【作者】 陈雪莉；

【导师】 孙兴怀； 余龙； 吴继红； 张圣海；

【作者基本信息】 复旦大学 ， 眼科学， 2009， 博士

【摘要】 对于婴幼儿型青光眼这一与遗传高度相关的疾病,目前已知的与发病相关的基因座有三个:GLC3A、GLC3B和GLC3C,而仅在GLC3A位点找到了致病基因----CYP1B1,该基因在不同人群患者中存在明显的遗传异质性。本研究将首先着眼于CYP1B1基因在中国汉族人群中的突变情况,进而揭示其基因型-表现型-手术疗效关系,来为临床诊断及治疗提供分子生物学依据。在排除这一已知基因致病的患者中,进一步分析疾病与其他两个候选位点的关系。不同于既往研究的是:1.在寻找基因突变的基础上,结合了临床发病特征的数据,构建出基因型-表现型关系,还结合手术疗效的随访数据,发掘出基因对临床预后的影响作用;2.全部采用了散发病例,验证了从家系病例定位的基因座,使其具有更高的可信度;3.尚未有中国汉族病例GLC3B和GLC3C位点的研究报道。第一部分CYP1B1基因与婴幼儿型青光眼表型及疗效关系目的:了解中国汉族人群婴幼儿型青光眼的CYB1B1基因突变情况并探寻CYB1B1基因型-表现型-疗效关系。方法:1.对122例散发的婴幼儿型青光眼患者外周血DNA采用PCR扩增、直接测序法,进行CYP1B1基因编码外显子的突变检测,序列图与正常人群数据库及120份正常对照样本序列比对;2.对238例患者进行基因型-表现型回顾性分析（结合本课题组前期己行突变筛查的116例实验数据）,分析因素包括:性别、单/双眼发病、发病年龄、起病眼压、角膜横径、角膜透明度评分;3.对其中在我院首次手术的192例（306只眼）进行影响手术疗效的多因素分析,分析因素包括:发病年龄、发病-手术时间、手术方式、术前眼压、角膜横径、角膜透明度评分、CYP1B1基因携带状态7个因素与手术成功疗效进行多元Logistic回归分析（Stepwise逐步回归法）、与手术成功时间进行Cox回归生存分析（Stepwise逐步回归法）。数据采用SPSS13.0统计学软件分析,以P＜0.05为差异具有统计学意义。结果:1.122份样本中的CYP1B1基因编码外显子区域一共检测出18种不同的突变,其中9种突变为本研究首次发现:g.3836T＞C（p.11W＞R）,g.4022delTC（p.730RFshift,P.221stop）,g.4151G＞T（p.116D＞Y）,g.4322G＞A（p.173E＞K）,g.4338T＞A（p.178V＞E）,g.4493G＞A（p.230E＞K）,g.4509T＞C（p.235V＞A）,g.8137T＞C（p.434W＞R）,g.8167C＞T（p.R444stop）。共21份病例样本检出突变,突变率为17.2%。2.综合本课题组前期实验数据,共238例（116+122）散发婴幼儿型青光眼患者,检测到致病突变的有41例（20+21）,总突变率仍为17.2%。其中男性患者的突变率为18.9%,女性患者的突变率为13.0%,差异无统计学意义。双眼患者的突变率为19.5%,单眼患者的突变率为12.7%,差异无统计学意义。携带突变患者的中位数发病年龄为2（0-6）月,不携带突变患者的中位数发病年龄为6（2-12）月,前者的发病时间明显的早于后者,差异具有统计学意义（P＜0.05）,角膜透明度评分在携带突变患者中更高（P＜0.05）。3.多元Logistic回归分析,得到CYP1B1基因为手术疗效的相关影响因素（P＜0.001）,其比值比为OR_CYP1B1=0.321。Cox回归生存分析,得到角膜透明度和CYP1B1基因为与手术成功时间的相关影响因素（P＜0.01）,其比值比分别为OR_Conea=1.846,OR_CYP1B1=0.468。结论:CYP1B1基因的突变与部分婴幼儿型青光眼的发病相关,基因编码外显子区域共检测出18种不同的突变,其中9种突变为本研究首次发现;携带CYP1B1基因突变的患者较无突变者发病年龄更小,角膜透明度更差;CYP1B1基因突变状态为手术疗效的保护性因素,相对无突变患者有更高的手术成功率和更长的手术成功时间。第二部分婴幼儿型青光眼候选位点的遗传学研究目的:探讨散发中国汉族婴幼儿型青光眼患者与候选位点GLC3C的关联关系;了解GLC3B位点中候选基因ANGPTL7的突变状况。方法:1.研究对象为中国汉族人群,患者为第一部分中排除携带CYP1B1基因突变者的152个核心家系（患者加正常双亲）,采用荧光标记复合扩增技术,进行覆盖GLC3C位点的12个STR分型,分型结果行传递不平衡检验;2.对获得的阳性区间,进一步挑选16个htSNP进行单倍型构建,采用基质辅助激光解析电离飞行时间质谱技术,对126个核心家系进行SNP分型,分型结果行传递不平衡检验;3.随机抽取96名患者的DNA制作混合样本池,对GLC3B区域内候选基因ANGPTL7的5个外显子行PCR扩增,直接测序后与数据库正常序列比对。结果:1.GLC3C区域内单个STR分型的传递不平衡检验结果显示,在D14S279、D14S555和D14S74的P值＜0.05（分别为0.021,0.009601和0.003421）,相距仅0.006cM;2.阳性区间内所有单个htSNP与疾病的传递不平衡检验P值均＞0.05;位于单倍型模块2（rs2111701-rs4020123-rs4903696-rs11159318-rs177216）的TAACG单倍型与疾病的传递不平衡检验P值＜0.05（P=0.001）,经过10000次的置换检测,该单倍型P值仍＜0.05（P=0.0135）;3.对于GLC3B区域内候选基因ANGPTL7的5个外显子序列,6份混合样本（含96人DNA）与数据库正常序列比对,无杂合峰发现。结论:GLC3C位点与中国汉族婴幼儿型青光眼的发病相关;在22kb区间内的5个htSNP单倍型与疾病发病相关,致病基因的确定有待进一步的候选基因筛查。GLC3B区域内候选基因ANGPTL7可能与中国汉族婴幼儿型青光眼的发病不相关。更多还原

【Abstract】 PartⅠ:Relationship of CYP1B1 Genotype-Phenotype and Operative EffectPurposes:To investigate the mutation condition of CYP1B1 gene in Chinese Han infantile glaucoma patients;and to explore the relationship of genotype-phenotype and operative effect.Methods:1.122 unrelated cases of infantile glaucoma patients were recruited in this study. Peripheral blood was collected and genomic DNA was extracted.The coding sequence in exons was amplified by polymerase chain reaction（PCR）from genomic DNA,then direct DNA sequencing was adopted to identify the variants exclusive in the patients.2.Combination with the previous experimental data of our team,we investigated the genotype-phenotype relationship of 238 cases by retrospective study. The analyzed factors included sex,single/both eyes involvement,onset age, intraocular pressure at onset,diameter of cornea,and score of cornea’s transparence.3.We reviewed the operative effect of 306 eyes in 192 consecutive patients by multiple factors analysis,which underwent initial surgery in our hospital.Seven factors of onset age,time between onset-operate,style of operate,preoperative intraocular pressure,diameter of cornea,score of cornea’s transparence,and mutant status of CYP1B1 gene were subjected to multivariate analysis by logistic regression. Cox proportional hazards regression modeling were used to analyze successful time and trace the survival curve.The SPSS13.0 statistical software was used for data analysis,and P value was less than 0.05 as the statistical significance.Results:1.18 different mutations were detected in the 122 cases’ coding exons of CYP1B1 gene.Among them,nine mutations were the first checked out in this study. They were g.3836T＞C（p.11W＞R）,g.4022delTC（p.73ORFshift,p.221stop）, g.4151G＞T（p.116D＞Y）,g.4322G＞A（p.173E＞K）,g.4338T＞A（p.178V＞E）, g.4493G＞A（p.230E＞K）,g.4509T＞C（p.235V＞A）,g.8137T＞C（p.434W＞R）, g.8167C＞T（p.R444stop）.There were 21 cases with the exclusive CYP1B1 gene variants in the 122 patients,and the mutation rate was 17.2%.2.Combined with the previous data,there were 41 cases with mutations of CYP1B1 gene in 238 cases.The mutation rate was 17.2%,too.The median onset age of patients with mutant CYP1B1 gene was 2（0-6）months,and that was 6（2-12） months in whom without mutation.The former was earlier than the latter statistical significantly（P＜0.05）.The carriers of mutant CYP1B1 gene were graded higher in corneal transparency（P＜0.05）.3.In multivariate logistic regression,the status of CYP1B1 gene showed statistically significance as independent protective factors for final outcome （P＜0.001）,and the odds ratio less than 1(OR_cyp1b1=0321).In Cox regression,the significant related factors were corneal transparency（P＜0.01）and the status of CYP1B1 gene（P＜0.01）,and the odds ratio were OR_Conea=1.846,OR_Cyp1b1=0.468.Conclusions:The mutant CYP1B1 gene is partly related with the infantile glaucoma. The carriers of mutant CYP1B1 gene may be at earlier onset and with poorer corneal transparency.Mutant CYP1B1 gene is the protective factor for operative effect,and the patients with it have higher success rate and longer successful time.PartⅡ:Genetic Study on Candidate Loci of Infantile GlaucomaPurposes:To investigate the association relation between the occurrence of infantile glaucoma and the GLC3C locus;and to find out the mutational condition of candidate gene ANGPTL7 in GLC3B locus.Methods:1.152 nuclear families（patients and their parents）without mutant CYP1B1 gene were recruited in this study.By using the Fluorescence Labeled Multiplex-PCR Technique,12 short tandem repeats（STRs）were genotyped covering the GLC3C locus,and transmission disequilibrium test（TDT）was performed.2.In the positive regions,16 haplotype tag SNPs（htSNPs）were choosed to construct haplotypes.By means of MALDI-TOF MS（Matrix-assisted laser desorption/ionization Time-of-flight Mass Spectrometry）,126 trios were genotyped, and transmission disequilibrium test was applied again.3.96 cases randomly selected to create DNA pools.Five exons of ANGPTL7 gene were amplified by PCR from the DNA pools,then direct DNA sequencing was adopted.Compare the sequence of the patients and the normal population in database.Results:1.By the single STR’s TDT,there were three markers existing transmission disequilibrium in GLC3C.They were D14S279（P=0.021）,D14S555（P=0.009601） and D14S74（P=0.003421）.The region was 0.006cM long.2.All the single htSNPs in the positive STR regions were no statistical difference in TDT.In the block2（rs2111701-rs4020123-rs4903696-rs11159318-rs 177216）,the haplotype TAACG was proved to be transmission disequilibrium （P=0.001）.After 10⁴ times of Permutation test,the haplotype was still significantly manifesting transmission disequilibrium（P=0.0135）.3.As for candidate gene ANGPTL7 in GLC3B locus,the sequence of five exons between the 6 DNA pools（containing 96 patients）and the normal ones in database was not any heterozygosis to be found.Conclusions:We confirmation the GLC3C locus is associated with the infantile glaucoma in Chinese Hans.A haplotype（including five htSNPs and 22 kilo-basepair long）is related on the occurrence of the disease,and further candidate genes screen should be carried out to dig out the pathogenic or susceptible gene.The candidate gene ANGPTL7 in GLC3B maybe not correlated with the Chinese Han infantile glaucoma patients.更多还原