Relationship Between The Polymorphism In The Third Intron Of Interferon-γ Gene And Susceptibility To Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune IFNlammatory disease characterized by demyelination and neurodegeneration within the central nervous system (CNS), which generally occurs in genetically susceptible people. MS is a multifactorial disease caused by the interactions of several genetic and environmental factors.Interferon gamma (IFN-γ) secreted from T-lymphocytes is believed to play an important role in immunological mediated disease. IFN-γwas known to induce myelin damage in patients. IFN-γgene is located on chromosome 12 and comprised of four axons and three introns. Recently it have been found that IFN -γintronⅠandⅢgene have polymorphisms, which were associated with high IFN-γproduction, suggesting the involvement of genetic predisposition to high IFN-gamma levels in the development of MS. The role of gene polymorphism in the third intron of IFN-γhas not been studied in MS, for the IFN -γintronⅠgene polymorphisms previous study have shown that they are unlikely to play a major role in MS susceptibility or disease course. To explore the possible relationship between polymorphism in the third intron of IFN-γgene and the susceptibility of MS, we detected +2118,+2707 and +3586 sites in the third intron of IFN-γgene among normal and MS groups.Objective: 1. To study the association between polymorphism in the third intron of IFN-γgene and the susceptibility to MS, and reveal the role of IFN-γin MS. 2. To investigate the changes of the serum and CSF levels of IFN-γin MS, to define a possible pathogenic role of IFN-γduring the progression of MS. 3. To analyze the differences of the MS about EDSS scores, the age at onset, the duration of disease, and the numbers of relapses.Methods: 1. A case-control research was performed on 58 MS patients and 40 healthy individuals as control group. All subjects are Han nationality of northern China. All patients fulfilled the criteria of MS proposed by Poser et al (1983). forty unrelated, healthy, randomly selected individuals from the same geographic area served as controls. None of the control subjects had a recorded personal or familial history of autoimmune disease. 2. DNA was extracted from peripheral blood leukocytes by improved fast procedure. 3. PCR was used to amplify the target sequence. The PCR products were separated on 8% SDS-PAGE stained with 0.5% ethidium bromide and visualized under ultraviolet light. 4. The gene polymorphism of IFN-γwere screened by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) assay. The PFLP products were 8% polyacrylamide stained with 0.5% ethidium bromide and visualized under ultraviolet light. 5. Serum and CSF samples were frozen in aliquots at -70℃within 1h of collection and repeated freezing and thawing of samples were avoided. Levels of IFN-γin test and control subjects were determined by means of sandwich enzyme-linked immunosorbent assay (ELISA), using IFN-γimmunoassay kit. 6. All statistical analyses were performed using SPSS 10.0 statistical analysis software. Genotype distribution underwent the Hardy-Weinberg (HW) equilibrium calculation, the two groups are presentative of the overall population. Theχ2 test was used to compare genotype and allele frequencies between the MS and control population. Relative risks (RRs) were calculated according to the methods of Woolf. The data of IFN-γlevels were expressed as Mean±S. Comparisons means in different groups were performed using Student t test or one-way ANOVA followed by Student-Newman-Keuls multiple range test. A P-value of less than 0.05 was considered statistically significant.Results: 1.The serum levels of IFN-γwas found to be significantly increased in the acute-couse MS group compared with the healthy control group, (10.1076±1.3279) pg/ml vs. (3.4321±0.5934) pg/ml, P<0.05, but not in the remitting-couse MS group (P>0.05). While the serum level of IFN-γin the patients with acute-couse MS were higher than in the patients with remitting-couse MS (10.1076±1.3279) pg/ml vs. (3.4106±0.8473) pg/ml, P<0.05.2. The CSF levels of IFN-γwere found to be significantly increased in the acute-couse MS group compared with the control group, (5.6862±1.0831) pg/ml vs. (1.0545±0.1592) pg/ml, P<0.05, but not in the remitting-couse MS group (P>0.05). While the CSF levels of IFN-γin the patients with acute-couse MS were higher than in the patients with remitting-couse MS,(5.6862±1.0831) pg/ml vs. (1.1404±0.2079) pg/ml, P<0.05.3. Compared with the control group or remitting-course MS,the increased levels of IFN-γin CSF is higher than that in serum in the acute-course MS group.4. No significant difference of genotypes and site frequencies of IFN-γintronⅢ(+2707,+3586) existed in MS group compared with in the control group. (P>0.05).5. The frequency of IFN-γintronⅢ+2118A allele increased significantly in MS group compared with it in the healthy control group. 81.03% vs. 66.25% P<0.05。6. There was significant positive correlation in the EDSS scores to the duration of disease, the number of relapses and the dye at inset. (r=0.3185, P=0.0201,and r=0.2917,P=0.0341,and r=0.3985, P=0.0021, respectively.)Conclusions: 1. The frequency of IFN-γintronⅢ+2118A allele in MS group increased significantly compared with the control group. The allele IFN-γintronⅢ(+2118A) might be a susceptive gene to MS.2. The IFN-γintronⅢ(+2707) gene polymorphism might not be associated with MS genetic susceptibility.3. The IFN-γintronⅢ(+3586) gene polymorphism studied is unlikely to play a major role in MS susceptibility. 4.The level of IFN-γin the patients with acute-couse MS, but not in the patients with remitting-couse MS. IFN-γmight play an important role in the development of MS.5. EDSS scores were positive correlation to the duration of disease, the age at onset and the numbers of relapses. (P<0.05)...