【作者】 刘伟；

【导师】 季建；

【作者基本信息】 天津医科大学 ， 眼科学， 2007， 硕士

【摘要】 目的检测成骨不全一家系基因突变及初步探讨其眼部特征方法抽取17名家系成员外周血，提取及鉴定其基因组DNA；选择与已知致病基因紧密连锁的微卫星分子标记，进行等位基因共享分析和排除性分析；利用LINKAGE软件对基因分型结果进行连锁分析，利用Cyrillie 2.0软件进行单体型构建；对候选区域进行基因克隆，PCR扩增，扩增产物进行测序：利用DHPLC对基因突变进行鉴定；对已确认的突变进行生物信息学分析。对家系成员V：3进行详细的眼部检查，并测量部分眼部参数，包括：视力、眼压、眼轴长度、中央角膜厚度、角膜曲率。结果等位基因共享分析将致病基因定位于COL1A1(17q21.3-22.1)，并排除了COL1A2基因；单体型分析显示微卫星分子标记D17S1293，D17S1180，D17S1319，D17S788在家系患者中出现共享；连锁分析显示，在θ=0时，致病基因与微卫星D17S1180，D17S1319紧密连锁(LOD值分别为2.91，2.20)；对COL1A1基因进行测序，发现第2464位碱基(位于36号外显子)由野生型C突变为T(C2464T)，所编码的氨基酸由谷氨酰胺(Gin)变为了终止子(Q644x)；利用DHPLC对该家系其他成员及100例正常人的COL1A1基因36号外显子扩增产物进行鉴定，发现突变仅存在于患者，非患者及100例正常人中未检测到突变。生物信息学分析显示COL1A1基因644位氨基酸位点(谷氨酰胺)位于一个高度保守的区域。临床检查发现家系成员中所有患者巩膜均呈淡蓝色；家系成员V：3的眼部参数测量结果为：视力OD 20／10，OS 20／10；眼压OD 14.7mmHg，OS 18.3mmHg；角膜曲率OD K1 43.3D，K2 43.6D；OS K1 43.0D，K2 44.0D；眼轴长度OD24.03mm，OS 24.23mm；中央角膜厚度OD 434μm，OS 441μm。结论1．COL1A1基因Q644X突变可以导致成骨不全。2．成骨不全患者中央角膜厚度偏薄。更多还原

【Abstract】 ObjectiveTo identify the genetic defect and to analyze the ocular presentation of OsteogenesisImperfecta in a large Chinese family of five generations.MethodsSeventeen members in an Osteogenesis Imperfecta (OI) type I family were recruited.The genomic DNA was isolated and identified from peripheral blood. All themembers were genotyped with microsatellite markers at loci associated with OI.Allele-sharing analysis and exclusive analysis was performed. Haplotype analysiswas performed using the Cyrillic 2.0. A two-point LOD score was calculated usingthe Linkage package, A mutation was detected by direct sequencing. The mutationwas confirmed by DHPLC. The bioinformatic analysis was performed about themutation.ResultsAllele-sharing analysis confirmed the linkage of the disease in the family withCOL1A1 and no linkage with COLA2. Haplotype analysis showed that all theaffected individuals shared a common haplotype with markers D17S1293, D17S1180,D17S1319 and D17S788 at 17q11. 2-22. Significant evidence of linkage wasobserved with microsatellite markers D17S1180 (LOD score [Z]=2.91, atrecombination fraction [0]=0.0) and D17S1319 (Z=2.20, at 0=0.0), respectively.Direct cycle sequencing of the amplified fragments of COLIA1 identified a singlebase alteration of C2464T in exon 36 of COL1A1, which resulted in a substitution of Gin at codon 644 to a stop codon (Q644X). Denaturing HPLC analysis confirmedthis mutation, which co-segregated with all affected individuals in the family, andwas not observed in any of the unaffected family members or 100 normal controls.Bioinformatic analysis showed that Q644 is located in a highly conserved region.The sclerae of all the affected individuals in the family have been blue since theywere born. The proband (V:3) was a 14-year-old boy with blue sclerae. Hisuncorrected visual acuity was: OD, 20/10; OS, 20/10. The IOP (Goldmann tonometry)were: OD, 14.7mmHg; OS, 18.3mmHg. The corneal curvatures were: OD, K1 43.3,K2 43.6; OS, K1 43.0, K2 44.0. The ocular axis lengths were normal: OD, 24.03mm;OS, 24.23mm, and his CCT were low: OD, 434μm; OS, 441μm.Conclusions1. This study is the first report that OI is associated with the mutation of Q644X ofCOL1A1.2. The CCT of the OI patients are lower than normal.更多还原