【作者】 高琳；

【导师】 刘玉峰； 沈柱；

【作者基本信息】 第四军医大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 银屑病是一种反复发作,具有特征性鳞屑性红斑的慢性自身免疫性皮肤病,其发病率高、危害大、治疗困难。在自然人群中的发病率为0.1%-3%,我国约有300万患者,发病率有逐年上升的趋势,国际卫生组织将其列为影响人类健康的重要疾病。目前银屑病的发病机制尚不清楚,近十几年来,关于银屑病的病因和发病机制的研究主要集中于基因异常、免疫炎症机制、环境因素等方面。流行病学研究表明银屑病具有家族聚集性,是一种多基因遗传病。但任何单一因素均不能完全解释银屑病的发病机理,有学者认为多因素、多基因累积共同导致了银屑病的发生,不同的个体可能存在不同的病因组合,其中遗传基因的易感性占据了重要的地位。寻找与疾病相关的致病基因的方法大致可以分为两种:连锁分析和关联研究。关联研究主要以病例对照研究为基础,通过研究患者和正常对照的基因多态性位点基因型和等位基因频率的差异,从而判断该基因与疾病易感性的关系。近年来的研究表明皮肤角质形成细胞异常及自身免疫失衡相关的细胞和分子在银屑病的发病机制中具有重要作用,然而目前对基因多态性与银屑病易感性的关系少有研究。为此,本课题选择了四个与皮肤角质形成细胞异常及自身免疫紧密相关的基因作为研究重点,试图寻找其基因多态性和银屑病发病的相关性。Foxp3叉状头/翅膀状螺旋转录因子在外周T细胞中表达的精密控制对于维持免疫系统的内环境稳定具有重要意义。转录因子Foxp3可特异表达于胸腺和外周淋巴组织中天然的CD4+ CD25+调节性T细胞亚群上,可以认为Foxp3是CD4+CD25+调节性T细胞发育的调控机制中的重要开关。CD4+CD25+Treg细胞与效应性T细胞的免疫失衡,导致自身反应性T细胞的活化是银屑病免疫学发病机制的中心环节。目前已证实Foxp3基因突变同多种自身免疫性疾病的发生相关,而在银屑病这种自身免疫性皮肤病的发生中, Foxp3基因的变化是否通过调控CD4+CD25+Treg细胞直接或者间接发挥作用尚未得到证实。在本研究中我们探讨了Foxp3基因-6054 (deletion/ATT, rs5902434)位点、-3279 (A/C, rs376158)位点、-924 (A/G, rs2232365)位点和IVS9+459 (A/G, rs2280883)基因多态性与银屑病易感性的关系。Fas/FasL系统通过凋亡及致炎作用途径与银屑病的发生紧密联系。研究结果表明银屑病皮损组织中Fas、FasL的强表达是导致患者表皮角质形成细胞异常凋亡的机制之一。Fas、FasL的表达异常使T淋巴细胞异常凋亡,进而导致免疫耐受失控,成为自身免疫性疾病的机理之一。Fas/FasL信号通路的另一路径是诱导炎症因子,尤其是肿瘤坏死因子(TNF-α)和白介素8(IL-8),该途径在表达抗凋亡分子如Bcl-xL水平较高的细胞中有明显优势。致炎的Fas信号通路通过活化的淋巴细胞介导银屑病的发生。国外已有研究证实Fas、FasL基因多态性与自身免疫性疾病的相关性,但Fas、FasL基因多态性与汉族银屑病患者的相关性目前未见报道。为此,我们对汉族银屑病患者和正常对照者进行Fas-1377、Fas-670、FASLG-844和FASLG-IVS2nt-124基因多态性检测,探讨其基因多态性在汉族人群中的分布及其与银屑病的相关性。儿茶酚邻位甲基转移酶(COMT)催化儿茶酚胺第3位羟基甲基化,是降解儿茶酚胺的主要代谢酶。文献表明COMT基因第4号外显子存在一个G>A点突变使158密码子从缬氨酸(Val)到蛋氨酸(Met)错义突变,导致COMT酶活性降低,神经末梢产生的儿茶酚胺类物质的增加可以直接损伤表皮细胞,同时不能阻止表皮细胞中毒物-o-醌的形成,诱发角质形成细胞氧化损伤,导致银屑病的发生和加重。有研究表明COMT-158(G>A)单核苷酸基因多态性与银屑病的发病有一定相关性。但目前COMT-158(G>A)单核苷酸基因多态性与汉族人群银屑病易感性的关系尚无研究。目的:分析Foxp3基因、Fas基因、FasL基因、COMT基因单核苷酸多态性与中国汉族人群银屑病发病的相关性,探讨银屑病的遗传学背景及发病机制。方法:对524例银屑病患者和549例正常对照抗凝血,抽提基因组DNA,使用PCR-RFLP和PCR-SSP方法分别检测Foxp3基因-6054 (deletion/ATT, rs5902434)、-3279 (A/C, rs376158)、-924 (A/G, rs2232365)和IVS9+459 (A/G, rs2280883)四个位点单核苷酸多态性。使用PCR-RFLP方法检测Fas-1377、Fas-670、FASLG-844和FASLG-IVS2nt-124四个位点基因单核苷酸多态性。使用PCR-SSP方法检测儿茶酚邻位甲基转移酶(COMT)基因第4外显子158密码子碱基单核苷酸多态性。对结果进行统计学分析,探讨Foxp3基因、Fas基因、FasLG基因、COMT基因单核苷酸多态性与中国汉族人群银屑病发病的相关性。结果:Foxp3-3279 AC (adjusted odds ratio [OR], 1.32; 95% CI, 1.01-1.74)和IVS9+459 GG(adjusted odds ratio [OR], 2.24; 95% CI, 1.41-3.58)基因型中国汉族银屑病的发病与发展存在相关性;Foxp3-3279变异联合基因型AC+AA显著增加男性人群(adjusted OR=1.60, 95% CI=1.11-2.31)罹患银屑病的危险性,同时与病情较重的银屑病发病(PASI score > 20)人群(adjusted OR=1.97, 95% CI=1.41-2.75)有显著相关性。IVS9+459变异联合基因型GA+GG与病情较重的银屑病发病(PASI score > 20)人群(adjusted OR=1.69, 95%CI=1.21-2.36)有显著相关性。Foxp3-6054位点ATT插入型等位基因频率在银屑病患者和正常对照组之间没有显著差异(32.1% vs. 29.3%, P = 0.174)。Foxp3-924位点G等位基因频率在银屑病患者和正常对照组之间亦无显著差异(42.6% vs. 40.0%, P = 0.238)。通过PCR-RFLP方法发现,所选择样本中Fas-1377位点A等位基因频率在银屑病患者和正常对照组之间没有显著差异(32.8% vs. 35.6%, P = 0.187)。Fas-670位点G等位基因频率在银屑病患者和正常对照组之间没有显著差异(37.2% vs. 39.1%, P = 0.389)。FASLG-844位点T等位基因频率在银屑病患者和正常对照组之间没有显著差异(26.5% vs. 23.4%, P = 0.100)。Fas-1377G/A和Fas -670A/G两点单倍体分析和单倍体联合分析结果发现,以没有突变的AG等位基因做为参照,GG与GA单倍体等位基因具有降低罹患银屑病危险性的趋势,AA单倍体等位基因降低罹患银屑病危险性的趋势具有统计学意义。联合分析发现以GA+AA/AA做为参照,其余三组GG/AA,GG/GG+GA, GA+AA/GG+AA显著降低罹患银屑病的危险性。通过PCR-SSP方法发现,COMT-158位点A等位基因频率在银屑病患者中无显著增高(24.7% vs. 23.7%, P = 0.624)。与COMT-158 GG基因型相比,-158GA (adjusted odds ratio [OR], 1.00; 95% CI, 0.78-1.29)和-158AA(adjusted OR, 1.23; 95% CI, 0.70-2.15)基因型不会增加罹患银屑病的危险性。联合基因型GA+AA与银屑病发病年龄和疾病严重程度之间均无显著相关性。结论:本研究首次分析了中国汉族人群中Foxp3、Fas、FasL、COMT基因单核苷酸多态性和单倍体基因型与银屑病发病的相关性,发现Foxp3-3279(A/C, rs376158)和IVS9+459 (A/G, rs2280883)位点基因多态性和汉族人寻常型银屑病发病相关,证实了Foxp3基因水平的变化与银屑病的关系,为研究汉族人群银屑病易感基因打下基础,也提示Foxp3可能会成为治疗银屑病的一个靶分子,为拓展银屑病的临床治疗途径提供基因学基础。Fas-1377、Fas-670、FASLG-844三个功能性多态性位点及COMT-158位点均未发现与银屑病易感性相关。Fas-1377G/A和Fas-670A/G两点单倍体分析和单倍体联合分析结果发现,GG与GA单倍体等位基因具有降低罹患银屑病危险性的趋势,AA单倍体等位基因降低罹患银屑病危险性的趋势具有统计学意义,合并基因型GG/AA,GG/GG+GA,GA+AA/GG+AA均显著降低罹患银屑病的危险性。此外,由于种族差异,仍有必要进行大样本量、不同种族的重复性研究。更多还原

【Abstract】 Psoriasis is a common skin disorder, characterized by focal formation of inflamed, raised plaques that constantly shed scales derived from excessive growth of skin epithelial cells. With high morbidity, severe damage to patients’health and great difficulty in treatments, psoriasis has long been listed as one of the important diseases threatening human health. 1-3% of population is afflicted with Psoriasis worldwide. In china it’s estimated that more than 3000,000 people are suffering from this disease, and the mobility still exhibits a increasing tendency. Epidemic researches has demonstrated that the psoriasis could be recognized as a polygenic disease with a strong tendency of familial aggregation. Nowadays, the pathomechanism of psoriasis has still not been completely clarified, although, in recent years, various possible hypotheses have been proposed, including the genetic factors, the immune-mediatedandthe genetic factors and the environmental factor. None of those factors mentioned above, however, could explain the development of psoriasis perfectly. Clinical evidence and experimental data suggest that psoriasis is an autoimmune disease, in which polygenic inheritance and suspect genes play an important role. There are two main methods to find susceptible genes related to disease: linkage analysis and association study. Association study is mainly based on case-control study, investigating the difference of genotypes and alleles frequency of genes in cases and controls and determining whether the polymorphisms of genes are associated with the suscepatbility of disease. In recent years, cellular and molecular components of keratinocyte damage and autoimmunity were found to play an important role in the pathology of psoriasis, however, only a few studies about polymorphisms of innate immunity related genes and the susceptibility of psoriasis were reported. In this study, we chose 4 candidate genes associated with keratinocyte damage and autoimmunity and explored the relationship between the functional polymorphisms of these genes and the susceptibility of psoriasis.The Foxp3 gene is primarily expressed in CD4+CD25+ T-regs in normal physiological conditions. the protein coded by Foxp3 gene is involved in the regulation of T cell activation, belonging to the forkhead/winged-helix family of transcription factors. The Foxp3 protein functions as a transcriptional repressor and down-regulates cytokine production in T cells. Further, some studies have suggested that gene transfer of Foxp3 confers a regulatory T cell phenotype on CD4+T cells that ameliorates autoaggression. While the exact pathogenesis of psoriasis remains unclear, several features of the disease suggest an immune system-mediated process; much evidence indicates that regulatory T cells (T-regs) are involved. Foxp3 deficiency may contribute to the occurrence of some autoimmune diseases, perhaps because of the lack of functional CD4+CD25+ T-regs. Here, we hypothesized that defects in the Foxp3 gene may be involved in the etiology of psoriasis. In this study, we examined four SNPs (-6054, deletion/ATT; -3279, A/C; -924, A/G; IVS9+459, A/G) in psoriatic patients, and assessed the association of genotype and allele frequencies between patients and normal controls.Fas/FasL signaling is associated with psoriasis by the pathway of apoptosis and the induction of inflammatory cytokines. For the pathway of apoptosis, Fas/FasL signaling can modulate the apoptosis of Keratinocytes. Some researches indicated that Fas/FasL could be secreted by psoriasis keratinocytes and be expressed at high level in psoriasis. The unbalance of the Fas and FasL expressed could render apoptosis abnormal of T lymphocytes. Therefore, Dysregulation of the expression of the Fas and FasL genes may be important in the development of autoimmune disease, because of pathological effects on immune system maintenance and disturbances in the balance between pro- and anti-inflammatory cytokines. Fas/FasL signaling has been best known for induction of apoptosis. However, there also exists an alternate pathway of Fas signaling that induces inflammatory cytokines, particularly tumor necrosis factor (TNF)-αand interleukin(IL)-8. This pathway is prominent in cells that express high levels of anti-apoptotic molecules such as Bcl-xL. Because TNF-αis central to the pathogenesis of psoriasis and inflammatory, Fas signaling mediates induction of psoriasis by activated lymphocytes. Fas/FasL gene polymorphism may be involved in some autoimmune diseases, but the associations between Fas/FasL gene and the susceptibility of psoriasis were not determined. In this study, we examined four SNPs (Fas-1377; Fas-670; FASLG-844; FASLG-IVS2nt-124) in psoriatic patients, and assessed the association of genotype and allele frequencies between patients and normal controls.Catechol-O-methyltransferase (COMT) is an enzyme that catalyses the O-methylation of biologically active or toxic catechols and plays an important role in the metabolism of drugs and neurotransmitters. A common single nucleotide polymorphism (SNP) in codon 158 of the COMT gene codes for a substitution of valine (Val) by methionine (met), resulting in reduced thermostability and activity of the enzyme. In keratinocytes, this SNP may inflence the activity of the enzyme, induce high level of toxic o-quinones and destroy the keratinocytes. COMT-158 G>A polymorphism proved to be related to the susceptibility of psoriasis, but the associations of it and the susceptibility of psoriasis in the Han Chinese population were not determined.Objective: We hypothesized the Foxp3, Fas, FasL gene SNPs and COMT-158 G>A polymorphism were associated with the risk of psoriasis in Han Chinese people.Methods: In a hospital-based case-control study of 524 patients with psoriasis and 549 psoriasis-free controls was made in frequency according to the age and gender. Foxp3-6054, -3279 , -924 and COMT-158 G>A polymorphism were genotyped by the PCR sequence specific primer (PCR-SSP) technique. Foxp3 IVS9+459 polymorphism and Fas-1377, Fas-670, FASLG-844, FASLG-IVS2nt-124 polymorphism were genotyped with the PCR-RFLP technique.Results: We found that an increased risk of psoriasis was associated with the Foxp3-3279 AC (adjusted odds ratio [OR], 1.32; 95% CI, 1.01-1.74) and IVS9+459 GG(adjusted odds ratio [OR], 2.24; 95% CI, 1.41-3.58) genotypes, compared with the -3279CC genotype and IVS9+459AA genotype, respectively. Consistently, the combined Foxp3-3279 AC+AA genotype was associated with a significantly higher risk of psoriasis in males (OR, 1.60; 95% CI, 1.11-2.31). The frequency of the -3279 AC+AA and IVS9+459 GA+GG genotypes was significantly higher among patients with severer psoriatic (PASI > 20) than among the controls. (45.9% versus 31.1%, 46.9% versus 36.1%, P < 0.001 and P = 0.002). Meanwhile, the Foxp3 -3279 AC+AA genotype and IVS9+459 GA+GG genotype were also associated with more severely psoriatic (PASI score > 20)patients (adjusted OR = 1.97, 95% CI = 1.41-2.75; adjusted OR = 1.69, 95% CI = 1.21-2.36). Significant difference could not be detected in the distribution of Foxp3-6054 ATT among the case and control subjects(32.1% vs. 29.3%, P = 0.174). Moreover, significant difference failed to be established among the distribution of Foxp3-924 G allele among the case and control subjects(42.6% vs. 40.0%, P = 0.238).By the PCR-RFLP techniques analysis, we didn’t find significant difference in the distribution of Fas-1377 A allele among the psoriasis and control subjects(32.8% vs. 35.6%, P = 0.187).Similarly, the frequency of Fas-670 G allele (37.2% vs. 39.1%, P = 0.389) and FASLG-844 T allele (26.5% vs. 23.4%, P = 0.100) among the psoriasis and control subjects didn’t suggest significant difference. Furthermore, in the haplotype analysis of Fas-1377 and -670, we noticed that the Fas haplotype genotypes GG and GA had a tendency to decrease risk of psoriasis. Haplotype genotypes AA were associated with a decreased risk of psoriasis. We also found combined genotypes of GG/AA,GG/GG+GA,GA+AA/GG+AA can decrease significantly the risk of psoriasis.Significant difference was not indicated in the distribution of COMT-158 A allele among the psoriasis and control subjects (24.7% vs. 23.7%, P = 0.624) by PCR-SSP techniques. We did not found the association between an increased risk of psoriasis and the COMT-158GA(adjusted OR, 1.00; 95% CI, 0.78-1.29) or -158AA (adjusted OR, 1.23; 95% CI, 0.70-2.15) genotypes, compared with the -158GG genotype. Consistently we found that psoriasis was not associated with the COMT-158 combined genotype GA+AA, compared with the -158GG genotype. Conclusions: Hitherto, this is the first known study about the association between the Foxp3 polymorphism, the COMT-158 polymorphism, the Fas vs FasL polymorphisms and the haplotypes genotypes and the psoriasis in the Han Chinese population. Our results demonstrated Foxp3-3279(A/C, rs376158) and IVS9+459 (A/G, rs2280883) polymorphism was associated with the susceptibility of psoriasis. It confirmed the relationship between the change of Foxp3 gene and psoriasis, and provided a possible way to use Foxp3 as a target in the treatment of psoriasis.However, larger, population-based studies are still necessary to confirm these findings.更多还原