【作者】 公小蕾；

【导师】 赵家良； 瞧瑞芳；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 【目的】应用连锁分析法对3个具有X连锁隐性遗传特点的中国人视网膜色素变性家系WYS、WXM、YYZ进行基因定位。从定位的染色体区域寻找候选基因,并对候选基因进行患病群体基因突变筛查。【材料和方法】1.收集门诊病例,通过家系调查、患者病史、眼科常规检查以及对部分受试者进行ERG检查确定了三个X连锁隐性遗传视网膜色素变性家系——WYS家系、WXM家系和YYZ家系。2.抽取WYS、WXM和YYZ家系成员的静脉血并提取DNA;选取分布在RPGR、RP2基因之间约20厘摩（centimorgan,cM）范围内的微卫星标记;利用PCR反应对微卫星位点进行扩增,通过聚丙烯酰胺变性凝胶电泳和银染法确定单倍体型;利用两点法计算微卫星位点与致病基因之间的LOD值。3.根据RPGR基因ORF15突变热区和RP2基因外显子DNA序列合成12对引物,以WYS家系基因组DNA为模板,PeR扩增出RPGR基因ORF15突变热区片断;以WXM、YYZ家系基因组DNA为模板,PCR扩增出RPGR基因ORF15突变热区片断以及包含RP2基因所有外显子的6个片段。扩增产物纯化后直接测序。4.通过比较病人和正常人相应的DNA序列,检测基因突变位点。对WYS家系进行RPGR基因序列分析;对WXM、YYZ家系进行RPGR、RP2基因序列分析。对发现突变的家系进一步行整个家系所有个体的突变筛查以及疾病无关个体的群体筛查。【结果】确定三个家系均为视网膜色素变性X连锁隐性遗传方式。根据计算而得的LOD值判断三个家系均不能排除与RPGR基因和RP2基因连锁的可能。在WYS家系首次检测到了RPGR基因上致病基因突变c.2336₂337insC,突变位于RPGR基因ORF15突变热区,为碱基插入的移码突变,造成翻译水平上多肽链氨基酸种类和顺序改变,蛋白质结构发生变化,引起发病。在WXM家系首次检测到了RP2基因上致病突变c.385 T＞A,c.392 G＞T,突变位于RP2基因的第2外显子,均为错义突变,造成氨基酸编码错误,引起发病。YYZ家系检测到以报道突变RPGR基因c.2233₂234delAG,碱基缺失造成移码突变,突变位于RPGR基因ORF15突变热区。【结论】首次检测到RPGR基因上致病基因突变c.2336₂337insC,RP2基因上致病突变c.385 T＞A,c.392 G＞T。发现RPGR基因上已报道突变c.2233₂234delAG。更多还原

【Abstract】 ObjectiveTo analyze the linkage status of three Chinese families with X-linked recessive retinitis pigmentosa with the known loci.To find the candidate genes and perform genetic study and mutation screening of these families.Materials and Methods1.Clinic cases were collected and three X-linked recessive retinitis pigmentosa families were identified by family survey,past medical history,ophthalmologic examinations,combined with ERG if necessary.2.Venous blood was collected from research subjects and genomic DNA were extracted.Several STRs ranging from RPGR gene to RP2 gene about 20 cM long were labeled and then amplified by PCR.The haplotype was then identified by SDS-PAGE and argentation.Linkage package was used to calculate LOD scores.3.Twelve pairs of primers designed from hot spot ORF15 of RPGR gene and exon sequences of RP2 gene were used to amplify all these exons.PCR were carried out with human genomic DNA as the template.The PCR products were sequenced after being purified.4.Mutation was identified by comparing DNA sequences of patients with those of normal controls.If there was any change in the family genome, the DNA sequences of all the family members were analyzed and colony screening was performed.ResultsThe genetic pattern of the three families was confirmed as X-linked recessive.There were possibilities that all three families were linked to RPGR and RP2 gene.Mutation c.2336₂337insC was detected on hot spot ORF15 of gene RPGR in family WYS.It was a single insertion which led to frameshift,resulting in structural changes of its coding protein and subsequent onset of retinitis pigmentosa.Mutation c.385 T＞A,c.392 G＞T were detected in the 2^nd exon of gene RP2 in family WXM.They changed the coding amino acids of the spots and induced the onset of retinitis pigmentosa.Mutation c.2233₂234delAG was detected on hot spot ORF15 of gene RPGR in family YYZ.ConclusionNovel mutations of c.2336₂337insC and c.2233₂234delAG on gene RPGR, and novel mutations of c.385 T＞A and c.392 G＞T on gene RP2 were identified,among which the mutation of c.2233₂234delAG on gene RPGR has already been reported.更多还原