Studies On The Susceptible Genes Of The Autoimmune Thyroid Diseases

Objective:Autoimmune thyroid diseases (AITD), which predominantly include Graves' disease (GD) and Hashimoto thyroiditis (HT), are recognized as a group of organ-specific autoimmune diseases. The interactions of genetic background and environmental factors play an important role in the etiology of the disease. The AITD patients often appear with a familial aggregation, and also was found a higher tendency of coincidence of the diseases in monozygotic twins. Therefore, the studies of AITD predisposing genes have been turned into hot spot by domestic and overseas researchers. To illuminate the susceptible genes of the diseases have great clinic significance, which can provide essential basis of biology for immunology and other function studies, and which can play a basic role for screening susceptible population , preventing the diseases and performing the gene therapy in the future.Cytokines, in common, produced by activated cells, are known as a kind of low molecule polypeptides or proteins, which have strong biologic function in immunoregulation and effective reaction. At present, studies have maken clear that a variety of cytokines, such as interferon (INF), interleukin-1 (IL-1) ,IL-4,IL-6 and tumor necrosis factor (TNF) et al, have become the important component in mechanism of AITD, which enhance the immune response between thyroid gland cells and immunocytes, regulate thyroid gland cells' reduplication, influence their hyperplasia, differentiation, synthesis and secretion, promote them to express cytokinereceptors, then synthesize and release various kinds of cytokines and participate in the apoptosis process of thyroid gland cells. The gene polymorphism of the above-mentioned cytokines must have an effect to their production. So we select IL-1 a ,IL-4, IL-6 and TNF- a as susceptible candidate genes of autoimmune thyroid diseases to carry on screening researches.Human leucocyte antigen (HLA) plays important roles in the development of T-cell, endogenous and exogenous antigen presentation, immune response priming and regulation. Thus, the normal expression of HLA on all kinds of cells membrane can make sure immune system to recognize itself and unitself and keep the stability of internal enviroment. HLA genes are the most complex human gene compounds up to now, most of them exist a great quantity of multiple allele, having weighted polymorphisms. So much polymorphisms in HLA genes result in the rate being greatly changed in conformation of antigen binding recess, binding and presentation antigens to T-cells, which cause to different HLA genetypes have differential susceptibility to diseases. HLA class I which adhere to CD8 accessory receptor and participate in CD8+CTL to recognize antigens mainly have the function of recognizing and presentating endogenous antigen. HLA class II ,however, which adhere to CD4 accessory receptor and take part in CD4+TH to recognize antigens mainly recognize and present exogenous antigen. HLA class I and class II genes are respectively the ligands to CD8 and CD4 molecule, it will stabilize and promote the interplay between immunocytes, will enhance the priming of immune response or effective function once they bind together. The interaction among cells cannot have a marked role unless the MHC molecules they expressed are consistent, that is MHC restriction, then will participate in immune regulation and the interplay among immunocytes. HLA molecules especially the products of HLA class II genes such as HLA-DP^ DQ> DR, which transfer antigens, combine with CD4+ or CD8+T cellreceptors in thyroid gland, then the T-cells are activated and produce lymphocyte factors to regulate immune reaction and generate autoantibody by activating B-lymphocytes, which induce the onset of AITD. Yanagawa and his partners had reported that HLA-DQAl *0501 gene had susceptibility to GD in American Caucasian. Because the frequency of HLA allele disposition is altered with racial specificity, disease incidence of diverse population of AITD has relation to certain specific HLA genotype. What HLA genes are related with GD in Chinese Han people are we interested in. So we have selected HLA-DQAl*0501, -DQBl*0201 > -DQBl*0303 as the candidate genes and analyze their relationship with GD.Object:1. GD group : referring to the diagnostic criteria of GD in and Metabology> as our selection conditions: ?They have the hyperthyroid symptoms, with or without ophthalmopathy, thyroid gland is swelling diffuse and soft. And thyroid function shows (by the means of time-resolved fluorescence staining) : high level of free T3 (FT3) and free (FT4), while low level of sensitive thyroid stimulating hormone (s TSH) .(2) Those who have been diagnosed as GD and accepted ATD therapy with or without remission except for the patients who turned into hypothyroidism after accepting the ATD to treat GD with middle or low dose. Meeting either of the above can be taken in the trial. The patients were divided into three subgroups according to gender, family history and thyroid associated ophthalmopathy. The objects are out- and in-patients in our hospital without relationship of Han people in Shandon area (There are 100 cases in cytokine gene group, among the total 80 cases are females, 20 are males. 103 cases in HLA gene group, among of them 79 cases are females while 24 are males).2. HT group: According to the patients whether they obtain hypothyroidism or high level of anti-thyroglobulin antibody (ATgAb ) or anti-thyroid microsomalantibody, with or without thyroid swelling, or diagnosis HT by the cytology result of fine needle aspiration, thyroid function shows (by the means of time-resolved fluorescence staining): FT3 4.57+ 1.5lpmmol/1, FT4 7.23±4.68pmmol/l,sTSH 20.13 + 7.85ulu/ml. Autoimmune antibody result shows: TgAb 40.72 ±8.06%,TmAb 35.89 + 9.07%. There are 100 cases who are out- and in-patients in our hospital without blood relationship of Han people in Shandon area (97 cases are females, 3 cases are males).3. Screening the healthy people without thyroid gland diseases, having no family history and other autoimmune history as control group.Methods:1. Abstraction of genome DNA: using the fast DNA abstraction kit to draw peripheral leukocyte DNA ,the steps as following(DDraw 1 ml peripheral blood, add 150 pL ACD to prevent concretion.CDTake out 300u L above blood and put it into 1.5m 1 Eppendorf tube, then add 900 u L RBC lysate and mixed evenly constantly , keep 10 minutes under room temperature, during the time put upside down 3 to 5 times to make it even fully. ?Centrifuge for 20 seconds at 15000 rpm under room temperature to make the karyocytes precipitate to the bottom of the tube. ?Discard the supernatant and keep cell precipitum about 30 U L, then turn it into cell suspension. ?Add 300 u L of WBC lysate and mixed evenly fully. Keep it calmly 10 to 15 minutes under room temperature till WBC split completely. ?Add 100 U L of protein precipitum then shock it fiercely for 20 seconds to make it even completely. ?After centrifuging for 3 minutes at 15000 rpm, protein precipitate to the bottom of the Eppendorf tube and become a block of compact black brown plaque. ?Take out of supernatant to a new Eppendorf tube and add equal volume of isoamyl alcohol, then mixed evenly completely. ?Centrifuge for 3 minutes at 15000 rpm. Then take out of the supernatant to a new Eppendorf tube and gently add 1 ml of precooling absolutealcohol through the tubal wall. Keep the tube on ice cubes for 2 to 3 minutes, slowly put it upside down till DNA precipitum separate totally. ?Centrifuge for 3 to 4 minutes at 15000 rpm, then discard the supernatant and use 70% alcohol to wash 2 times, add 50 u L hydration liquid to dissolve DNA precipitum. Put the tube at 4°C for pre-emergency.2. Apply polymerase chain reaction sequence specific primers (PCR -SSP) and according to the ways of Mullighan> Fanning and Olerup amplify gene segments about IL-1 a (-889)> IL-4(-590), IL-6(+3247), TNFa (+488, -308), DQAl*050K DQB1*020 k DQB 1*0303. PCR products stained by ethidium bromide and separated by agarose gel electrophoresis, then amplification of product gene and genotype can be certained. During the process of determinating cytokine genes, all the mixing reaction systems contain HLA-DRBl or human growth hormone as control primer to make sure the dependability of all the PCR reaction. If amplified gene segments<600bp, we select HLA-DUB1 as control; if amplified gene segments>600bp, we select human growth hormone as control. The PCR heat cycle parameter of IL-lA(-889)^ IL-4(-590)> IL-6(+3247)^ TNFa (+488, -308) is: degeneration at 94°C for 1 minutes; reannealing at 55°C for 55 seconds; extension at 72 °C for 1 minutes; 30 circulations are in total. Before the first circulation pre-degenerating will profit to arise the quantity of gene production. After the final circulation extension at 72 "C for 7 minutes to make the reaction complete. The PCR heat cycle parameter of DQA1 *050K DQB l*020k DQB 1*0303 PCRis: Before the first circulation pre-degenerating at 94 °C for 4 minutes; others degeneration at 94 °C for 1 minutes; reannealing at 62°C for 1 minutes; extension at 72°C for 1 minutes; 35 circulations are in total. After the final circulation extension at 72°C for 7 minutes.3. Tatistic analysis: input the observation results to computer, use the SPSS 10.0 statistic software to analysis, at first read respectively the individual sample's allelesand genotypes, then calculate each group's frequency of alleles and genotypes. By the Hardy-Weinberg balance test to check whether the frequency of the two groups' alleles and genotypes do have representation of population. Numeration data between two groups and three subgroups make use of chi square test and the result is P < 0.05. Results:1. In HT group: the comparison of frequency distribution of IL-4 (-590) t allele and IL-4 (-590) c/t genotype is both lower than normal control group (P value < 0.05).2. HT group compare to control group shows that the comparison of frequency distribution of IL-1 a (-889X IL-6 (+3247X TNF a (+488> -308) alleles and genotypes has no notable difference.3. IN GD group: the comparison of frequency distribution of IL-4 (-590) t allele and IL-4 (-590) c/t genotype is both lower than normal control group (P value < 0.05).4. GD group compare to control group shows that the comparison of frequency distribution of IL-1 a (-889X IL-6 (+3247.) TNF a (+488^ -308) alleles and genotypes has no notable difference.5. Futher hierarchy analysis shows the frequency of IL-1 a (-889X IL-4 (-590)> IL-6 (+3247>) TNF a (+488 ^ -308) alleles and genotypes by gender , thyroid associated ophthalmopathy and GD family history : in GD patients has no significant difference among the subgroups of Shandon Han people in Chinese.6. Compared to Caucasian, there are no significant difference of the frequency distribution of IL-4 (-590) alleles and genotypes in GD patients in Shandong Han people of China.7. The comparison of frequency distribution of HLA-DQAl*0501 and -DQB 1*0201 alleles in GD group is both obviously lower than control group (P=0.002), RR=0.38A and P=0.001, RR=0.27), but there are no difference of the frequency of DQB 1*0303 (P=0.189, RR=0.64).8. In GD group there is no difference of HLA-DQA 1*0501 and -DQBl*0201 alleles by gender. Conclusions:1. There are significant difference between frequency distribution of IL-4 (-590) allele and genotype of AITD patients and normal control group in Shandong Han people of China. The frequency of IL-4 (-590) t allele in promoter loci and c/t genotype in AITD patients is both cower than normal contrl group So IL-4 is likely contribute to protectant factor of AITD.2. The comparison of frequency distribution of IL-1 a (-889X IL-6 (+3247O TNF a (+488n -308) alleles and genotypes has no significant difference between AITD patients and normal control group in Shandong Han people of China. Above-mentioned genetic polymorphisms have no relation to the AITD by gender, AITD family history and thyroid associated ophthalmopathy.3. The frequency distribution of HLA-DQAl*0501 and -DQBl*0201 is obviously lower in GD patients than control group . So HLA-DQA 1*0501 and -DQB1 *0201 are probably protectant factors for the Han people.