| Multiple sclerosis (MS) is an autoimmune inflammatory disease characterized by demyelination and neurodegeneration within the central nervous system (CNS), which generally occurs in genetically susceptible people. MS is a multifactoral disease caused by the interactions of several genetic and environmental factors. Recent study has shown that distinct differences exist in the immunogenetic background as well as in the magnetic resonance imaging(MRI) and clinical features between the Western and Asian subtypes of MS.Tumor necrosis factor (TNF) which contains TNFαand TNFβis believed to play an important role in immunologically mediated disease. TNFαis secreted from activated monocytes and TNFβis secreted from induced T-lymphocytes, and they have similar biologic functions. TNF genes are closely linked to HLA genes and have been mapped to human chromosome 6. Recently it has been found that TNF genes have polymorphisms, which were associated with high TNF production, suggesting the involvement of a genetic predisposition to high TNF levels in the development of MS. TNF was known to induce myelin damage and degeneration of axons in patients. For the important role of TNF in immunology and the special genetic location,we investigated the gene polymorphisms of TNFαand TNFβand the relations of the TNFα/βgene polymorphisms to the two subtypes of MS in different courses.Objective: 1. To study the relations between the susceptibility to Western-type MS and Asian-type MS, two subsets of MS, and the frequencies of TNFα/βgene polymorphysims. 2. To investigate the changes of the serum levels of TNFα/βin different MS courses of the two subtypes, and to define a possible pathogenic role of TNF in MS patients during the phases of the disease. 3. To investigate the associations between TNFα/βgene polymorphisms and EDSS scores, the duration of disease, and the number of relapses in MS patients. 4. To analyze the correlations of EDSS scores to the serum levels of TNFα/β, the duration of disease, the number of relapses,and the age at onset. 5. To analyze the differences of the two subtypes about EDSS scores, the age at onset , the sex ratio,the duration of disease, and the number of relapses.Methods: 1. A case-control research was performed on 58 MS patients and 79 healthy individuals as control group. All subjects came from Han nationality of northern China. All patients fulfilled the criteria of MS proposed by Poser et al(1983). These patients were classified into two groups: Western-type MS 22 cases and Asian-type MS 36 cases, according to corresponding diagnostic criteria proposed by Kira et al(1996). All patients were also divided into two different groups: acute-course MS 37 cases and remitting-course MS 21 cases. Seventy-nine, 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 saltingout. 3. PCR was used to amplify the aim genetic sequence. The PCR products were separated on 2% agarose gel stained with 0.5% ethidium bromide and visualized under ultraviolet light. 4. The gene polymorphisms of TNFαand TNFβwere screened by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) assay. The RFLP products were separated on 2 %～3 % agarose gel stained with 0.5 % ethidium bromide and visualized under ultraviolet light. 5. Serum samples were frozen in aliquots at–70°C within 1h of collection and repeated freezing and thawing of samples were avioded. Levels of TNF in test and control subjects were determined by means of sandwich enzyme-linked immuno- sorbent assay (ELISA), using TNF immunoassay kits. 6. The neurologic impairment status of patients was scored according to the Expanded Disability Status Scale (EDSS) , meanwhile collecting clinical data——the duration of disease, the age at onset, sex-ratio,and the number of relapses. 7. All statistical analyses were performed using SAS 6.12 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 populations. Theχ2 test was also used to compare the sex ratio of the two subtypes. Relative risks (RRs) were calculated according to the methods of Woof. The clinical data such as serum TNF levels , EDSS scores , the age , the duration of disease, and the number of relapses were expressed as Mean±SD. Comparisons between means in different groups were performed using Student t test or one-way ANOVA followed by Student-Newman-Keuls multiple range test. Chitest was used to compare the relation of EDSS scores to TNF levels, the age , the duration of disease, and the number of relapses . A P-value of less than 0.05 was considered statistically significant.Results: 1. No significant difference of genotype and allele frequency of TNFαexisted in MS group as well as in the two clinical substypes of MS compared with the control group (P>0.05). 2. The frequencies of TNFβ1 allele increased significantly both in MS patients and in the Asian-type MS patients compared with those of healthy controls (45.69% vs. 27.85%,RR=2.18, and 51.39% vs. 27.85%, RR=2.74,P<0.05, respectivelly), while Western-type MS and control groups showed similar distribution in the frequencies of TNFβ1 alleles(P>0.05). 3. There was no significant difference in the EDSS scores, the age at onset, the duration of disease and the number of relapses among MS group with TNFα1/1, TNFα1/2 and TNFα2/2 respectively(P>0.05). 4. There was no significant difference in the EDSS scores, the age at onset, the duration of disease and the number of relapses among MS group with TNFβ1/1,TNFβ1/2 and TNFβ2/2 respectively,the same results were found in the Western-type MS and Asian-type MS groups(P>0.05). 5. The serum levels of TNFαand TNFβwere found to be significantly increased in the acute-course MS group compared with the healthy control group respectively ((26.89±14.65)pg/mL vs(.11.17±8.41)pg/mL, (607.92±289.13) pg/mL vs(.308.45±206.19)pg/mL, P <0.05, respectively), but not in the remitting-course MS group(P>0.05), while the serum level of TNFαwas found significantly increased in the remitting-course patients of Western-type MS group compared with the healthy control group((19.87±17.97)pg/mL vs.(11.17±8.41)pg/mL ,P<0.05). The serum levels of TNFαand TNFβin the patients with acute-course MS were higher than in the patients with remitting-course MS respectively (P<0.05), while the serum level of TNFαwas no significant difference in the patients with acute-course of Western-type MS compared with the patients with remitting-course of Western-type MS (P>0.05), and the serum level of TNFβwas no significant difference in the patients with acute-course of Asian-type MS compared with the patients with remitting -course of Asian-type MS (P>0.05). 6. There was significant positive correlation in the EDSS scores to the duration of disease, the number of relapses and the age at onset (r=0.3185, p=0.0201, and r=0.2917, p=0.0341, and r=0.3985, p=0.0021,respectively). 7. There was no significant difference in the EDSS scores, the age at onset, sex-ratio,the duration of disease and the number of relapses between the two subtypes (P>0.05).Conclusions: 1. The frequency of TNFβ1 allele in MS group increased significantly compared with the control group , especially in the Asian-type MS group, but not in the Western-type MS group. The allele TNFβ1 might be a susceptical gene to MS, especially to Asian-type MS. 2. TNFαgene polymorphism might not be associated with MS genetic susceptibility. 3. The serum levels of TNFαand TNFβelevated in the patients with acute-course MS , but not in the patients with remitting-course MS. TNF might play an important role in the development of MS, furthermore TNFαand TNFβmight play more important roles in the development of Asian- and Western-type MS respectively. 4. There was no significant relationship of TNF gene polymorphisms to EDSS scores, the age at onset , the duration of disease and the number of relapses. 5. There was no significant difference in the EDSS scores, the age at onset, sex-ratio,the duration of disease and the number of relapses between the two subtypes. 6. EDSS scores were positive correlation to the duration of disease, the number of relapses and the age at onset.
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