Association Study Between The Genetic Variations Of Gene SLC30A8 And Polycystic Ovary Syndrome And The Study On The Regulatory Effect Of Estrogen On B Cell Function

PART 1 ASSOCIATION STUDY BETWEEN THE GENETIC VARIATIONS OF GENE SLC30A8 AND POLYCYSTIC OVARY SYNDROMEBackground Polycystic ovary syndrome(PCOS) is a highly heterogeneous endocrine disorder of women that always results in infertility.It is characterized by oligomenorrhea or amenorrhea,hyperandrogenism,insulin resistance,infertility, acne,obesity and multiple small subcapsular cystic follicles in the ovary on ultrasonography.It affects about 5%-10%of women of reproductive age.Recently, it was demonstrated that PCOS was also a metabolic disorder.The etiopathogenisis of PCOS is still unknown.PCOS displays evident familial aggregation.The interaction between genetic susceptibility and environmental factors plays a possible role in the pathogenesis of PCOS.PCOS and type 2 diabetes mellitus are both diseases of multifactorial inheritance.The genetic backgrounds of these two disorders are complex.They share many common features in both clinical metabolism manifestations and epidemiology characters.Insulin resistance and hyperinsulinemia is the link between PCOS and type 2 diabetes.The risk of developing type 2 diabetes is significantly higher in women with PCOS than in women without PCOS.Therefore, the known susceptibility genes for type 2 diabetes are reasonable candidates for investigation of the genetic basis of PCOS.Recently,a study in a French case-control cohort identified several novel risk genes for type 2 diabetes.Single nucleotide polymorphism(SNP) rs13266634 in gene encoding the solute carrier family 30(zinc transporter),member 8(SLC30A8) stood out(with an odds ratio[OR]of 1.53±0.31).The gene product of SLC30A8 is a member of zinc transporter called ZnT-8.Its expression is limited to the secretory granules of pancreatic beta cells.Zinc plays a pivotal role in insulin synthesis, storage and secretion.Therefore,this pancreas-specific zinc transporter may impact insulin secretion.We hypothesized that rs13266634 in SLC30A8 may also contribute to the risk for PCOS.Objective To test whether the SNP rs13266634 in gene SLC30A8 associated with PCOS.To analyze the associations between the genotype distribution of rs13266634 and the clinical features of PCOS patients.MethodsSubjects A total of 894 women with PCOS were recruited from the outpatient clinic of Shandong Provincial Hospital.Recruitment was based on the revised Rotterdam diagnostic criteria.We recruited 622 healthy nondiabetic women with regular menstrual cycles as a control group.None of the controls had hyperandrogenism or other endocrine disorders related to PCOS.All patients and controls were Han Chinese women and were recruited from the same area.After undergoing a physical examination,including measurement of abdominal and hip circumferences and the measurement of hormone,an oral glucose tolerance test (OGTT) was performed in PCOS patients.Body mass index(BMI) was used to evaluate obesity.Insulin sensitivity in the fasting state was estimated by the homeostasis model assessment of insulin resistance(HOMA-IR). Genotyping Peripheral blood from participants was collected and the genomic DNA was isolated.Genotype was identified by single-tube allele-specific polymerase chain reaction(PCR) and the fluorescence melting curve analysis.Both the PCR reaction and the melting curves analysis were performed on the ABI 7500 real-time PCR system.Statistical analysis The Hardy-Weinberg equilibrium test was performed using the chi-square test.Genotype and allele frequencies for the case and control groups were compared using the chi-square test.Analysis of covariance(ANCOVA) was used to analyze the associations between the genotype of rs13266634 and the clinical features of PCOS patients in PCOS group,introducing BMI as the covariate. P＜0.05 was considered statistically significant.Results1.Genotype distribution of SNP rs13266634 in case and control groups showed no deviation from the Hardy-Weinberg equilibrium.2.No significant differences in genotype and allele frequencies were found between PCOS patients and healthy controls.3.No significant differences in genotype and allele frequencies were found between PCOS patients with or without insulin resistance.4.No significant differences in genotype and allele frequencies were found between PCOS patients with or without obesity.5.No associations were observed between genotype of rs13266634 and the quantitative clinical features of PCOS patients in PCOS group after adjustment for BMI.Conclusions The risk allele of rs13266634 in gene SLC30A8 has no associations with PCOS or PCOS-related clinical features. PART 2 STUDY ON THE REGULATORY EFFECT OF ESTROGEN ON B CELL FUNCTIONBackground Systemic lupus erythematosus(SLE) is an autoimmune disease that always results in the damage of several tissues and organs,such as skin and kidney.It is characterized by the appearance of variety of immune complexes and autoantibodies that react with the autoantigens(such as histone,nuclear ribonuclear protein and DNA) of human body.The autoantibodies and immune complexes can injure the tissues directly or indirectly.About ninety percents of SLE patients are women.Approximately,the incidence of SLE in Chinese is 0.07%.The etiopathogenisis of SLE remains unknown.The interactions between genetic susceptibilities and various environmental factors(virus infection,hormone abnormality,and so on) may contribute to the immune dysfunction of SLE patients. Several immune dysfunctions play important roles in SLE.The abnormal activation of autoreactive T and B lymphocyte result in the production of autoantibodies.The decreased capability of the negative-regulated function to immune reaction of inhibitory immunocytes leads to the overreaction of immune effector cells. Excessive secretion of a number of proimflammatory cytokines,such as interferon-α(IFN-α) and interleukin-10(IL-10),may exacerbate the imflammation of SLE patients.The core event in the development of SLE is the overreaction of B lymphocyte to several self molecules and the aberrant interaction between B and T lymphocytes.The the autoantigens that presented by B lymphocyte and the autoantibodies and proimflammatory cytokines that produced by B lymphocyte may contribute to the development and progression of SLE.Systemic lupus erythematosus displays a conspicuous women's bias.This bias may mainly due to the function of estrogen,the main female hormone in women. Since estrogen can block the deletion of immature autoreactive B lymphocyte in bone marrow and break the tolerance of B lymphocyte.Estrogen plays a regulatory role in immune system.The development of immune organs(thymus and spleen) was influenced by estrogen through its natural receptors.The differentiations and functions of several immune cells,such as mature T and B lymphocytes, macrophages,dendritic cells,natural killer cells and granulocytes,were also influenced by estrogen.Objective In order to investigate the regulatory effect of estrogen on B cell function,we detected the effect of estrogen on the proliferation,activation, apoptosis,differentiation and antibody production of splenic B lymphocyte that isolated from female BALB/c mice.Materials and MethodsAnimals and reagents Female BALB/c mice aged 8-12 weeks which maintained under specific pathogen-free conditions were used for the isolation of splenocytes.Endotoxin-free,cell culture grade 17β-estradiol(E2),phenol red-free RPMI 1640 medium,charcoal-stripped fetal bovine serum,anti-mouse monoclonal antibodies for flow cytometry assay,carboxy fluorescein diacetate,succinimidyl ester(CFSE),mouse IgG ELISA kit and Annexin V-FITC/propidium iodide(PI) apoptosis assay kit were purchased.Cell culture splenotyes were isolated and cultured in complete phenol red-free RPMI 1640 medium and incubated at 37℃in a humidified atmosphere with 5%CO₂.Proliferation,apoptosis,activation and differentiation assay The apoptosis, profiferation,activation and differentiation of splenic B lymphocyte were detected by dual fluorescence flow cytometry.Antibody production assay The total IgG antibody produced by mouse splenocytes was detected by enzyme-linked immunosorbent assay(ELISA).Results1.E2 decreased the expression of CD80 molecule on splenic B lymphocyte in vitro.2.E2 decreased the serum-deficiency-induce apoptosis of B lymphocyte in vitro.3.E2 enhanced the total IgG antibody production of splenocytes without promoting the differentiation of B lymphocyte in vitro.4.E2 had no influence on the proliferation of B lymphocyte in vitro.Conclusions Estrogen promotes the activity of B lymphocyte. Down-regulation of CD80 expression by estrogen may contribute to the activation of splenic B lymphocyte.These results imply the possible role of estrogen in the overreaction of B lymphocyte of SLE patients.