【作者】 蒋劲；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学， 2008， 博士

【摘要】 随着当代眼科学手术技术不断改进和基础研究的飞速发展,先天性白内障的诊断、治疗及预后都有了很大的提高,但它仍然是导致儿童盲的首要原因。如果不能得到及时和正确的治疗,混浊的晶状体会阻碍物体在视网膜上的清晰成像,影响视网膜皮质突触连接的发育,最终导致不可逆的弱视。大约50%的先天性白内障与遗传有关,其中最常见的遗传方式是常染色体显性遗传,该遗传方式患者下一代的发病概率为50%,因此会影响整个家族的生活质量。迄今为止,研究人员已发现至少19个候选位点以及14个白内障疾病相关基因与常染色体显性先天性白内障（autosomal dominant congenital cataract,ADCC）有关,这些基因可以归为4类:（1）编码晶状体蛋白的基因,包括CRYAA、CRYAB、CRYBA1、CRYBB1、CRYBB2、CRYGC、CRYGD以及CRYGS;（2）编码膜转运及通道蛋白的基因,如MIP,GJA3以及GJA8;（3）编码细胞骨架蛋白的基因,如BFSP2;（4）编码发育调节因子的基因,如PITX3以及HSF4。目前对先天性白内障疾病相关候选基因的研究主要从两方面着手。一方面从白内障形成相关的功能蛋白出发,寻找其编码基因作为突变研究中的重要侯选基因;另一方面对先天性白内障的大样本家系资料进行基因组扫描和连锁分析确定染色体定位,再筛选定位区域内的热点基因经测序发现突变点。后者是目前研究先天性白内障疾病相关致病基因的主要方法之一。本实验对一个具有特殊表型（蜗牛壳样混浊）的先天性白内障家系进行了研究,该家系共24人,患者分布于四代中共13人,现存活12人,其中男7人,女5人,均为双眼发病。家系中患者均在出生后不久发病,白内障形态基本一致。绝大多数患者伴有双眼眼球震颤,未手术患眼视力最差为手动最好为0.25。另外,家系成员中未发现其他眼部异常及全身疾病,可基本排除药物和环境的影响。因该家系白内障表型独特,国内、外文献均未见报道,本实验拟对该先天性白内障家系进行疾病相关候选基因的定位和克隆研究,以期找到与此独特先天性白内障表型相关的候选基因,并进一步探讨先天性白内障的发病机制及认识人眼晶状体发育和代谢的基因编码及遗传规律,为先天性白内障的产前诊断和基因治疗提供理论依据。本实验获浙江大学伦理委员会同意,并且所有家系成员均签署知情同意书。第一部分先天性蜗牛壳样白内障家系所有家系成员均接受病史调查,并行眼科检查,包括视力、裂隙灯、散瞳眼底检查等。根据病史、白内障手术史或裂隙灯检查所见确定成员患病与否。该家系共24人,患者分布于四代中共13人,现存活12人,其中男7人,女5人,均为双眼发病。病史及裂隙灯检查:家系中患者均在出生后不久发病,白内障形态基本一致,呈蜗牛壳样混浊,个别息眼还伴有皮质骑子。绝大多数患者伴有双眼眼球震颤,未手术患眼视力最差为手动最好至0.25。家系成员均为足月顺产,妊娠史均正常,可排除药物和环境的影响,无其它眼部和全身病变。根据该家系遗传特点,无近亲结婚、每一代男女都有患者、且男性患者的男性下一代也可患病,我们可以判断该家系遗传模式为常染色体显性遗传。第二部分疾病相关候选基因的定位在家系调查的基础上,应用短串联重复序列（微卫星位点,short tandemrepeats sequence,STRS）为遗传标记的基因扫描和遗传连锁分析技术,进行疾病相关候选基因的定位分析。采集包括12例患者在内的家系成员血样22份,使用3-Spin Blood DNA Isolation（上海申能博彩生物科技有限公司）试剂盒提取基因组DNA。选取与已知18个常染色体显性遗传先天性白内障相关位点邻近的微卫星位点共41个,分别用荧光染料标记的引物进行聚合酶链反应（polymerasechain reaction,PCR）扩增,ABI Prism^TM377XL型DNA测序仪电泳,Genescan3.0软件和Geno typer 2.1（Perkin-Elmer Applied Biosystem）软件完成等位基因的分型,运用Linkage 5.10软件MLink程序进行家系两点连锁分析。两点连锁分析得出在STRS标记D12S368和D12S83处,当重组率θ=0时其两点LOD最大值(Z_MAX)分别为1.66和2.01,支持连锁。而ADCC相关基因MIP所在区域恰好位于这两个标记点之间。在D12S368和D12S83之间,再选取3个微卫星标记D12S1586、D12S1632和D12S1691进行连锁分析,其中最高LOD值3.08（θ=0）在STRS标记物D12S1632处获得,属肯定连锁关系。第三部分疾病相关候选基因的突变筛选通过对该家系先天性白内障疾病相关候选基因MIP（编码水通道蛋白0,aquaporin0,AQP0）基因筛选测序,显示在MIP基因第3号内含子3’最后一个碱基发生了杂合的G→A置换（c.607-1G＞A）。在发现了此改变后,我们对所有的家系成员以及100个与该家系无血缘关系的正常个体进行了限制性片断长度多态性分析（Restriction Fragrnent Length Polymorphism Analysis,RFLP）。该点突变使原有的一个BstSFⅠ限制性内切酶酶切位点消失,此改变与白内障表型完全共分离:家系中所有患者均有此改变,而健康者均无此改变,并且100个不同遗传背景的正常对照样本亦无此改变,提示MIP基因为此独特表型白内障疾病相关基因。第四部分MIP基因3号内含子隐性受体剪接位点的预测因该突变位点恰好位于MIP基因的3号内含子受体剪接位点处,将导致MIP前体mRNA无法正常剪接。通常剪接位点突变会出现外显子丢失、替代性剪接位点的激活、内含子中出现假外显子及内含子残留等改变。用NNSPLICE程序,版本0.9（http://www.fruitfly.org/seq_tools/splice.html）对该剪接位点的临近序列进行隐性受体剪接位点评分（分值范围0～1.0）,预测可能存在的替代性受体剪接位点（评分大于0.4）。结果表明除原有正常受体剪接位点外（评分1.0）,另有两个隐性剪接位点评分分别为0.44和0.88。当G→A突变发生时,原正常剪接位点AG变为从,评分也从1.0降至0.0,因此预测的两个隐形剪接位点有可能被激活。第五部分异常剪接产生突变型MIP蛋白（AQP0）模型的计算机构建和分析水通道蛋白0（AQP 0）是水通道蛋白家族中的一员,其主要功能为跨膜快速转运水分子。其主要在晶状体终末分化纤维中表达,并占总膜蛋白的50%以上。AQP0三维结构高度保守,对维持人晶状体透明性和屈光性具有重要作用。运用SWISS-MODEL软件以牛AQP0蛋白结构为模板预测野生型和异常剪接型AQP0蛋白结构,用SWISS-Pdb浏览器进行观察与分析。SWISS-MODEL软件蛋白建模后发现MIP基因（c.607-1G＞A）突变后如激活隐性受体剪接位点,产生突变型蛋白,将导致其第六段跨膜α-螺旋及后续的羧基端丢失,破坏其正常的沙漏结构及其在细胞膜上的锚定。结论1、本研究首次报道了一个中国特殊表型（蜗牛壳样）先天性白内障大家系,该家系的遗传方式为常染色体显性遗传;2、首次报道受体剪接位点突变引起的常染色体显性遗传性先天性白内障。MIP基因第3号内含子受体剪接位点突变（c.607-1 G＞A）与该先天性白内障表型共分离,MIP为该先天性白内障家系的疾病相关基因。3、MIP基因3号内含子隐性受体剪接位点激活后产生的异常剪接蛋白,将导致AQP0第六段跨膜α-螺旋及后续的羧基端丢失,破坏其正常的沙漏结构及其在细胞膜上的锚定。4、本实验研究结果进一步拓展了先天性白内障的基因突变谱,支持了AQP0在人晶状体发育以及正常功能形态维持中具有重要作用的理论。更多还原

【Abstract】 Although surgical techniques and visual prognosis have been improved recently,congenital cataracts remain the leading cause of visual disability in children worldwide.Without prompt treatment,cataracts can occlude clear imaging on the retina,resulting in failure to develop normal retinal-cortical synaptic connections and finally irreversible amblyopia.Approximately 50%of congenital cataracts are inherited,with the most common being the autosomal dominant form.The frequrent autosomal inheritance provides a tool to identify the genes involved in lens development and cataract formation. To date,more than 19 candidate loci have been identified and 14 cataract-related genes characterized.These genes can be divided into four groups:(1)Genes encoding crystallin:Mutations in 8 crystallin genes have been identified as the cause of autosomal dominant congenital cataract (ADCC)including CRYAA,CRYAB,CRYBA1,CRYBB1,CRYBB2 ,CRYGC,CRYGD and CRYGS;(2)Genes encoding membrane transport proteins including:MIP,GJA3,and GJA8; (3)Genes encoding cytoskeletal proteins such as BFSP2;(4)Genes encoding transcription factors such as PITX3 and HSF4.At present,there are two major methods used in cataract candidate disease-associated gene research:one is based on major functional protein associated with cataractgenesis,mapping the candidate gene location by hybridization;the other is mapping the locus for congenital cataract in typical family using genomic genescan,candidate gene linkage analysis and candidate gene screening to identify the disease-associated gene.Here,we studied a large,four-generation Chinese family containing members affected by special congenital snailshell-like cataract.Opacification of the lens was bilateral and happened very early after birth in all the affected cases.Most of the patients had nystagmus with visual acuity in the unoperated eye ranging from 0.25 to hand move.There was no family history of other ocular or systemic abnormalities.Based on the presence of affected individuals in each of the four generations,and male-to-male transmission, autosomal dominant inheritance of the cataract was demonstrated.We studied the candidate disease-associated gene of this special phenotype congenital cataract,clarifying the congenital cataract mechanisms in human. Informed consent in accordance with the Declaration of Helsinki and Zhejiang Institutional Review Board approval was obtained from all participants.PartⅠThe snailshell-like congenital cataract family22 individuals participated in the study:12 affected individuals and 10 unaffected individuals of whom 7 were spouses.Affected status was determined by a history of cataract extraction or ophthalmologic examination, which including slit-lamp examination under dilated pupils,visual acuity testing and fundus examination.The cataract phenotype of the patients who had had cataract extraction was learned from the history records.Opacification of the lens was bilateral and happened very early after birth in all the affected cases.Most of the patients had nystagmus with visual acuity in the unoperated eye ranging from 0.25 to hand move.The appearance of the opacification was quite unique and at first glance under diffuse illumination,it gave the appearance of a snailshell.In many cases,there was a wedge-shaped opacity from the inferotemporal cortex with the apex pointing to the central nucleus,and in the slit section it appeared as a rider.There was no family history of other ocular or systemic abnormalities.Based on the presence of affected individuals in each of the four generations,and male-to-male transmission,autosomal dominant inheritance of the cataract was demonstrated.PartⅡCandidate disease-associated gene mappingBlood specimens(5ml)were collected in EDTA and leukocyte genomic DNA was extracted.The initial strategy consisted of screening 18 known loci related to ADCC formation and 41 fluorescent short tandem repeat polymorphic markers(ABI PRISM Linkage Mapping Set,Version 2.0)were used.Mutiplex PCR products were resolved using a ABI PrismTM 377XL DNA sequencer and the data were collected and analyzed using these computer programs:(ABI PrismTM Genescan 3.0 and Genescan analysis(?)2.1). Two-point Iod scores between the cataract locus and markers were calculated by the MLINK program of LINKAGE package(version 5.1)for a full range ofθvalues.Positive two point lod scores were obtained at markers D12S368 (Zmax=1.66 atθ=0.0)and D12S83(Zmax=2.01 atθ=0.0).The MIP gene was just flanked by these two markers.Three additional markers very near to the MIP gene were subsequently used for further confirmation.All of these 5 markers received significant scores,and the maximum score was obtained with marker D12S1632(Zmax=3.08 atθ=0.0)PartⅢMutational analysis of MIP geneA strong candidate gene,the MIP gene,is composed of 4 exons.Gene specific PCR primers were designed flanking each exon and intron-exon junction.PCR products were sequenced commercially.Three affected and three unaffected individuals were compared.By sequencing of exon4 of the MIP gene,we found a single base substitution in the splice acceptor site of intron3(c.607-1G>A)in the affected.This site in the unaffected individual was homozygous for a G,while in the affected was heterozygous G,A.The mutation abolished a BstSF I restriction site that segregated with all of the affected members,but didn’t exist in unaffected family members or 100 unrelated normal Chinese controls.It indicated that the MIP gene might be the disease-causing gene.PartⅣThe prediction of cryptic acceptor splice site in intron3 of MIP geneThis mutation just located in the acceptor splice site of intron3,so it would cause abnormal splicing.Splice site mutations may result in exon skipping, activation of cryptic splice sites,creation of a pseudo-exon within an intron or intron retention,among which exon skipping is the most frequent outcome. The sequence environment of this mutation was analyzed with the NNSPLICE program,version 0.9(http://www.fruitfly.org/seq_tools/splice.html).The minimum score for the functional 3’ splice site(between 0 and 1)was set at 0.4.There were two possible cryptic splice sites in the normal sequence which scored 0.44 and 0.88,respectively,besides the normal acceptor splice site scored 1.0.When the G→A mutation happened,the normal splice site AG turned to AA with the score reduced to 0.00.Thus one of the cryptic splice sites might be activated.PartⅤComputer prediction of mutated protein formed after abnormal splicingThe major intrinsic protein(MIP,AQPO;OMIM+154050)is a member of the aquaporins,a family of membrane water transport proteins that confer rapid transport of water across the cell membrane.AQPO is only expressed in terminally differentiated fibers in the lens and constitutes approximately 50% of the total membrane protein.The tertiary structure of AQPO is highly conserved and it plays an important factor in human lens development and cataract formation.Both wild type and mutant AQPO structure predictions were done by SWISS-MODEL using bovine AQPO as templates.Models were viewed in Swiss-Pdb Viewer.The mutant AQPO structure predicted by SWISS-MODEL showed that the sixth transmembraneα-helix and the following carboxy-terminal of the mutant were both removed,with the result that the integrality of the hourglass structure and the stability of the protein anchoring in the membrane were both lost. Conclusion1.This is the first report about the special snailshell-like congenital cataract family in China.Autosomal dominant inheritance was demonstrated in this family.2.It’s the first acceptor splice site mutation reported in ADCC.The c.607-1G>A in MIP gene co-segregated with the presence of the snailshell-like congenital cataracts and was not observed in 100 unrelated controls.3.The mutated protein forming after the abnormal splicing would cause the removal of the sixth transmembraneα-helix and the following carboxy-terminal,with the result that the integrality of the hourglass structure and the stability of the protein anchoring in the membrane were both lost.4.This study widened the mutation spectrum of congenital cataract and further supported the notion that alterations to AQPO played an important factor in human lens development and cataract formation.更多还原