Studies On The Relationship Between Single Nucleotide Polymorphism Of FOXP3 Intron-1 And Psoriasis Vulgaris

With the completion of the human genome project, more and more attention is being paid on the study of relationship between individual genetic variation and diseases. The single nucleotide polymorphisms （SNPs）, which is among the most common types in terms of the genetic variation in human genome, is an important factor in producing various clinical features of different individuals. Therefore, the studies on SNP in connection with complex diseases is of great importance concerning early diagnosis, premunition, and treatment of these disease.Psoriasis （commonly referred to as scaly tetter） is a common dermatological disorder with characteristics of high incidence of disease, frequent fluctuations and difficulty in treatment. Although in most cases it will not threat to the lives of patients, it still brings serious side effects on their quality of life, work efficiency as well as physical and mental health.It was previously considered that the cause and pathogenesis of psoriasis relate to various reasons, such as inheritance, infection, metabolic disorder, internal secretion, nerves, spiritual state, immunization, and so on. It was found later that the environmental factors also count a lot in causing the disease or do its work during the process of treatment. Now it is widely recognized that psoriasis is a kind of chronic inflammatory and proliferative disease due to dysimmunity, which is both controlled by polygenic inheritance and incited by a variety of environmental factors. In other words, the diversity of people’s genetic background will affect their gene functions. Under the influence of the environmental factors, the cellular immunity will be stimulated and make a response, the autoimmunity mediated by CD4+T cell will then be thrown into disorder, which directly causes psoriasis.Several studies have shown that FOXP3 genes and CD4+CD25+FOXP3+ regulatory T cells（ Tri-Treg） which are affected by the former play important roles in the pathogenesis of psoriasis caused by the disorder of autoimmunity. The reduction in the number of Tri-Treg, especially the immune imbalances between Tri-Treg and effectiveness T Cell （Te）, both lead to the excessive activation of Te. It is the core link during the whole process of disease development concerning severe psoriasis and psoriasis vulgaris（PV）.It has been recently reported in China that the individual site in SNP of FOXP3 genes does relate a lot to the onset of psoriasis. However, in regard to the complex autoimmune disease affected by multiple genes, we need to arrange relative tests with large samples in reference to multi-site SNP, which will provide fundamental information for us to carry on studies about the transcriptional regulation of FOXP3.For this reason, we choose in our study the FOXP3 genes to conduct our preliminary experimental research considering their close relations with the onset of immune-related diseases. We plan to investigate the distribution and frequency of occurrences among the patients with PV in China. At the same time, the biological significance of positive- correlated SNP sites is also our focus in our study.Based on our collection of information of 408 patients with PV and 363 healthy people, we use questionnaire survey and logistic regression analysis involving multiple factors to find out the risky factors that have something to do with the onset of PV.We analyzed four SNPs （rs2232365, rs3761547, rs3761548 and rs3761549） of FOXP3 genes in a case–control study using the method of testing the polymerase chain reaction and ligase detection reaction （PCR-LDR）.The allele frequency is directly calculated through the genotypes. The equilibrium of Hardy-Weinberg is tested based on the test ofχ². We regard the result of p>0.05 as one which is in accordance with the equilibrium of Hardy-Weinberg. The difference between these two groups is tested by the 2×2 and 2×3 contingency tables. For the linkage disequilibrium, we employ the software of linkage disequilibrium analyzer （LDA） to analyze it. Interaction among sites are tested on the test ofχ², we regard the result of p<0.05 statistical significance. All calculations were carried out using Statistical Analysis System （version 9.0, SAS Institute Inc, Cary NC）We make progress with the preliminary analysis on functions of the FOXP3 gene locus （rs2232365,rs3761547,rs3761549） that are positively related with PV. In other words, we use the method of PCR（reverse transcription PCR,RT-PCR） to analyze the different expression of FOXP3 mRNA in those patients with PV before and after we use Interleukin 2 （IL-2） to make external intervention. In addition, we also analyze the influence on the genetic transcription and expression of FOXP3 genes caused by the different genotypes in FOXP3 gene intron.The main results and conclusions are summarized as follows:1. It is found in this study that family history （p<0.001, OR=3.12, 95% CI= 2.40- 4.07）as well as early onset age（p<0.001,OR= 1.12, 95% CI= 0.92-1.38）is also acutely effect PV. At the same time some environmental factors are very risky ones concerning the onset of PV. Those that show positive association with the onset of psoriatic are as follows: smoking （ p<0.001, OR =3.91, 95% CI=2.68-5.71）, drinking （occasionally: p= 0.037, OR=1.46, 95% CI=1.02-2.09; alcohol withdrawal: p<0.001, OR=5.37, 95% CI = 3.21-8.98 ;continuously: p<0.001, OR=11.90, 95% CI=7.08-19.98） ; unemployment （p= 0.0175, OR=3.72, 95% CI=1.26-10.97）, singlehood（p<0.0001, OR=3.93, 95% CI= 2.80 -5.52 ） , insufficient sleep（time<5h: p<0.0001, OR=56.61, 95% CI= 13.56- 236.36; 8h>time>5h : p<0.0001, OR= 10.30, 95% CI=7.16-14.82） and dwells outside of the city （p<0.001, suburban area: OR=3.72, 95% CI= 2.06-6.72; county: OR=40.56, 95% CI= 6.27- 101.14; village: OR=26.80, 95% CI= 10.65-67.47）, menstrual disorder （p=0, OR=3.88, 95% CI= 1.88-7.92）.2. As to the comparison based on the case-control study between 408 patients with PV and 363 healthy people, we firstly classify the types of four SNPs sites（rs2232365, rs3761547, rs3761548, rs3761549）of FOXP3 genes in association with PV by PCR-LDR, and then draw a conclusion that the allele frequency of rs2232365, rs3761547 and rs3761549 differ a lot in these two groups（all p<0.05）.The minority homozygote（low frequency transduction isoloci homozygosis genetype）（rs2232365 GG, rs3761547 GG, rs3761549 TT）and combined genotypes （rs2232365 AG+GG, rs3761547 AG+GG and rs3761549 CT+TT） are conservanct genotypes with which individuals will elude from the increased risk of getting infected with PV: rs2232365 GG: p=0.0206, adjusted OR= 0.32, 95% CI=0.17-0.86; rs3761547 GG: p= 0.0288, adjusted OR=0.301,95% CI=0.10-0.88; rs3761549 TT: P=0.0399,adjusted OR = 0.33, 95% CI = 0.12- 0.95。3. Combined with stratified analysis on the genotypes of different samples, we study the association between gene polymorphism and clinical genotype of PV. The results show that the condition of carrying the genotypes like rs2232365 AG+GG,rs3761547 AG+GG, and rs3761549 CT+TT can obviously decrease the risk of suffering PV in patients whose onset age <40 years old , the genotypes like combined genotype ,heterozygote, minority homozygote can obviously decrease the risk of suffering PV in females .Furthermore , minority homozygote in these three SNP loci can also decrease the risk of PV in people without company disease comparing with patients with complication .4. This study first discovers that the functional SNP site of FOXP3 genes named rs3761548 has no correlation with the onset of PV.5. We detect the linkage disequilibrium among the three SNPs when rs2232365 is regarded as site 1, rs3761547as site 2 and rs3761549 as site 4. The analysis showed that the three SNPs are interlocked to a certain degree ( they are D’_1,2=0.8794, r²= 0.3927; D’_1,4=0.9924, r²= 0.4223; D’2,4=0.9306, r²= 0.9455 respectively).6. We performed a combined reciprocation analysis regarding the genotype of polymorphic sites of FOXP3 genes. The joint type between each other among these three loci :（rs2232365 and rs3761547, rs2232365 and rs3761549、rs3761547 and rs3761549） also differ a lot in the group of patients and healthy people. （p value is 0.0356,0.0004 and 0.0043 respectively）. Among them, only the combined genotype named FOXP3-rs2232365 AA+GG -rs3761547AG+GG increases the risk of suffering PV by 2.90 times （p=0.0393, adjusted OR=2.90 95%CI =1.05–1.97）, the others named respectively rs2232365AG+GG-rs3761547AG+GG（p=0.0019, adjusted OR=0.59, 95% CI= 0.42-0.82）、rs2232365AG+GG-rs3761549CT+TT（p=0.0012, adjusted OR=0.57, 95% CI= 0.41-0.80）、rs3761547AG+GG- rs3761549CT+TT （p=0.0027, adjusted OR=0.62, 95% CI= 0.45-0.85） can decrease the risk of PV. All these point out that there would be combined enhancement effect between heterozygote and low frequency transduction isoloci homozygote .To mentiion that there are combination enhancement effect betwwen each other of all sites.7. When we employ the method of joining them in a pair and test on non-independent samples, we do statistical comparison between the expression and C_T value of mRNA in FOXP3 genes concerning the peripheral white blood cells before and after the intervention of IL-2. We find the expressive scale of FOXP3 mRNA in the samples’blood will increase a lot when the allelomorphic genes are changing toward the variant pattern. Accordingly, this provides us a satisfactory explanation about our research results, that is, the safety genotypes of three SNP positive sites of FOXP3 genes will definitely decrease the risk of suffering PV.Given all that, through the case study and comparison among the people with PV across the country, we find that the polymorphism of rs2232365, rs3761547,and rs3761549 of FOXP3 genes is interrelated with the onset of PV in Chinese population. All of low frequency transduction allele in these loci are protective to people by freeing from PV. The three sites have certain linkage. The combined genotype rs2232365 AA -3761547AG+GG can obviously increase the risk of suffering PV. Additionally, the altofrequency allellotype of the previous three sites have some influence on the transcriptional level of FOXP3 mRNA. Therefore, it is possible that the altofrequency allellotype of FOXP3 increase the number of risky factors through reducing the expression of FOXP3 genes. Simultaneously, this research also gives us the clue about the close relations between some environmental factors and the onset of PV. Those people who expose themselves to the infected areas, singlehood ,unemployment, dewell outside of city,or are experiencing the state of alcohol withdrawal, insufficient sleep and female sex hormone disorder, are testified to undergo an easier trend of suffering PV. It is worth mentioning that those who quit smoking and drinking during illness are also easier to catch PV.In a word, this study shows that a number of environmental factors as well as SNPs of FOXP3 are closely associated with the onset of PV. We support the theory that the interaction of physical inheritance and external environment is one of the most important pathogenesis which cause the disease -- psoriasis.