah Teaching Hospital between February and April of 2019; healthy subject samples were obtained from private labs.While the other portion of the sample was allowed to coagulate at room temperature before being centrifuged at 3000 rpm for 10 minutes, one component of the sample was divided into two and put in an EDTA tube to extract DNA for genetic analysis.Sera samples were then put into eppendrof tubes and maintained in a deep freezer until required..

Laboratory investigations :
A.Immunological assays : IL-6 levels were determined manually using ELISA kits from the UK's Bioassay Technology Laboratory to estimate their levels.

B. PCR amplification
Genomic DNA was recovered from the blood of scabietic patients and controls using a DNA micro kit (favergen,korea).Using amplification refractory mutation  (1).The master mix for the PCR amplification of the IL-6 polymorphism (-174 G/C) was composed of 5 l of genomic DNA, 1 l of each primer (10 pmol/1 l), and 11 l of nucleasefree water.The reaction was conducted at 95 °C for two minutes, followed by 35 cycles of 95 °C for one minute of denaturation, 63 °C for one minute of annealing, and 72 °C for one minute of extension, with the final two minutes of each cycle being conducted at 72 °C.The initial annealing temperature for touchdown reactions was 72°C.Once the annealing temperature was reached, the temperature was decreased by 1°C per cycle until the end of the cycles, when it was kept there.After the PCR result had been stained with ethidium bromide and electrophoresed on a 2% agarose gel, a picture had been taken.

4.Statistical Analysis:
Utilizing the statistical system SPSS version -18, the data were examined [7].Standard deviation (SD) and means were used to express the data.Least significant differences were used to evaluate differences between patient and control groups (L.S.D) [ 8].

Results and Discussion
The levels of IL-6 in the patient and control groups were significantly different, as shown in table 2, with the levels of the first infestation and re-infestation groups reaching 83.8810.58ng/L and 230.3072.10ng/L, respectively, and the healthy group reaching 85.449.35ng/L.The data showed that there were significant variations in the levels of IL-6 between the patient and control groups (P>O.O5); nonetheless, it was hypothesized that because IL-6 inhibits TNF-, it operates as an anti-inflammatory cytokine rather than a proinflammatory cytokine in scabietic cases]9 [ .Meanwhile, the interleukin IL-6 has both pro-and anti-inflammatory properties, and it exerts its anti-inflammatory effects by inhibiting TNF-α in individuals with atopic dermatitis ] 10[ .While the major disparities between scabietic patients and healthy groups were explained by the fact that IL-6 does not inhibit TNF-α, acting instead as a pro-inflammatory cytokine, Since TNF-α is inhibited by IL-6, this means that IL-6's anti-inflammatory cytokine activity is mediated via TNF-α]11[ .
The inhibitory actions of IL-6 on TNF-α mediate its anti-inflammatory cytokine activity.asevidenced by the significant differences between scabietic patients and healthy groups, in which they explained that the due to its inability to stop TNF-α, IL-6 works as a pro-inflammatory cytokine rather than an anti-inflammatory cytokine.In the meantime, the first infestation, re-infestation, and control group patients' GC genotype frequencies were 33.3%, 33.3%, and 35.5%, respectively, with a nonsignificant difference in OR of 1.909; table 3 also showed non-significant differences with OR of 0.909 between the first infestation, re-infestation, and control group patients.In the patient (original infestation, re-infestation) and control groups, the GG genotype frequencies were 6.7%, 63.3%, and 61.3%, respectively.This is not statistically significant, although there are small significant differences between the two groups (OR=0.105).76.7%, 30.0%, and 19.76% of the first infestation, re-infestation, and control groups, respectively, had the IL-6 (-174 G/C) genotype, respectively.This allele was significantly distributed with a high OR between the first infestation and control groups, but not between the re-infestation and control groups, where the OR was 1.000 and the distribution was non-significant.
While the frequencies of the G allele were 23.3%, 80.0%, and 80.3% in the initial infestation, re-infestation, and control groups, respectively, the distribution of the IL-6 (-174 G/C) allele was significant without an OR., in the other groups.The IL-6 levels for the first infestation, re-infestation, and control groups were, respectively, 69.175, 258.27, and 90.60 ng/L; 64.137, 182.00, and 75.49ng/L for GG and GC genotypes.However, the IL-6 levels for the first infestation, re-infestation, and control groups were all statistically higher in patients who carry the G allele.In the first infestation, re-infestation, and control groups, respectively, the serum levels of IL-6 in patients with the C allele (CC homozygote mutant type) were tested and found to be slightly higher at 64.959, 34.5 ng/L, and 60.2 ng/L.

Figure1:
Figure1: Image from a single experiment investigating the IL-6 (-174) (G/C) gene polymorphism using agarose gel electrophoresis.2% agarose at 100V for 1 hour, with M. (100-1500bp).The internal control product was discernible at 326 bp in all samples.The GG wild type homozygote genotype lanes were visible at 205bp ARMS PCR product size.The homozygote genotype lanes of the CC mutant type were visible at a 176bp ARMS PCR product size.The G and C alleles, however, were amplified at 205 and 176 bp product sizes, respectively, in the G/C heterozygote genotype lanes.
transcriptional regulation and cytokine levels, has an impact on inflammatory phenotypes]12[.Psoriasis susceptibility can be determined by the IL6-174 G/C polymorphism, with the GG genotype carrying a nearly two-fold greater chance of developing the condition ] 13[,Additionally, a single nucleotide change from G to C influences gene transcription, and people with the -174GG genotype have plasma levels of IL-6 that are twice as high as people with the homozygous CC allele ] 12[ .The IL6 (-174G/C) polymorphism's G allele causes an increase in transcriptional activity that raises the serum level of IL-6.; ]14 and 12[.IL-6 blood levels have been linked to an SNP in the IL-6 gene's -174 promoter region, albeit few studies have linked allele C to high IL-6 production]15[;.]16[On the other hand, research has also shown that allele C leads to a reduced production of IL-6 , additionally, research on the IL-6 SNP revealed that the G allele at position -174 may contribute to an inflammatory response that is worsened in homozygous G allele genotypes, such as in AD patients]17[.The G allele and GG genotype of the IL-6 -174C/G polymorphism were associated with an increased risk of AD]18[,Although there is no association between the polymorphisms nt565A/G and IL-6 -174C/G and the risk of AD]19[,Nevertheless, IL-6 174C/G and nt565A/G polymorphisms and the risk of AD]20[.
PCR, the interleukin-6 gene was amplified for IL-6(-174 G/C)]7[ .Table summarizes the primers used to amplify different types of polymorphism

Table 4 : The relationship between IL-6 genotype (-174 G/C) and IL-6 ng/L levels in patient and control groups products' various sizes.
It was used in the scabies investigations since IL-6 (-174 G/C) polymorphism for people with psoriasis and atopic dermatitis as dermatologic disorders had not previously been explored.The most studied SNP at position -174 (IL-6 -174G/C), which modifies