【作者】 赵晨；

【导师】 汤欣；

【作者基本信息】 天津医科大学 ， 眼科学， 2007， 博士

【摘要】 一个新的视网膜色素变性的连锁位点RP33，以及RP11连锁家系中PRPF31基因的完整突变筛查研究目的：分析两个常染色体显性视网膜色素变性(autosomal dominant retinitis pigmentosa，adRP)家系AA、AB的临床表型特征并研究其遗传学病因。通过全基因组连锁分析首先进行家系疾病的遗传学定位，然后在连锁区内进行候选基因突变筛查。1．研究两个adRP家系AA、AB的临床表型特征。2．通过全基因组扫描连锁分析确定两个adRP家系致病基因的染色体定位。3．通过基因序列分析对连锁区内一系列可能的候选基因进行突变筛查，寻找致病基因。4．通过新技术多重连接依赖探针扩增(Mutiplex Ligation-Dependent Probe Amplication，MLPA)反应和单核苷酸多态性(Single nucleotide polymorphism，SNPs)检测对RP11连锁家系AB进行候选基因PRPF31基因巨大缺失突变筛查。研究方法：1．临床研究方法：1．1眼科常规检查包括询问病史，屈光状态、眼前节裂隙灯检查，直接和间接眼底镜检查以及眼底照相等。1．2特殊检查方法包括视野检查(Humphrey 750)，明、暗适应视网膜电图(electroretinogram，ERG)以及多焦视网膜电图(multifocal ERG)检查等。2．分子遗传学研究方法：2．1取家系成员外周静脉血5～8ml，采用Roche Biochemical公司的DNA分离试剂盒提取基因组DNA。2．2遗传连锁分析2．2．1全基因组扫描连锁分析在全部常染色体范围内选取370对荧光微卫星标记物进行全基因组扫描，相邻微卫星标记物之间的平均距离为10厘摩(Centimorgan，cM)。利用聚合酶链反应(Polymerase Chain Reaction，PCR)扩增全部370对荧光微卫星标记物。利用ABI 377XL DNA测序仪读取370对微卫星标记物等位基因片段大小并利用Genescan 3.1和Genotyper 2.0软件进行单体型分析。运用Sim Walk2 2.89，Version 3.35软件计算计算疾病表型与多个微卫星位点之间的多点最大优势对数LOD值。2．2．2精确连锁定位在通过全基因组连锁分析取得疾病基因在染色体上的初步定位以后，在取得最大正数LOD值的初步定位区域内进一步选取荧光标记微卫星标记物，以缩小并精确连锁区域临界范围。标记物之间的平均距离为1cM。2．3基因序列分析根据连锁分析的结果，分别在AA和AB两个家系所定位的染色体候选区内选取侯选基因，并用ABI 3730 Genetic Analyzer对侯选基因进行直接基因序列分析。2．4 PRPF31基因的巨大突变筛查在AB家系中对PRPF31基因进行SNPs和MLPA检测以筛查潜在的巨大基因缺失突变即基因半合子状态(hemizygousity)。研究结果：1．AA家系临床上，AA家系中的患者以夜盲起病，发病年龄较晚，表现为进行性视力下降和周边视野缺损。RP临床表现在家系患者之间甚至同一患者的双眼之间均具有较大的变异，致病基因外显不全。通过全基因组连锁分析在排除了所有已知adRP连锁位点之后，在2号染色体长臂上确定了一个新的adRP连锁位点RP33(OMIM％610395)。在微卫星标记物D2S2222取得最大多点LOD值4.69，通过家系中的两个正常同胞身上发生的重组(recombinant)可将RP33位点定位于微卫星标记D2S2159和D2S1343之间约4.8cM的区域(染色体带2cen-q12.1)内。对RP33区域中的候选基因SEMA4C，CNGA3和HNK1ST以及紧邻RP33区域的一个已知arRP相关基因MERTK进行序列分析未发现任何致病突变。2．AB家系AB家系在临床上属弥漫型RP表型且具有较高外显率。此外，家系中有13名患者还同时具有皮质型白内障表型。通过全基因组连锁分析和进一步的精确定位将此家系致病基因定位于染色体区19q13.4(RP11)(OMIM #600138)，微卫星标记物D19S924和D19S880之间约5.71cM区域内。对连锁区内的已知adRP基因PRPF31进行完整的突变筛查基本排除了PRPF31全部阅读框架范围内的突变。进一步在连锁区域内选取其它候选基因PRKCG、TFPT、TSEN34、RDH13和U2AF2进行直接测序未发现致病性突变。结论：1．AA和AB家系属于常染色体显性遗传视网膜色素变性家系。2．通过AA家系定位了一个新的adRP连锁位点RP33于染色体区域2cen-q12.1，这是首个由中国报道的RP新位点。SEMA4C、CNGA3、HNK1ST和MERTK基因均不是AA家系的致病基因，在RP33区域内存在的新adRP致病基因还有待发现。3．AB家系连锁于染色体区域19q13.4(RP11位点)。RP11基因PRPF31不是AB家系的致病基因。在连锁区域19q13.4内，除了PRPF31基因之外，我们推测还存在另一个新的adRP相关致病基因导致了AB家系中弥漫型RP表现且可能还合并有外显不全的白内障表型。更多还原

【Abstract】 RP33. a novel locus of autosomal dominant retinitis pigmentosa；and a complete mutation screening of PRPF31 gene in RP11 linked familyObjectiveTo study the clinical manifestations of two Chinese families with autosomal dominant retinitis pigmentosa(adRP)and investigate their genetic pathogenesis．A genome wide linkage analysis was applied and followed by a complete mutation screening of candidate genes．1. To clinically characterize two families with adRP(kindred AA and AB)．2. To identify the chromosomal locations of the disease loci in the two adRP families by genome-wide linkage screening．3．To search the Causative mutation of cardidate genes within linked regions in the two families respectively．4．To screen any substantial big deletion mutation in PRPF31 gene in family AB by mutiplex ligation-dependent probe amplication (MLPA) reaction and single nucleotide polymorphism(SNPs)testing．Methods：1. Clinical investigation1.1 Routine ophthalmologic examinationsOphthalmologic examinations including disease history, best correct visual acuity, slit-lamp examinations, direct funduscopy and photographphic fundus．1.2 Electrophysiological examinations Humphrey threshold perimetry (Humphrey 750), full field electroretinogram (ERG) and multifocal ERG (mfERG) were performed in all patients of the two families.2. Molecular genetic study2.1 Human genomic DNA was extracted from peripheral blood leukocytes using DNA Isolation Kits for Mammalian Blood (Roche Biochemical, Inc.).2.2 Linkage analysis2.2.1 Genome-wide linkage analysisA genome-wide linkage screening was conducted by genotyping 370 microsatellite markers representing all autosomes at an average resolution of approximately 10 cM (Weberset 6.0; Research Genetics). The polymerase chain reactions (PCR) were carried out to amplify all 370 markers. The PCR products were appropriately pooled according to allele sizes and labelling, and were run in ABI 377XL for fluorescent detection. Linkage analyses were carried out by calculating multipoint LOD scores using the LINKAGE software package of SimWalk2 2.89, Version 3.35.2.2.2 Fine mappingBased upon the genome-wide linkage analysis, more fluorescent microsatellite markers located within the initial linked regions were genotyped to refine the critical interval.2.3 Sequence analysisCandidate genes were selected from linked regions of the two families respectively and direct sequencing was performed using ABI 3730 Genetic Analyzer to evaluate all the exons and flanking introns sequences of the candidate genes.2.4 Detection for big deletion in PRPF31 geneSNPs testing and MLPA reaction were applied in family AB to detect any substantial big deletions (hemizygousity) ofPRPF31 gene.Result: 1. AA familyClinically, AA family presented relatively late onset of night blindness, gradually decreased visual acuity and progressive loss of peripheral visual field. Variable clinical features and incomplete penetrance were found in the affected members of AA family.After excluding all known adRP loci by genome-wide linkage analysis, a novel disease locus RP33 (OMIM %610395) was assigned to the long arm of chromosome2. From meiotic recombinations in two unaffected members, RP33 was further refined to a 4.8 cM (9.5 Mb) interval flanked by D2S2159 and D2S1343 in chromosomal region 2cen-q12.1.No disease-associated mutations were detected in the candidate genes SEMA4C,CNGA3 or HNKlST from within RP33 region. MERTK, a known disease gene for autosomal recessive RP located close to RP33 was similarly excluded.2. AB familyAB family presented a form of diffuse adRP phenotype with high penetrance. In addition, cortex cataract was also observed in 13 affected members with adRP phenotype.AdRP in family AB was mapped to chromosomal region 19q13.4 (RP11 locus, OMIM #600138) , a 5.71 cM interval flanked by microsatellite markers D19S924 and D19S880. A maximum multi-point LOD score of 7.46 was reached at marker D19S927.After a relatively complete mutation screening throughout the open reading frame of PRPF31 gene, it has been excluded that the PRPF31 gene is the causative gene of AB family. No disease-associated mutations were detected in other candidate genes PRKCG, TFPT, TSEN34 or RDH13 from within the linked region. U2AF2, an essential pre-mRNA splicing factor coding gene located close to the critical interval was similar excluded.Conclusions: 1. Pedigrees analysis demonstrated that the RP conditions in kindred AA and AB are both inherited as an autosomal dominant (adRP) trait.2. A novel adRP locus RP33 (OMIM %610395) was assigned to chromosomal region 2cen-q12.1 in AA family. It’s the first novel RP locus reported by China. SEMA4C, CNGA3, HNK1ST and MERTK genes are not the causative gene in AA family. There is a new gene in RP33 region which is responsible for adRP phenotype.3. Family AB was linked to chromosomal region 19q13.4 (RP11 locus). PRPF31 gene is not the causative gene for the adRP phynotype in AB family. In addition to PRPF31, there may be a new adRP related gene in RP11 locus, which caused the diffuse form of adRP phenotype in AB family, or might be also causative for the cataract phenotype.更多还原