【作者】 王旭；

【导师】 王擎； 刘木根；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 基因最重要的特征是其从亲代到子代高度保守的复制能力,从而保证物种遗传的稳定性。但在这一复制过程中,有时会发生错误,导致突变的发生。突变可以是大型突变,如染色体变异(chromosomal anomalies),包括染色体数目或结构的变化(易位,反转,三体等),大片段DNA缺失,重复,插入等。突变也可以是比较小的基因突变,包括点突变,微小缺失,插入等。许多突变由于其所处的位置重要,对重要人体发育和生理功能起关键作用的基因的表达和功能有影响,产生的结果往往直接影响人类健康,导致各种各样人类疾病。本课题对两种单基因显性遗传病家系进行分子遗传研究,涉及家系分析,致病基因的定位,克隆,和基因突变分析。第一研究项目是关于指关节粘连(Proximal symphalangism, SYM1)致病基因的定位及突变检测。指关节粘连是一种常染色体显性遗传病,其特征是近端指关节强直,腕骨和跗骨骨性融合。我们确诊了两个具有SYM1症状的五代中国家系。对这两个家系的全基因组扫描表明该致病基因与微卫星标记D20S112显著连锁,LOD值为4.32(LOD值≧3支持连锁)。通过DNA测序,在两个家系中均在GDF5基因(编码骨形态发生蛋白家族的成员)上发现了一个新的E491K杂合突变。E491K突变位于GDF5的TGF-β功能域的高度保守区。该突变在这两个家系中与患者共分离,而且在200个正常人中没有出现过。本研究中两个家系的临床症状与NOG基因上的一些突变导致的SYM1以及GDF5基因上R438L突变导致的SYM1具有一些共同特征(例如2-4手指指关节的骨性融合)。但是,他们同时也表现出了一些独特的临床表型,包括骰骨缺失,第一和第五掌骨正常,以及扁平足。E491K这一突变是目前发现的GDF5基因上的导致SYM1的第二个突变,也是中国人群中发现的第一个GDF5突变。这些结果表明在中国人群中,GDF5的突变可以导致SYM1。该研究丰富了GDF5基因突变导致的临床症状。第二研究项目是关于短指A1(Brachydactyly type A1, BDA1)致病基因的定位及突变检测。短指A1是由位于染色体2q35-36的Indian hedgehog gene (IHH)突变引起的。本研究中,我们确诊了一个中国短指A1的5代大家系。所有的家族内成员临床表型相似,同时也表现出了一些与已经报道IHH基因上的突变引起的短指A1临床症状明显不同的地方。连锁分析表明该家系致病基因与微卫星标记D2S126显著连锁,LOD值为4.74。DNA测序分析发现了一个新的杂合突变,该突变位于第461碱基处,导致苏氨酸被异亮氨酸取代(T154I)。T154I在这个中国家系中与患者共分离,而且在200个正常人中没有出现过。这些结果表明该研究中发现的突变是引起短指A1的一个新突变。该研究丰富了IHH基因突变导致的临床症状和IHH基因的突变谱。更多还原

【Abstract】 One of the most important features of a gene is its accurate replication when transmitted from one generation to the next. However, during the replication process, errors occasionally occur and lead to the generation of mutations. Some mutations are gross changes, including chromosomal anomalies (changes of the number or structure of chromosomes; for example, translocations, inversions, aneuploidy, deletions, duplications) and deletions, insertions, and duplications of large DNA fragments. Some mutations are small changes within or around genes, including point mutations, small deletions, and insertions. Mutations may affect the function and expression of critical genes that are important for the normal human development and physiology, which will cause various human diseases. Recent development of human genetic technologies has allowed the identification of disease genes and mutations based on their chromosomal locations (positional cloning). The current study used the human genetic approach to characterize three Chinese families with autosomal dominant diseases affecting the bone system. This thsis consists of two major sections:First, to identify a novel disease-causing mutation in GDF5 in two Chinese families affected with proximal symphalangism (SYM1). SYM1 is an autosomal dominant disorder characterized by ankylosis of the proximal interphalangeal joints and fusion of carpal and tarsal bones. We identified and characterized two five-generation Chinese families with SYM1. The two families share some similarities (e.g. osseous fusion of interphalangeal joints of the 2-4 fingers) with SYM1 families with mutations in the NOG gene or the family with mutation R438L recently reported in the GDF5 gene (encoding a bone morphogenetic protein family member). However, they show some unique features including the absence of cuboid bone, the lack of shortness of the first and fifth metacarpal bones, and manifestation of flat feet. Genome-wide linkage analysis of the two families linked the disease gene to marker D20S112 with a combined LOD score of 4.32. Mutational analysis revealed a novel E491K mutation in the GDF5 gene in both families. The mutation occurs at a highly conserved residue in the TGF-βdomain of GDF5 and represents the second GDF5 gene mutation identified for SYM1 to date. The E491K mutation co-segregated with the affected individuals in the two families, and did not exist in unaffected family members or 200 normal controls. These results indicate that defects in GDF5 can cause SYM1 in the Chinese population, and expand the spectrum of clinical phenotypes associated with mutant GDF5.Second, to identify a novel mutation in IHH that caused brachydactyly type A1 (BDA1). BDA1 is caused by mutations in the Indian hedgehog gene, IHH on chromosome 2q35-36. In this study, a large five-generation Chinese family with BDA1 was identified and characterized. All affected family members demonstrated significant intrafamilial homogeneous phenotype and some unique clinical features different from those associated with the reported BDA1 mutations in IHH. Linkage analysis showed that the causative mutation in the family was linked to marker D2S126 close to IHH with a LOD score of 4.74 at a recombination fraction of 0. DNA sequence analysis revealed a heterozygous C to T transition at nucleotide 461 of IHH, resulting in a novel T154I substitution. The T154I mutation co-segregated with all affected individuals in the family, and was not present in normal family members or 200 normal controls. These results identify a novel IHH mutation associated with BDA1 and expand the spectrum of clinical phenotype associated with IHH mutations.更多还原