382例溃疡性结肠炎患者维生素D受体基因多态性及单倍型分析

Analysis on the genetic polymorphisms and haplotypes of vitamin D receptor in three hundred and eighty-two patients with ulcerative colitis

目的 探讨浙江汉族人群维生素D受体（VDR） FokⅠ、BsmⅠ、ApaⅠ、TaqⅠ及其构成的单倍型与UC的关系.方法 收集2004年1月至2012年9月的UC患者382例.同期收集489名年龄、性别匹配的健康体检者作为对照.取研究对象空腹外周静脉血2mL,提取全基因组DNA.采用微测序技术,经多重PCR扩增、PCR产物纯化、延伸反应和二次纯化、毛细管电泳检测基因型,测定VDR基因多态性.用卡方检验分析对照组VDR FokⅠ、BsmⅠ、ApaⅠ、TaqⅠ的基因型分布是否符合HardyWeinberg平衡定律.校正年龄、性别、吸烟因素后,采用非条件Logistic回归分析法比较UC组和对照组间VDR FokⅠ、BsmⅠ、ApaⅠ、TaqⅠ的等位基因和基因型频率差异,以及其对UC患者临床病理特征的影响.用Haploview 4.2软件进行连锁不平衡和单倍型分析.结果 对照组中VDRFokⅠ、BsmⅠ、ApaⅠ、TaqⅠ的基因型分布均符合Hardy-Weinberg平衡定律（P均＞0.05）.校正年龄、性别和吸烟因素后,非条件Logistic回归分析显示,UC组和对照组VDR FokⅠ、BsmⅠ、ApaⅠ、TaqⅠ的等位基因和基因型频率差异均无统计学意义（P均＞0.05）.轻度和中度UC患者VDRFokⅠ位点的突变等位基因C和TC＋CC基因型的频率[54.14％（353/652）和81.60％（266/326）]均高于重度UC患者[39.29％（44/112）和57.14％（32/56）,OR=1.825和3.322,95％CI分别为1.212～2.747和1.828～6.061,P均＜0.01].DR BsmⅠ、ApaⅠ和TaqⅠ与UC疾病严重程度无关（P均＞0.05）.UC远端结肠炎和广泛结肠炎患者VDR FokⅠ、BsmⅠ、ApaⅠ、TaqⅠ的突变等位基因频率和基因型频率差异均无统计学意义（P均＞0.05）.VDR BsmⅠ、ApaⅠ和TaqⅠ之间存在连锁不平衡;BsmⅠ与ApaⅠ的连锁不平衡系数（D ’）=0.982,相关系数（r2）=0.156;ApaⅠ与TaqⅠ的D＇=0.929,r2=0.141;BsmⅠ与TaqⅠ的D＇=0.826,r2=0.675.在8种单倍型（GAC、ACC、GCC、AAT、ACT、GCT、GAT、AAC）中,仅UC组AAC单倍型的频率[3.80％ （29/764）]低于对照组[5.93％（58/978）],差异有统计学意义（OR=0.631,95％CI 0.394～1.013,P=0.039）.结论 VDR FokⅠ基因突变可能影响UC患者的疾病严重程度.携带BsmⅠ、ApaⅠ、TaqⅠ构成的AAC单倍型的个体罹患UC的风险可能较低.

Objective To investigate the relationship between polymorphisms and haplotypes of vitamin D receptor （VDR） genes FokⅠ,BsmⅠ,ApaⅠ,TaqⅠ and ulcerative colitis （UC） in Han population of Zhejiang province.Methods From January 2004 to September 2012,382 patients with UC were collected and 489 age and gender matched healthy individuals were collected at the same period as controls.Two milliliter fasting peripheral blood of subjects was obtained for whole-genome DNA isolation.Microsequencing technology was applied.Polymnorphisms of VDR were determined through multiplex polymerase chain reaction （PCR） amplification,PCR products purification,extension,second purification and capillary electrophoresis genotype.Whether the distribution of VDR FokⅠ,BsmⅠ,ApaⅠ,Taq Ⅰ genotype of control group met Hardy-Weinberg equilibrium was analyzed by chi-square test.After age,gender and smoking factors adjusted,the differences of VDR FokⅠ,BsmⅠ,ApaⅠ,TaqⅠ allele and genotype frequencies between UC group and control group were analyzed by non-conditional Logistic regression analysis,and their effects on clinic-pathological features of patients with UC were also analyzed.Linkage disequilibrium and haplotype analysis was made by Haploview 4.2 software.Results The distributions of VDR FokⅠ,Bsm],ApaⅠ,TaqⅠ genotype met Hardy-Weinberg equilibrium （all P〉0.05）.After age,gender and smoking factors adjusted,the results of non conditional Logistic regression analysis indicated that there were no significant differences in VDR FokⅠ,BsmⅠ,ApaⅠ,TaqⅠ allele and genotype frequencies between UC group and control group （all P〉0.05）.However,the frequencies of mutant allele （C） and genotype （TC＋CC） in VDR FokⅠ of patients with mild and moderate UC （54.14%,353/652 ; 81.60%,266/326） were significantly higher than those of patients with severe UC （39.29%,44/112; 57.14%,32/56; OR=1.825 and 3.322,95%CI 1.212 to 2.747 and 1.828 to 6.061,both P〈0.01）.There was no correlation between VDR BsmⅠ,ApaⅠ,TaqⅠ and severity of UC （all P〉0.05）.There were no significant differences in VDR FokⅠ,BsmⅠ,ApaⅠ,TaqⅠ allele and genotype frequencies between UC patients with distal colitis and extensive colitis （all P〉0.05）.There were linkage disequilibrium in VDR BsmⅠ,ApaⅠ and TaqⅠ.Linkage disequilibrium coefficient （D＆#39;） between BsmⅠ and ApaⅠ was 0.982 and the correlation coefficient （r2） was 0.156; D＇ between ApaⅠ and TaqⅠ was 0.929 and r2 was 0.141; D＇ between BsmⅠ and TaqⅠ was 0.826 and r2 was 0.675.Among eight haplotypes （GAC,ACC,GCC,AAT,ACT,GCT,GAT and AAC）,the frequency of AAC haplotype of UC group （3.80%,29/764）was lower than that of control group （5.93%,58/978） and the difference was statistically significant （OR=0.631,95%CI 0.394-1.013,P=0.039）.Conclusions The genetic mutation of VDR FokⅠ may influence severity of patients with UC.The risk of developing UC is low in the individuals carrying AAC haplotype of VDR BsmⅠ,ApaⅠ,TaqⅠ suffering UC.