【作者】 张天晓；

【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】 目的先天性白内障为较常见的眼病之一,是儿童失明的主要原因。在先天性白内障患者中,8%-25%是遗传性的。利用我国的遗传资源优势,发现新的白内障致病基因并揭示其突变规律和特点,用于指导产前基因诊断和遗传咨询,将有效地预防和杜绝白内障患儿出生,提高人口质量。对于遗传性白内障的研究已有悠久的历史,随着遗传学和分子生物学的迅猛发展,目前遗传性白内障已有39个基因座被确定,其中19个遗传性白内障致病基因已被发现。这19个致病基因分别是10个晶状体蛋白基因(CRYAA、CRYAB、CRYBA1、CRYBA4、CRYBB1、CRYBB2、CRYBB3、CRYGC、CRYGD和CRYGS),4个膜蛋白基因(缝隙连结蛋白基因GJA3/GJA8、水通道蛋白基因MIP和内源性膜蛋白基因LIM2),3个转录因子基因(HSF4、PITX3和MAF),中间丝样细胞骨架蛋白BFSP2和染色质修饰蛋白基因CHMP4B。但由于先天性白内障种类多,表型又多有交叉,研究较为困难。虽然已经确定了多个致病基因,仍然有很多致病基因尚未发现。本研究收集了5个遗传性白内障家系,利用先进的分子遗传学技术及人类基因组计划的成果,开展了白内障致病基因研究。诺里病(Norrie disease,ND)是罕见的X连锁隐性遗传病,以视网膜出血、剥离导致儿童失明为其主要特征。因没有很好的治疗办法,ND预后极差。ND致病基因为定位于染色体Xp11.3的NDP基因。迄今为止,在NDP基因相关视网膜病变(ND、X连锁隐性遗传的家族性渗出性玻璃体视网膜病变、Coats病和早熟性视网膜病)中,至少已经发现NDP基因的80种突变。本研究收集了一个中国人诺里病家系,对该家系进行了致病基因突变的鉴定。材料和方法收集5个遗传性白内障家系,根据家族史、临床症状及体征进行准确的临床诊断,绘制系谱图。与患者及其家属签署知情同意书后,采集家系成员外周血5-10ml,-70℃冻存备用。使用常规酚氯仿法提取家系成员基因组DNA。在目前遗传性白内障已知的基因座中,选择包括19个已知常染色体显性遗传性白内障致病基因的共15个位点及其它10个遗传位点(1p36、1p22.3、1q21-25、2p12、2p24-pter、2q33-36、3p22-24.2、3q21-25、3q27.3、10q25、11q22.1-23.21、12q13-14、13q12.11、14q22-23、15q21-22、16q23.1、16q22.1、17p13.3、17q11-12、19q13.33、20p11.23-p12.1、20p12.1、20q11.22、21q22.3和22q11.2-12.1),在其区段内共计选取62个多态性微卫星标记,设计并合成微卫星标记的引物。应用聚合酶链式反应(PCR)扩增各家系全部成员的基因组微卫星片段,通过8%聚丙烯酰胺变性凝胶电泳分离PCR等位片段,硝酸银染色检测片段大小,确定各家系成员的基因型。应用LINKAGE软件(version5.2)中的MLINK程序进行微卫星标记两点间连锁分析,计算两点连锁LOD值。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,人工构建家系成员的单体型。根据美国国立图书馆数据库中的标准序列,对染色体22q11.2-12.15区域内的4个已知白内障致病基因(CRYBB1、CRYBB2、CRYBB3和CRYBA4)、染色体16q22.1区域内的1个已知白内障致病基因HSF4、染色体1p36.23-36.21区域内的7个候选基因(PTCHD2、KAZRIN、CASP9、SPEN、RSC1A1、PRMD2和PAX6)进行PCR引物设计,扩增这些基因的全部外显子及外显子/内含子交界区。PCR产物纯化回收后,直接测序。对于家系4中发现的PTCHD2基因可能致病突变,PCR扩增家系成员及268个无关正常个体的该基因第20个外显子与部分内含子,限制性内切酶ApalⅠ消化PCR产物,琼脂糖凝胶电泳检测酶切片段;使用高保真DNA聚合酶和位于PTCHD2基因外显子18及外显子21的一对引物,PCR扩增患者基因组DNA片段,克隆到pcDNA3.1质粒并构建PTCHD2微基因(minigene),转染COS-7细胞和HeLa细胞后提取总RNA,利用RT-PCR检测RNA剪接情况。对于在家系5中发现的可能致病突变HSF4基因c.356G＞A(p.Arg119His),设计针对突变的错配引物,采用巢式PCR扩增家系成员及100名无关正常对照个体的HSF4基因外显子3中含有突变点的DNA片段,限制性内切酶NsbⅠ消化PCR产物,8%聚丙烯酰胺凝胶电泳检测酶切片段。收集1个诺里病家系,根据家族史、临床体征及眼科B型超声波检查进行临床诊断。采集家系成员外周血标本,常规酚氯仿法提取基因组DNA。设计并合成3对PCR引物,通过PCR扩增患者及其母亲NDP基因的全部3个外显子与外显子/内含子交界区,PCR产物直接测序。针对可能致病突变,通过PCR产物限制性内切酶分析和PAGE分析验证对家系所有13名成员进行突变鉴定。确定致病突变后,对家系中一名怀孕的突变携带者,采集其胎儿绒毛、提取胎儿DNA。在NPD基因附近染色体区域选择2个微卫星标记,设计PCR引物扩增胎儿及其他家系成员微卫星片段,通过8%聚丙烯酰胺变性凝胶电泳及银染分析PCR等位片段基因型。最终通过单体型分析及限制性内切酶分析确定胎儿是否携带致病基因,进行产前基因诊断。结果临床检查表明5个遗传性白内障家系的表型包括多形态性白内障,后极性白内障及核性白内障。调查确定五个家系成员共计212人,其中64人患病(男性患者34人,女性患者30人)。现存家系成员共计201人,患者55人(男性患者30人,女性患者25人)。5个家系均符合常染色体显性遗传。25个位点的微卫星标记两点连锁分析结果表明,家系1和2在全部检测位点均未发现连锁。家系3在微卫星标记D22S689可获得最大LOD值2.71(θ=0时),提示该家系表型可能与染色体22q11.2-12.1区域连锁。家系4在微卫星标记D1S1151可获得最大LOD值3.36(θ=0时),提示该家系表型与染色体1p36.2的位点连锁;通过单倍型分析确定致病基因在微卫星标记1pTTTC(基因组位置8956145)和1pCAAA(基因组位置16294562)之间7.3Mb的区域内;参考已报道的致病基因范围,可以将该位点的致病基因范围缩短至微卫星标记D1S228与1PCAAA之间2.4Mb的区域内。家系5在微卫星标记D16S3085可获得最大LOD值2.63(θ=0时),提示该家系致病基因在染色体16q22.1区域内。针对各家系致病基因定位范围内的已知或候选基因进行突变筛查。在家系3中,未发现CRYBB1、CRYBB2、CRYBB3和CRYBA4四个已知致病基因的致病突变。在家系4中,对PTCHD2、KAZRIN、CASP9、SPEN、RSC1A1、PRMD2、PAX6七个候选基因进行测序,发现PTCHD2基因第20个内含子中第5个碱基由G变为T,即INV20+5G＞A。其它基因没有发现有意义的改变。限制性内切酶分析结果显示,家系4所有患病成员都携带该变化,家系成员中的正常个体与268个无关个体均不含该改变。患者PTCHD2微基因克隆后,转染HeLa和COS-7细胞,RT-PCR显示PTCHD2基因的INV20+5G＞A改变不影响RNA的剪接。在家系5中,患者HSF4基因测序结果表明该基因第三外显子发生c.356G＞A,使蛋白上高度保守的119位精氨酸替换成组氨酸。限制性内切酶分析显示该家系中所有患病成员都携带这个突变,100个无关正常个体均不含有该突变。诺里病家系成员现存13名,患者1名(先证者)。疾病传递符合X连锁隐性遗传。先证者及其母亲的NDP基因测序结果显示二人都携带NDP基因突变c.220C＞T(p.Arg74Cys),且先证者为该突变的半合子,其母为该突变的杂合携带者。酶切鉴定表明家系中另外4名表型正常的女性个体(Ⅲ3、Ⅳ3、Ⅲ5和Ⅱ5)均为该突变的携带者。微卫星标记单体型分析及限制性内切酶分析显示胎儿为女性,且为致病突变携带者。结论1、本文收集到常染色体显性遗传的白内障家系5个。其白内障表型不仅在不同家系间有不同,同一家系的不同个体间也有不同,表明国人中常染色体显性遗传性白内障存在表型异质性。同时,不同基因与遗传性白内障的发生有关,表明了遗传性白内障的遗传异质性。2、在家系1与家系2中,通过连锁分析排除了全部19个已知遗传性白内障致病基因的15位点及其它10个位点,提示存在未知白内障致病位点,有待进一步研究。3、家系3的两点连锁分析结果提示其致病基因可能定位在染色体22q11.2-12.1区域内,但在4个已知致病基因内均未发现致病突变,提示可能存在未知突变机制(包括存在其它位点)。4、家系4的致病基因定位于染色体1p36区域,参考已报道的致病基因范围,将致病基因所在的范围缩短至微卫星标记D1S228与1pCAAA之间2.4Mb的区域内;对该区域内PTCHD2、KAZRIN、CASP9、SPEN、RSC1A1、PRMD2和PAX6七个可能候选基因测序尚未发现致病突变。5、在家系5中,HSF4基因发生的c.359G＞A(p.Arg119His)突变是该家系的致病突变,此突变是HSF4基因上新的致病突变。再次证实了HSF4基因与遗传性白内障的相关性。6、首次在中国人诺里病家系中发现了NDP基因错义突变c.220C＞T,该突变与国外报道的一个诺里病致病突变一致;并且成功地为一例诺里病高危胎儿做了产前基因诊断。更多还原

【Abstract】 ObjectiveCongenital cataract is the most common cause of treatable childhood visual impairment and half of them are estimated to have genetic cause.Inherited cataract is a lens disease with remarkable phenotypic and genetic heterogeneity.It shows considerable inter and intra familial clinical variation but high penetrance,and it most often presents as an autosomal dominant Mendelian trait.To date,at least 39 mapped loci including 19 causative genes associated with isolated congenital cataracts were reported.These genes can be grouped as crystallins(CRYAA,CRYAB,CRYBA1, CRYBA4,CRYBB1,CRYBB2,CRYBB3,CRYGC,CRYGD,CRYGS),membrane transport proteins(GJA3,GJA8,MIP),transcription factors(HSF4,PITX3,MAF), cytoskeletal proteins(BFSP2) and chromatin modifying protein 4B(CHMP4B).The identification of genes causing inherited cataract will improve our understanding of the mechanisms underlying cataractogenesis and will also provide further insights into development and physiology of normal lens.In this study,we have collected five Chinese families with congenital bilateral cataract and tried to identify the genetic defect leading to the cataract in these families.Norrie disease is a rare X-inked recessive condition characterized by congenital blindness in males due to degenerative and proliferative processes in the neuroretina. The NDP gene is the Norrie disease causative gene.We have collected a four generation Norrie disease family and tried to find the pathogenic mutation in this family.In addition,we planed to perform prenatal diagnosis for a fetus with high risk in this family.Materials and MethodsProbands and family members from 5 Chinese families with congenital cataracts underwent detailed ophthalmic examination.After informd consent was obtained, peripheral blood samples(5 -10ml per individual) were collected from family members (affected and unaffected).Genomic DNA was extracted from peripheral blood lymphocytes using standard phenol/chloroform-proteinase K method.25 of the known ADCC loci was chosen to be screened by linkage analysis using 62 microsatellite markers at chromosome 1p36、1p22.3、1q21-25、2p12、2p24-pter、2q33-36、3p22-24.2、3q21-25、3q27.3、10q25、11q22.1-23.21、12q13-14、13q12.11、14q22-23、15q21-22、16q23.1、16q22.1、17p13.3、17q11-12、19q13.33、20p11.23-p12.1、20p12.1、20q11.22、21q22.3、22q11.2-12.1.These markers were selected and their PCR primers were designed according to the genomic sequences on the UCSC Genome Browser on Human Mar.2006 assembly.PCR were performed using standard conditions.The PCR products were separated by electrophoresis on 8% denaturing polyacrylamide gel and allele fragments were detected by routine silver staining.Two-point linkage LOD scores were calculated using the MLINK program in the LINKAGE software package(version 5.2).Haplotypes were constructed for each family according to the allele information.To detect pathogenic mutations in the gene CRYBB1、CRYBB2、CRYBB3、CRYBA4、HSF4、PTCHD2、KAZRIN、CASP9、SPEN、RSC1A1、PRMD2、PAX6, PCR primers were designed to amplify all coding exons and their flanking intronic sequences.PCR products were examined on 2%agarose gels to confirm the amplification and purified using the TaKaRa Agarose Gel DNA Purification Kit.The purified PCR fragments were submitted for direct automatic sequencing.In family 4,to confirm the mutation in PTCHD2 gene(INV20+5G＞A),the exon 20 of PTCHD2 gene and its flanking intronic sequences were amplified from their genomic DNA of all family members and 268 unrelated normal controls,and the resulted PCR fragments were analyzed by ApalI digestion and 2%agarose gels electrophoresis.The mutation INV20 + 5G＞A will introduce a ApalI restriction site.In order to determine whether this change(INV20 + 5G＞A) affects mRNA splicing, primers were designed to amplify genomic region spanning exon 19 to 21 and the PCR products from a patient were cloned into pcDNA3.1 vector to construct a PTCHD2 minigene expressing vector.COS-7 cell and HeLa cell were cultured and transfected with the vector;then the total RNA was extracted;reverse transcription- PCR was carried out using primer located in exon 19 and exon 21 of PTCHD2 gene respectively.To confirm the c.356G＞A(p.Arg119His) mutation of HSF4 gene in family 5,a mismatch primer was designed to introduce a NsbI restriction site into the normal allele by nested PCR,and the resulted PCR fragments of 188bp were analyzed by NsbI digestion and polyacrylamide gel electrophoresis.In the Chinese family with Norrie disease we collected,the clinical diagnoses were made according to familial history,clinical signs and B ultrasonic examination.After informed consents were obtained,peripheral blood samples were collected from all available family members.Genomic DNA was extracted from peripheral blood lymphocytes using the standard SDS-proteinase K-phenol/chloroform method.The three exons and all intron-exon boundaries of the NDP gene were amplified by PCR using three pairs of primers from the proband and his mother,and subjected to automatic DNA sequence.The causative mutation was confirmed by restriction enzyme analysis and PAGE analysis in all family members.For the fetus pregnant in a mother carring the causative mutation,Genomic DNA was extracted from chorionic villi.Molecular examinations in two aspects were carried out for precise prenatal diagnosis:two-point linkage analysis using microsatillite markers and Restriction analysis.ResultsThe pattern of phenotype transmission in all 5 families with congenital cataract suggested a mode of autosomal dominant inheritance.Of the 5 families,2 were diagnosed as nuclear cataract,2 as posterior polar cataract,and 1 as polymorphic cataract.Microsatellite polymorphic markers 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 1p22.3、1q21-25、2p12、2p24-pter、2q33-36、3p22-24.2、3q21-25、3q27.3、10q25、11q22.1-23.21、12q13-14、13q12.11、14q22-23、15q21-22、16q23.1、17p13.3、17q11-12、19q13.33、20p11.23-p12.1、20p12.1、20q11.22、21q22.3 in each of the 5 families.However,we obtained a maximum lod score of 2.71 atθ=0 with marker D22S689 at chromosome 22q11.2-12.1 in family 3,indicative of linkage.Haplotypes were constructed for the markers analyzed on chromosome 22 and cosegregation was observed in all affected individuals with all markers.It suggested possible involvement of the CRYB gene cluster in the cataractogenesis in the family 3.A maximum LOD score of 3.36 was obtained with marker D1S1151 atθ=0 in family 4 that was the evidence of the linkage.Haplotypes were constructed for the markers analyzed on chromosome 1p36.IndividualⅢ8 was recombinant at marker 1pTTTC andⅢ6 was recombinant at marker 1pCAAA and cosegregation was observed in all affected individuals between marker 1pTTTC and 1pCAAA.The cataract locus was thus mapped to the 7.3cM region bound by 1pTTTC and 1pCAAA,which overlaps with the smallest interval reported so far.A maximum LOD score of 2.63 was obtained with marker D16S3085 atθ=0 in family 5,and haplotype analysis suggested possible involvement of the HSF4 gene in the cataractogenesis in the family 5.Mutation profiling of the candidate genes at the cataract loci was done in the 3 families which had been linked to these loci.In family 3,mutation analysis of CRYBB1,CRYBB2,CRYBB3 and CRYBA4 showed no disease associated changes. While in family 4,DNA sequencing of the 7 candidate genes(PTCHD2,KAZRIN, CASP9,SPEN,RSC1A1,PRMD2,PAX6) revealed a G＞T substitution in intron 20 of PTCHD2(INV20 + 5G＞A).Restriction enzyme analysis showed the same substitution in all affected family members,but not in the 268 normal individuals.RT-PCR analysis of PTCHD2 transcripts showed that the INV20 + 5G＞A substitution does not affect mRNA splicing.Sequencing of HSF4 in family 5 revealed a c.356G＞A change in exon 3 which leads to the substitution of Arg by His at the 119th coden.Restriction enzyme analysis showed cosegregation of the same substitution with all affected family members,but not with the 100 normal individuals.In the family suffering from Norrie disease,sequencing of the affected male proband and his mother revealed a missense mutation in NDP(c.220C＞T,p.Arg74Cys) in both of them,while the proband is a hemizygote for this mutation and his mother is a heterogenous carrier.Restriction enzyme analysis showed 4 female family members (Ⅲ3,Ⅳ3,Ⅲ5 andⅡ5) with normal phenotype also carrying the same mutation. Linkage analysis using microsatillite markers and restriction analysis showed the fetus is a female mutation-carrier. ConclusionsHere we report five Chinese families with congenital cataract,which were inherited in autosomal dominance pattern in these families.Both the phenotypic and the genetic heterogeneity were obvious in these families,since there are 2 families suffering from nuclear cataract,2 families showing posterior polar cataract,and the rest one manifesting the polymorphic cataract;and they were mapped at least 3 different genetic loci.Linkage analysis of family 1 and 2 has excluded all known candidate genes and most of known candidate loci,which indicated that other loci may be responsible for congenital cataract in these two families.In family 3,linkage analysis suggested the phenotype possibly link to chromosome 22q11.2-12.1 and definitely not link with all other loci;however,no disease-associated mutations were detected in all four candidate genes,which indicate the possibility of unknown pathogenic mechanisms including novel genetic locus.In family 4,the linkage analysis proved a definite link between the disease phenotype and the chromosome 1p36 region;our haplotype analysis has narrowed the disease interval into a 2.4 Mb region,which is smaller than the known 7.3 Mb smallest interval in this locus;and sequencing showed no disease-causing mutation in PTCHD2, KAZRIN,CASP9,SPEN,RSC1A1,PRMD2 and PAX6 genes.The cataract phenotype in family 5 was suggested to link to chromosome 16q22.1 and sequencing of HSF4 gene in patients revealed a novel pathogenic mutation, c.356G＞A,which leads to the substitution of Arg by His at the highly conserved 119th coden.In the Chinese family suffering from Norrie disease,we identified a pathogenic mutation in NDP gene,c.220C＞T,the same causative mutation in a foreign Norrie disease patient.This is the first report of mutation detection in a Chinese Norrie disease family.In addition,we performed prenatal diagnosis for a high-risk fetus and found it is a female fetus carring the causative mutation,c.220C＞T.更多还原