【作者】 杨健康；

【导师】 张亚平； 何黎；

【作者基本信息】 云南大学 ， 遗传学， 2014， 博士

【摘要】 重型痤疮是一种常见的慢性炎症性皮肤病,表现为颜面、前胸及后背部的囊肿、结节,易形成瘢痕,损害重、病程长、遗留的瘢痕临床疗效不佳。虽然皮肤疾病很少危及生命,重型痤疮对患者外观的损容、毁容所引起的心理影响不容低估,疾病可使患者产生如焦虑、抑郁等心理疾病,对青少年这一疾病高发群体的社交、就业及婚育产生严重负面影响。目前痤疮相关基因的研究仅限于雄激素代谢相关通路及炎症和免疫反应相关基因,而雄激素的分泌和代谢还受肾上腺皮质激素等多种其它信号或代谢通路的影响。鉴于痤疮是一种多基因疾病,是由多个基因的共同作用而导致的,候选基因研究策略的局限性限制了对细胞内其它多种复杂的代谢通路及信号传导通路是否与痤疮相关的研究,而明确致病易感基因是早期控制该病的重要前提。我们通过3个方面的研究：全基因组关联研究(GWAS)、易感基因与临床表型关系的研究、基因通路研究,寻找重型痤疮的易感基因及相关基因通路并研究他们与临床表型的关系。结果如下：(1)采取全基因组关联研究的方法,在中国汉族人(2,916个病例和4,716个健康对照)中寻找重型痤疮的易感基因。采取两阶段研究策略,初筛阶段包括1,056例重型痤疮病例和1,056例健康对照,使用HumanOmniZhongHua-8BeadChip基因芯片进行基因分型,每个样本可以检测900,015个SNPs位点。然后选取101个位点进行验证,包括初筛阶段最显著的86个SNPs位点以及来自于9个候选基因的15个SNPs位点。验证阶段包括1,860例病例和3,660例对照,采用的分型平台为Sequenom MassArray系统。最后将初筛和验证阶段的结果合并分析,最终发现2个易感基因座位与重型痤疮相关：11p11.2(DDB2,rs747650,P=4.41×10-9,OR=1.24,和rs1060573,P=1.28×10-8,OR=1.23)和1q24.2(SELL,rs7531806, P=1.20×10-8,OR=1.22)。这两个易感基因座位与雄激素代谢及炎症和瘢痕形成相关。本研究成功发现了与重型痤疮相关的新的遗传易感因素及其所属生物学通路。(2)在重型痤疮GWAS的基础上,对重型痤疮的发病年龄、有无萎缩性瘢痕、有无增生性瘢痕以及有无家族史进行分层,探讨GWAS找到的易感基因座位1q24.2(SELL)和11p11.2(DDB2)与中国汉族重型痤疮4种临床表型的相关性,同时也探讨这4种临床表型之间的相关性,为进一步研究重型痤疮的发病机制奠定基础。研究方法主要使用卡方检验比较不同临床表型间3个SNPs(rs7531806、rs747650、 rs1060573)等位基因频率的差异,使用Spearman相关系数(rs)计算各临床表型之间的相关性。结果发现11p11.2(DDB2)与疾病家族史显著相关(P<0.05)。发现增生性瘢痕和萎缩性瘢痕正相关(rs=0.315)。本研究揭示了llpll.2(DDB2)与痤疮疾病家族史这一临床表型相关,而与重型痤疮其它临床表型不相关,同时还发现几个临床表型间存在着关联性。上述结果使我们进一步理解了11p11.2(DDB2)在重型痤疮发病中的作用,也加深了我们对疾病相关的临床表型之间关系的了解。(3)我们将全基因组基因通路研究方法应用于重型痤疮GWAS数据(900,015个SNPs,1,056病例和1,056对照),期望能找到与重型痤疮发病相关基因通路。本研究主要采取了两种基因通路研究方法：VEGAS基因通路研究和Top-SNP基因通路研究。选择了2个常用的基因通路数据库(KEGG、BioCarta)。两种基因通路分析方法共发现了15个基因通路与重型痤疮相关(PEDR<0.05)。其中的5条基因通路主要是参与了免疫应答和炎症反应,包括：“凝集素诱导补体途径”、“参与急性炎症反应的细胞和分子”、“细胞粘附分子”、“IL3信号通路”、“细胞因子与炎症反应”。这5条通路的发现与以前对于重型痤疮的认识是相符的,重型痤疮本身就是一个以炎症反应密切相关的疾病。同时,还发现了多条以前没有认识到的基因通路与重型痤疮显著相关,如通路“酪氨酸代谢”和“钙离子信号转导”。研究结果为我们揭示了与重型痤疮相关的新的生物信号通路,为推动对重型痤疮发病机制的认识,同时也为疾病的预测和治疗提供了潜在的治疗新靶点。同时这些新发现的基因通路也为以后进一步的验证和功能研究打下了基础。更多还原

【Abstract】 Severe acne is a common chronic inflammatory skin disease and characterized by cysts, nodules and potential scarring, which generally distributed in the face, upper chest, and back. It’s a disease with long duration and can cause extensive damage to the skin. However, the treatment to the scarring usually has poor results. Severe acne is rarely life-threatening, but it can cause anxiety or depression, which further influence psychological health. Severe acne peaks in teenage years and have a seriously negative impact on the social, future employment, and marriage for teenagers.At present, most of the study of acne is limited to genes that are involved in androgen metabolism, immune response and inflammation processes. Moreover, the secretion and metabolism of androgen is also regulated by adrenal cortical hormone, and other metabolic or signaling pathways. Because severe acne is a polygenic disease resulting from the combined action of multiple genes, the strategies of candidate gene study has some limitations in that a variety of other metabolic pathways or signal transduction pathways are excluded. Indeed, identifying the causative susceptibility genes is an important prerequisite to early control the disease. In this paper, we performed three studies:genome-wide association study (GWAS), genotype-phenotype correlations study and gene pathway study, to identify susceptibility genes and related gene pathways of severe acne and to study their relationship with the clinical phenotypes.(1) We performed the genome-wide association study for severe acne that is to search the susceptibility genes in a Han Chinese population comprising2,916cases and4,716controls. We used a two-stage study strategy. In initial GWAS stage,1,056cases and1,056controls were genotyped using the Illumina HumanOmniZhongHua-8BeadChip which could detect900,015SNPs. In the replication stage, we genotyped the101SNPs in an independent cohort of1,860cases and3,660controls using Sequenom MassArray. The101SNPs included86SNPs with the most significant P value in the discovery stage and15SNPs with nominal association evidence located within or close to nine susceptibility genes. We combined the initial GWAS and replication stages together and identified two new susceptibility loci at11p11.2(DDB2, rs747650, Pcombined=4.41×10-9, OR=1.24and rs1060573, Pcombined=1.28×1O-8, OR=1.23) and1q24.2(SELL, rs7531806, Pcombined=1.20×10-8, OR=1.22) that are involved in androgen metabolism, inflammation processes and scar formation in severe acne. These results point to new genetic susceptibility factors and suggest several new biological pathways related to severe acne.(2) Two novel susceptibility loci1q24.2(SELL)和11p11.2(DDB2) for severe acne have been identified in our GWAS of a Han Chinese population. We further investigated their relationships with clinical phenotypes, including onset age, atrophic scarring, hypertrophic scarring and family history, as well as investigated correlations between the four clinical phenotypes. This study will lay a foundation for further study of pathogenesis of severe acne. We used the Chi square test to compare the allele frequency of3SNPs (rs7531806, rs747650, rs1060573) among different clinical phenotypes. Spearman’s Correlation Coefficient (rs) is calculated to measure the relationship between different clinical phenotypes. Significant association was found for the11p11.2(DDB2) with disease family history (P<0.05). Hypertrophic scarring was positively correlated with atrophic scarring (rs=0.315). The study suggested that susceptibility loci11p11.2(DDB2) is associated with disease family history of severe acne and not correlated with the other phenotypes of severe acne, while there are also correlations between these different phenotypes. These results not only shed more light on the role of llpll.2(DDB2) in the pathogenesis of severe acne, but also deepen our understanding of the relationship between disease-related clinical phenotypes.(3) In this study, we applied genome-wide pathway analysis to GWAS data of severe acne (900,015variants,1,056cases and1,056controls) and expected to discover gene pathways associated with the severe acne. We applied two customized of pathway-based analysis methods, VEGAS and Top-SNP, to the GWAS data in order to evaluate enrichment of pathways for their contribution to severe acne. Pathways used in this study are retrieved from two databases including KEGG and BioCarta. Fifteen significant pathways are identified by the two pathway analysis methods using a FDR of P<0.05. Of these, at least five pathways participate in immune response and inflammation processes (lectin Induced complement pathway, cells and molecules involved in local acute inflammatory response, cell adhesion molecules, IL3signaling pathway, cytokines and inflammatory response). The finding is consistent with previous understanding of pathogenesis of the disease:severe acne is a disease closely related to inflammatory reaction. Moreover, other pathways that are less known for their association with severe acne were identified in this study, such as tyrosine metabolism and calcium signaling. Our study not only provided more insight into biological signaling pathways associated with severe acne and promote understanding of the pathogenesis, but also provided some potential new targets for prediction and therapy of severe acne. These findings will also facilitate future follow-up and functional studies for severe acne.更多还原