【作者】 肖伟；

【导师】 张劲松；

【作者基本信息】 中国医科大学 ， 眼科学， 2008， 博士

【摘要】 目的先天性白内障是儿童常见的致盲性眼病,全世界有近4000万儿童患先天性白内障,其中有1/3是遗传性先天性白内障。遗传性白内障有常染色体显性遗传、隐性遗传和性连锁遗传。常染色体显性遗传性先天性白内障(Autosomal dominantcongenital cataract,简称ADCC)是遗传性白内障中最常见的类型。ADCC随着致病基因代代相传,对患者家系后代的生活和健康危害巨大,也给社会带来负担。如何降低或消除先天遗传性疾病是提高我国人口健康状况的关键问题之一。我国是人口大国,充分利用我国人类基因组和遗传资源的优势,发现新的白内障致病基因,找到其突变规律和特点,并用于产前基因筛查、基因诊断和遗传咨询,将有效地预防先天性白内障患儿的出生,从而使遗传性白内障家族成员从视觉残疾的困境中走出来,并可以与健康人、与社会和谐共处。在分子水平上揭示遗传性白内障及ADCC基因缺陷的本质,将为遗传干预和基因治疗提供理论依据。本文就我们收集的生活在我国东北辽宁地区的四代10人患病的大家系,就遗传性白内障发病的相关基因,进行了一系列的分子遗传学研究。材料与方法家系收集,直接调查我们收治的ADCC家系4个,排除眼部和全身其他遗传性疾病,对其所有家系成员眼前节散瞳检查拍照,并对患病成员做表型认定,绘制系谱图。经知情同意后,采集先证者及家系成员外周血4ml,-70℃冷冻保存备用。酚氯仿法提取家系成员外周血基因组DNA,在染色体上的1q21-25、2q33-36、3q21-25、3q27.3、10q25、11q22.1-23.21、12q13-14、13q12.11、16q23.1、16q22.1、17q11-12、19q13.33、20p12.1、21q22.3、22q11.2-12.1区段内分别选取26个多态性微卫星标记进行连锁分析。通过含7mol/L尿素的8%聚丙烯酰胺变性凝胶电泳分离PCR等位片段,硝酸银染色显色,根据凝胶上DNA片段大小确定微卫星标记等位基因及家系中各成员的基因型。应用LINKAGE软件(version 5.2)中的MLINK进行微卫星标记与该家系致病位点的两点间连锁分析,计算LOD值。判断标准:Lod值≥3,两个位点肯定连锁;Lod值≤-2,两个位点不连锁;-2＜Lod值＜3,提示连锁。根据个体的等位基因型,家系成员间的亲缘关系及孟德尔遗传定律,人工构建该家系单体型。通过国际互联网,在美国国立图书馆数据库(网址:www.ncbi.nlm.nih.gov)下载人类基因组中GJA8基因的全部外显子标准序列。根据基因的结构,将预扩增的DNA序列向两端延伸100bp左右,根据引物设计原则进行引物设计并合成。以先证者和家系中一名正常人的基因组DNA为模板,以引物对GJA8F/GJA8R扩增GJA8基因的外显子。PCR产物纯化后回收,纯化后的PCR产物25μl,每个测序反应提供10μmol/L的引物8～10μl,测序过程由奥科生物有限公司完成,使用美国ABI PRISMTM 377XL DNA自动测序仪,应用双脱氧末端终止法进行序列分析。根据片段长度决定测序反应的个数,小于500bp测一个反应,大于500bp则进行双向测序反应并检查测序图,观察是否有杂合峰出现。对疑为突变的位点,进一步做突变的酶切分析验证。结果追溯调查家系成员共计71人,有26人患病,男性患者10人,女性患者16人;现存家系成员共计66人,患者25人,其中男性患者10人,女性患者15人。共采集外周血49人份,其中患者血样25人。除家系1外其余3个家系均为先天性白内障在家系中连代相传,男女均有发病,符合常染色体显性遗传特点。2个家系为绕核白内障,2个家系为核性白内障。根据微卫星标记等位基因在8%聚丙烯酰胺尿素变性凝胶中的分布情况,分析家系成员的基因型组成。根据连锁分析软件LINKAGE(version5.2)的要求,建立每个微卫星标记连锁分析的家系档案。通过对家系成员进行微卫星标记的连锁分析,发现位于染色体2q33-36、3q21-25、3q27.3、10q25、11q22.1-23.21、12q13-14、13q12.11、16q23.1、16q22.1、17q11-12、19q13.33、20p12.1、21q22.3、22q11.2-12.1区段的20个微卫星标记与常染色体显性白内障大家系均无连锁关系,LOD值为负值。但家系3位于1q21-25的微卫星标记LOD值存在正值(θ=0);对于微卫星标记D1S1156,当θ=0时,最大LOD值为2.36。PCR产物经2%琼脂糖凝胶电泳鉴定,能满足测序反应要求。家系3先证者GJAB基因纯化后PCR产物直接测序,结果GJAB基因外显子2的测序图显示第773位碱基处出现一个杂合峰。通过与正常序列进行比较,我们发现先证者的一个等位基因第773位碱基C突变为T,导致258位丝氨酸密码子突变为苯丙氨酸(p.Ser258Phe),提示该突变为该家系的致病突变。以错配引物GJA8MF和GJA8R扩增家系3的20名家系成员和100个正常人群体的DNA,扩增出169bp的DNA片段。PCR产物以限制性内切酶HinfI消化,8%的PAGE分析酶切产物,结果显示所有白内障患者都含有与先证者相同的突变,即:酶切产物电泳后产生169bp,149bp和20bp的三个片段,由于切胶时,20bp的DNA片段丢失,所以所有正常人仅有一个149bp的片段,而所有患者都能见169bp和149bp的两个片段,即GJA8基因突变导致遗传性白内障得到了证实。结论1、本文发现并收集到的4个先天性白内障家系,系谱分析表明均为常染色体显性遗传。2、本文报告的常染色体显性遗传性白内障在不同家系中表型不同;2个家系为绕核性白内障,2个家系为核性白内障。3、在家系3的全部患病成员中,排除了与染色体2、3、10、11、12 13、16、17、19、20、21和22上侯选白内障致病基因区域微卫星多肽标记连锁。4、家系3两点连锁分析结果提示与染色体1q21-25区域标记连锁。5、在常染色体显性遗传性白内障家系3中发现了一个新的GJA8基因致病性突变[S258F(733C＞T)]。6、对各家系的表型研究表明中国人常染色体显性遗传性白内障具有遗传异质性。家族遗传性先天性白内障是常见的家族遗传性疾病之一。先天性白内障是儿童常见的眼病,也是儿童致盲的主要原因。我国发病率为0.05%,欧美国家发病率为0.01-0.06%。约有三分之一的先天白内障是遗传性的,而常染色体显性遗传是常见的一种遗传方式。遗传性先天性白内障在临床上可为单纯晶状体混浊,或晶状体混浊伴有眼部其它组织发育异常,或表现为遗传性疾病综合征的一部分。随着分子遗传学的发展,新的导致先天性遗传性白内障的基因突变不断被发现,但由于先天性白内障分类多,表型交叉重叠,研究较为困难,致病基因尚未确定。对于遗传病而言,一个大家系在致病基因的定位上有特殊优势,利用一个多世代、多个体的大家系进行连锁分析定位,能有效的去除多个小家系组合时常有的座位异质性和等位基因异质性的干扰。我国先天性白内障临床表现型复杂多变,因而进行基因分型的难度也比较大。对先天性白内障临床表型的分析,从多方面入手,探讨先天性白内障的致病基因将为其它先天性遗传性疾病的研究提供值得借鉴的方法,这也是遗传学研究的一个重要发展趋势。我们收集到的3个单纯常染色体显性遗传性白内障大家系和一个小家系临床表现不尽相同,因此需要进行全面的临床遗传学分析。更多还原

【Abstract】 ObjectiveCongenital cataract is a common blindness threatening disease among children. About 40 million children suffer from the disorder worldwide,among which 1/3 are hereditary cataract.Hereditary cataract includes autosomal dominant congenital cataract (ADCC),autosomal recessive cataract and X-linked ones.ADCC is the most common type.It spreads generation by generation in the family,and brings about a poor living condition for the descendants.The elimination of hereditary diseases is the targeted proposition of the national health care.This is a populational country which is rich of hereditary resources.To find out novel causative genes and to better understand their mechanism is our goal to benefits human being and prenatal investigation and inquiry. ADCC is gene defect,which is being workout in molecular level.The outcomes of the research work in this field will fully support the hereditary interference and genomic therapy theoretically.The present study introduces a Northeasten resident family which covers 4-generation and 10 members with hereditary cataract.A series of research work had been done in screening and identifying the novel gene mutation.Materials and MethodsWe investigated 4 hereditary cataract families and gave them physical examination to exclude their general diseases and other ocular abnormalities.Pupil had been dilatated and anterior segment photographies had been documented.Phenotypes of each family had been identified clinically and pedigrees had been drawn carefully.After signed the written consent,Peripheral blood samples(4 ml per individual)were collected from the family members including non-affected members.Genomic DNA was extracted from their whole blood using standard phenol/chloroform-proteinase K method.A total 26 microsatellite polymorphic markers at chromosome 1q21-25、2q33-36、3q21-25、3q27.3、10q5、11q22.1-23.21、12q13-14、13q12.11、16q23.1、16q22.1、17q11-12、19q13.33、20p12.1、21q2.3、22q11.2-12.1 were selected and genotyped for linkage analysis.Sequence information for PCR primers were obtained from the NCBI website.Primers were commercially synthesized.All family members were subject to PCR genotyping.The PCR fragments were separated though 8% denaturing polyacrylamide gel electrophoresis.Two-point linkage analysis between the cataract phenotype and genetic markers were performed using the MLINK component of the LINKAGE package.Haplotype was constructed using allele information of family3.To calculate LOD score:LOD≥3,the two-point linkage exists.LOD≤-2,fail to linkage.LOD score between- 2 and 3,indicates linkage relation.Acording to the allele and the relation between family member,artificially construct the family haplotype.From NCBI download web site complete exon sequence of human GJAB.To amplify the DNA sequence by elongate the either side of DNA to 100bp.Design and synthesize primers.Amplify the exon of GJA8 by using primer GJASF/GJASR and using DNA of proband and an affected family member.Purify and collect PCR product.To purified PCR fragments were submitted for direct automatic sequencing.Reaction cycle according to fragment length.A less-than-500bp tested a reaction,a greater-than-500bp took sequence reaction bidirectionally.For the suspected locus, further restriction analysis is needed to identify the mutation.ResultsA total of 71 individuals,including 26 patients(10 males and 16 females),from the 4 families with congenital cataract,were investigated retrospectively.And 66 individuals were available for examination.Blood samples were collected from 49 individuals,including 25 patients.The pattern of phenotype transmission in all families suggested a mode of autosomal dominant inheritance.Of the 4 families,2 were zonular and another 2 were nuclear cataract.Microsatellite polymorphic markers,mainly of tetranucleotide repeats,were successfully genotyped by polyacrylamide gel electrophoresis and silver staining. Two-point linkage analysis did not provide evidence for linkage to markers from chromosome regions 1q21-25、2q33-36、3q21-25、3q27.3、10q25、11q22.1-23.21、12q13-14、13q12.11、16q23.1、16q22.1、17q11-12、19q13.33、20p12.1、21q22.3 and 22q11.2-12.1.However,a maximum LOD score of 2.36 were obtained at 1q21-25 for marker D1S1156(θ=0)in family 3,suggesting possible involvement of the GJA8 gene cluster in the cataractogenesis in this family.PCR direct sequencing of the proband genes in the GJA8 gene cluster revealed a missenss mutation of the GJA8 gene,S258F(773C＞T),leading to the substitution of serine for phenylalamine,which suggested that the novel mutation is pathogenic mutation.Mismatch primer GJA8MF and GJA8R amplified DNA of 20 family members from family 3 and 100 nomal individuals,which produced 169bp DNA fragments.PCR products were digested by Hinf I and analysed by 8%PAGE.The result showed that all the affected individuals contained the mutation which was exactly the same as the proband,but all nomal individuals did not.Conclusion1、Four families with congenital cataract were found and autosomal dominance was indicated as the mode of inheritance.2、The phenotype of ADCC in all families was different.Two families expressed zonular cataract and the other two were nuclear cataract.3、Two-point linkage analysis using 26 microsatellite polymorphic markers from 12 candidate chromosome regions excluded chromosome 2,3,10,11,12,13,16,17, 19,20,21 and 22 in family 3.4、Two-point linkage analyses showed the linkage relation with 1q21-25region in family 3.5、A novel GJA8 mutation was found in family3[S258F(773C＞T)].6、The phenotype of Chinese ADCC families exist hereditary hetrogenicity.更多还原