【作者】 唐朝晖；

【导师】 王擎；

【作者基本信息】 华中科技大学 ， 遗传学， 2009， 博士

【摘要】 视网膜色素变性是一类遗传性视网膜疾病，世界范围内平均约有1／3500的患病率，是成年人失明的主要原因之一。视网膜色素变性患者通常在早期出现夜盲，渐行性视野丧失；到末期阶段，往往在中年之后，患者的中心视野严重受损时，可导致永久性失明。眼底检查中通常可见视网膜血管变细，眼底分布有色素沉着，视盘呈苍白色等典型特征。目前，临床上对视网膜色素变性尚没有有效的预防与治疗方法。视网膜色素变性表现出高度的临床以及遗传异质性。遗传上，主要可表现为常染色体显性（30-40％），常染色体隐性（50-60％），X染色体连锁（5-15％）等三种方式的遗传，少数视网膜色素变性的病例不属于孟德尔遗传模式，如双基因遗传以及线粒体遗传等。有些基因的不同突变，在不同的家系中还可以表现为不同的遗传模式。虽然已经有很多相关基因或位点被报道，但是，迄今为止，仍然有60％的视网膜色素变性病例没有找到致病基因。发现新的视网膜色素变性基因，对于进行基因诊断、理解疾病发展的分子机制以及发现有效的治疗方法有着积极意义。本课题主要对两个中国人视网膜色素变性家系的遗传因素进行研究。通过连锁以及单倍型分析先在染色体上确定相关致病基因的位置；接着运用DNA直接测序法来分析该基因是否含有导致疾病的突变，以及突变是否在家系中共分离；再应用高分辨率熔解曲线分析等方法，分析突变是否存在于正常人群中。我们首先对一个来自山东的常染色体隐性遗传的家系进行研究。通过连锁分析和单倍型分析，我们发现该家系的致病基因可能与新发现的arRP位点RP26紧密连锁，而排除了其它隐性遗传RP基因致病的可能性。通过对RP26基因CERKL的整个编码序列，以及外显子与内含子交接区的测序，发现病人的第一个外显子中存在一个插入突变，即在CERKLcDNA的第156位核苷酸和157核苷酸之间插入了一个T（c．156₁57insT）；病人另一条染色体上的第5外显子中则发现缺失了一个T，即CERKL cDNA的第758位核苷酸T缺失（c．758delT）。进一步对整个家系的测序分析结果显示先证者的母亲含有c．156₁57insT突变，先证者的父亲则含有c．758delT突变，两个姐妹则分别从其父母中遗传到两个正常的等位型，而先证者及其两个兄弟均得到其父母两个突变的等位型，提示CERKL两个复合型杂合突变c．156₁57insT和c．758delT是该家系三位患者患有视网膜色素变性的遗传基础。在随后进行的HRM结果表明，这两个突变在100多位正常人群中不存在。CERKL的c．156 157insT和c．758delT突变均属于移码突变，显著改变了其编码蛋白的读码框。c．156 157msT由于T的插入，正好产生一个密码子TGA，使得该基因的编码产物提前终止，产生只有52个氨基酸的异常蛋白；而c．758delT突变由于第758位T的缺失，也改变了其后的阅读框，不仅将原来的第253-258氨基酸METDRI序列，改变为异常的RKQTES，还在第259位产生一个终止密码子TGA，因而产生一个只有258个氨基酸的异常蛋白，而不是正常CERKL基因编码的含532个氨基酸的正常蛋白质。目前为止，CERKL中只有三个突变被报道，并都是在同血缘关系的家系中发现的。而我们发现的这两个新突变不仅是在中国人RP家系中的首次报道，而且是世界上第一次发现，CERKL的复合型杂合突变，可以在非血缘关系的家庭中，引起隐性遗传的视网膜色素变性。我们的研究丰富了CERKL突变引起视网膜色素变性的遗传突变谱，对于视网膜色素变性的基因诊断，以及认识该基因的功能，和视网膜色素变性发病的分子病理机制具有一定的意义。为探讨这两个CERKL突变引起视网膜色素变性的分子机制，我们构建了含野生型和突变型CERKL的pEGFP-CERKL载体，对突变的细胞学效应进行分析。将重组载体转染HeLa细胞，用共聚焦显微镜观察野生型和突变型CERKL的亚细胞定位，发现突变后的CERKL蛋白在细胞中的定位有明显变化。本论文还对一个在来自湖北省的常染色体显性遗传家系进行分子遗传学研究，通过连锁分析我们排除了其它常染色体显性遗传的RP基因，发现该家系的致病基因与RP4紧密连锁；运用DNA直接测序法对RP4基因RHO整个编码区的测序，发现患者的RHO基因中有一个碱基替换突变，即：c．C5271T，该突变导致RHO蛋白第347个氨基酸由脯氨酸变为亮氨酸（p．P347L）。该突变在家系中与疾病共分离，提示RHO的p．P347L为该家系患有adRP的遗传基础。我们还对家系中两个尚未发病的个体进行了基因诊断。和其他RHO的p．P347L引起的RP的临床症状不同，该家系的部分患者还患有白内障。我们的研究对于理解RHO突变型和临床表现型的关系具有一定意义。更多还原

【Abstract】 Retinitis pigmentosa is a set of hereditary retinal diseases affecting about 1 in 3500people worldwide,and considered to be the main cause of adult blindness.Patients withretinitis pigmentosa usually show the early onset of night blindness followed by aprogressive loss of the visual field.As a result,the grossly constricted central visioncauses irreversible blindness,often after midlife.In addition,perivascular pigmentation,attenuation of the retinal arterioles,and pale appearance of the optic disk are commonfindings during a fundus examination.To date,there is still no efficient prevention andtherapy for retinitis pigmentosa.Retinitis pigmentosa exhibits extreme heterogeneity both clinically and genetically.Itcan be inherited most frequently （50-60%） as an autosomal recessive trait,followed by anautosomal dominant pattern （30-40%） and an X-linked （5-15%） pattern.A small proportionof retinitis pigmentosa cases present with non-Mendelian inheritance patterns,such asdigenic or mitochondrial inheritance patterns.In some cases,different mutations in the samegene may involve in different inheritance traits.To date,a number of loci or genesresponsible for retinitis pigmentosa have been reported,but for about 60% of RP cases,thegenetic basis has not been found.Localization and identification of more pathogenic genesfor retinitis pigmentosa is crucial for genetic diagnosis,understanding the molecularmechanism by which the disease develops,and for finding efficient therapeutic methods.In this thesis project,we studied two Chinese retinitis pigmentosa families,including oneautosomal dominant family and one autosomal recessive family.By linkage and halpotypeanalyses,we localized the pathogenic genes onto specific chromosomal locations,and thenused direct DNA sequence analysis to find the disease-causing mutations and to demonstrate ifthe mutations co-segregate with the disease in the family.The High Resolution Melt （HRM）analysis was used to verify if these alterations exist in normal controls.In the first section of the thesis,we started our studies from a Chinese family withautosomal recessive RP from Shandong Province.Linkage analysis linked the diseasecausing gene of the arRP family to the RP26 locus,and DNA sequence analysis revealedtwo compound heterozygous mutations in the newly identified RP26 disease-causing gene, CERKL.One mutation is c.156₁57insT in exon 1,and the other is c.758delT in exon 5.The affected male individuals in the family inherited both mutations,whereas twounaffected sisters inherited normal alleles from their parents.The two mutations werenot detected in more than 100 unrelated normal controls by HRM.Both mutations are predicted to cause frameshift of CERKL protein.Thec.156₁57insT mutation introduces a stop codon at position 53 of CERKL,causing aprematurely truncated CERKL protein with only 52 amino acids.Mutation c.758delTcauses frameshift and substitutes 6 amino acids 253-METDRI-258 by abnormal RKQTES,and produces a truncated CERKL protein with only 257 amino acid residues.To date,there are only three RP causing CERKL mutations that have been reported,and all of them were identified in consanguineous families.The two novel CERKLmutations identified in this project are from a non-consanguineous Chinese family,andrepresent the first report that compound heterozygous mutations of CERKL cause retinitispigmentosa.These studies expand the spectrum of CERKL mutations causing autosomalrecessive retinitis pigmentosa.In order to usderstand the mechanism by which mutations c.156 157insT andc.758delT in CERKL causing retinitis pigmentosa,we generate the compound mutationsby the method of mutagenesis,fusing the wildtype and two mutant CERKLs withpEGFP-C1 vector,and transfected them to HeLa cell line.We investigated if the twomutations could alterate the normal subcellular localization of CERKL protein.Asignificant difference was identified between the two mutant CERKL proteins and thenormal wild type protein.Altered subcellular localization may be a mechanism the twomutations causing retinitis pigmentosa.We also investigated a Chinese family with autosomal dominant retinitis pigmentosa,inthe second section of the thesis.The patients of this family display some unique character ofsymptom of RP,such as early onset,and combining with cataract.Linkage analysis excludedall RP loci except for RP4,sequencing of the whole coding area of RHO revealed a Pro347Leumutation in exon 5 of this gene.We further performed gene diagnosis study for twoasymptomatic young members in the RP family.Our studies expand the clinical spectrum ofp.P347L mutation in RHO,and may help to understand the correlationship between thegenotypes of RHO mutations and phenotypes of retinitis pigmentosa.更多还原