Studies On Association Between Behcet's Disease And Polymorphisms Of HLA Class Ⅰ And Class Ⅱ Genes, Serum Leptin And Soluble Leptin Receptor

IntroductionBehcet's disease (BD) , namely Behcet's syndrome is a chronic multisystem autoimmune disease that was first desribed in 1937 by Turkish dermatologist Hulusi Behcet. It is mainly characterized by recurrent oral ulcerations,genital ulcerations,ocular lesions and skin lesions,also affects multiple organs including joints, vessels,central nervous systems,gastrointestinal tract, heart, kidneys, lungs, epididymis, etc. Patients with BD are found in the world,especially in the countries of Eastern Asia,Middle East and Mediterranean with high incidence,So it is also called Silk Road Disease. The incidence is no less than 0. 0014 % in northen China. Although the main pathology of BD has been known to be the vasculitis,the exact etiology of BD is still uncertain. The genetic epidermiological investigations at home and abroad indicate that BD is characteristic of higher heritable predisposition and familial aggregation, in the mean time infectious factors,enviromental factors,psychological factors and immunologi-cal factors play an important role in the onset and development of BD. The pathogenesis of BD is belived to be triggered by immune dysregulation under the involvement of infectious and other factors in individuals with a particular heritable predisposition.Human major histocompatibility complex (MHC, Human leukocyte antigen (HLA) gene or HLA gene complex) located in the short arm of human chromosome 6 (6p21. 31) is closely chained gene groups with the length of 3600 kb. It is known to be the most complicated human domi-nant polymorphic genetic system so far. At present 224 gene loci and 1500 alleles have been found in this genetic system. The encoded products — HLA antigens play an important role in immune response. HLA gene consists of 3 classes: HLA class I gene locates in the side of telomere of chromosome, including HLA—A,B,C and other genes and pseudogenes which have uncertain functions. HLA class I antigens are on surface of all the karyocytes?and its main functions are to recognize and present endogenous antigen peptide to CD8+ T cells. HLA class II gene locates in the side of centromere of chromosome, including HLA—DR,DQ and DP with the most complicated structure. HLA class II antigens are on surface of some immune cells such as antigen presentation cells,mature B cells,active T cells and so on,and its main functions are to recognize and present extrinsic antigen peptide to CD4+ T cells. HLA— DR is the most important HLA class II molecules. The speciality of HLA—DR is depended on the highly polymorphic DRB1 alleles. HLA class III is between HLA class I and class II, mainly including some genes encoding complements and inflammation cytokines. The studies showed that the association of HLA gene with many autoimmune disease overtook any other regions of human gene group,as a mainly heritable region of susceptibility to disorders.Leptin,the product of the obese gene is a 16000 peptide hormone secreted primarily by adipocytes. It affects many physiological and metabolic process,mainly including the controls of appetide and food intake,energy expenditure,inhibition of fat synthesis,regulation of body weight and reproductive function through all types of leptin receptor expressed on central and peripheral tissues. It is closely relevant to the diseases such as o-besity, hyperlipemia, hypertensive disease, diabetes mellitus, coronary heart disease. As an important member of neuroendocrine immune regulation network,recent studies suggested that leptin also participated in immune regulation by stimulating the proliferation and secretion of interleu-kin (IL)—2 of natural T lymphocytes and stimulating secretion of inter-feron (IFN)— 7 of memory CD4+ T lymphocytes, and inhibiting its secre-tion of IL—4. Therefore,leptin can promote Thl cell—mediated immune response and suppress Th2 cell — mediated immune response, and may play an important role in the onset of autoimmune diseases.Leptin performs biological functions through its specific receptor. Leptin receptor belongs to the class I cytokine receptor family. Cytokine receptor is characterized by producing soluble receptor,and moving in circulation in the type of binding protein, and carrying out its physiological functions. Leptin receptor consists of 6 subtypes including ob —Ra,Rb, Rc,Rd,Re and Rf. Ob —Rb,the main receptor is the long isoform of the leptin receptor which has all the functional regions of leptin receptor,five other receptors are the short isoform of leptin receptor. Ob—Re is the soluble type of leptin receptor which can bind free leptin and dicrease its concentration in circulation. As a biological regulation factor signaling trans-membrane receptor and a carrier protein with leptin which passes blood brain barrier to function at the level of the hypothalamus and a factor controlling reduction and clearance of leptin through regulating glomerular filtration, soluble leptin receptor (sLR) has a feedback with leptin in body. Its production is affected by ob —Ra,Rb and serum levels can reflect expression of leptin receptor gene in tissues. So sLR is an useful instrument to test biological activity of leptin and expression of leptin receptor.This study was involved in the etiology and pathogenesis of BD from genetic and immune aspects. At first, 40 Han patients with BD in Liaoning Province and 100 ethnic group— and region— matched healthy controls were studied for HLA—A,B,DRB1 alleles by LABType?SSO method, the most advanced HLA gene typing method in the world in order to explore the associations between HLA class I and class II genes and genetic susceptibility and clinical manifestations of BD. Then 40 patients with BD and 30 sex—, age— and body mass index (BMI)— matched healthy controls were studied for concentrations of serum leptin and sLR by radioim-munoassay (RIA) and enzyme linked immunosorben assay (ELISA) respectively in order to explore the role of leptin and sLR in immunopatho-genesis of BD.Materials and Methods1. Subjects1. 1 patients: 40 patients with BD who admitted to Department of Dermatology,No. 1 Affiliated Hospital of China Medical University from 2002 to 2005 satisfied the criteria of International Study Group for BD in 1990. All the patients,22 male and 18 female patients,aged 12 —62,mean age 32. 39 years were unrelated Han Chinese in Liaoning Province. The clinical manifestations of patients were as follows: 40 patients with recurrent oral ulcerations,28 patients with genital ulcerations,19 patients with ocular lesions, 16 patients with erythema nodosum—like lesions, 22 patients with folliculitis—like lesions,29 patients with positive pathergy reaction, 19 patients with joint involvement, 16 patients with gastrointestinal involvement, 14 patients with renal involvement,5 patients with family history of recurrent oral ulceration. 13 patients under 20 at the onset of disease,22 patients between 20—40 at the onset of disease,5 patients over 40 at the onset of disease. 21 patients in active period, 19 patients in inactive period.1. 2 controls: 100 unrelated Han health blood donors at Liaoning Provincial Blood Center were selected randomly. They are not significantly different from patients in sex,age and BMI. There are not BD patients in their families.2. MaterialsBlood Genome DNA Extraction Kit (TaKaRa Biotechnology Co. , Ltd. , Dalian),LABTypeSSO A locus,B locus and DRB1 locus typing test for DNA typing of HLA class (One Lambda, Canoga Park, CA, USA) , Human Leptin RIA Kit (North Biotechnology Institute,Beijing) and Human Leptin Receptor ELISA Kit (Boster Biotechnology Co. , Ltd. , Wuhan).3. Methods3.1 LABType? SSO: Genomic DNAs of patients and controls were extracted applying Blood Genome DNA Extraction Kit, then Generic HLA—A,B,DRB1 typing was performed using LABTypeSSO A locus,B locus and DRB1 locus typing test for DNA typing of HLA class according to the manufacturer's instructions.3. 2 RIA: Serum leptin concentrations of patients and controls were measured using Human Leptin RIA Kit according to the manufacturer's instructions.3. 3 ELISA: Serum sLR concentrations of patients and controls were measured using Human Leptin Receptor ELISA Kit according to the manufacturer 's instructions.4. Statistical Analysist — test was used for quantitative materials expressed as mean ± standard deviation (x±s),Pearson test was used for correlation between different variables, y? — test was used for qualitative materials. All statistical calculation were performed using the spss 12. 0 program for windows software,P0. 05).1. 2 The gene frequency of HLA—B * 51 was significantly higher in BD patients than those in controls, the difference was statistically significant (P0. 05).1. 3 The results of study on association between HLA—A alleles and clinical manifestations of BD show that HLA — A * 02 was significantly decreased in BD patients with folliculitis—like lesions (P0. 05).2. 2 The results of study on association between HLA—DRB1 alleles and clinical manifestations of BD show that HLA—DRB1 * 15 was significantly decreased in.BD patients with genital ulcerations (P0. 05). The patients with arthritis had significantly lower leptin concentrations when compared with patients with non — arthritis,the difference was statistically significant (P0. 05). The active periodfemale patients had lower sLR concentrations when compared with inactive period female patients, but the difference was not statistically significant (P>0. 05). The male patients with arthritis had significantly lower leptin concentrations when compared with male patients with non — arthritis, the difference was statistically significant (P