哮喘易感基因的克隆及两个单基因遗传性疾病的遗传学分析研究

【作者】 章海兵；

【导师】 孔祥银；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2005， 博士

【摘要】 哮喘是一种多基因遗传性疾病,是由一些基因与环境因素共同作用而致病,其遗传度为7O～8O%。不同基因一基因和基因一环境相互作用导致哮喘发生。随着人类基因组学与分子生物学技术的发展,哮喘的遗传学研究已成为国际热点。迄今为止,至少有22个分布于15条常染色体的不同位点被不同的研究组证明与哮喘或及相关性状连锁,由于细胞因子在哮喘炎症的触发和炎症持续的过程中起到非常重要的作用,而许多编码炎症细胞因子的基因位于染色体5q31-33区域,因此细胞因子基因是倍受关注的候选基因.我们根据对哮喘发生的分子机制的研究分析,选择了IL13与IL4为关键作用因子的分子免疫通路中的细胞因子基因作为研究对象,我们首先确定了位于染色体5q31-33区域的IL13,IL4,IL5,IL9,IL3,CD14,IL12B基因,以及位于染色体16p12区域的IL4和IL13基因的共同受体IL4R基因作为我们进行哮喘相关性研究的候选基因,我们随机选择了14个哮喘病人样本和10正常人样本进行多态性位点的检测,通过对上述基因的编码区以及转录起始位点上游的1500bp的DNA序列进行直接测序,发现多态性位点,然后利用高通量SNP检测技术Pyrosequence以及直接测序相结合的方法对大样本量进行逐个多态位点的基因分型,我们共发现42个SNP位点,其中15个是NCBI中尚未报道的,我们选择了30个杂合率较高的SNP对全部样本进行了与哮喘疾病的关联分析及单倍型分析,所有SNP位点的基因型分布均符合Hardy-Weinberg平衡,我们对所有SNP位点与疾病关联分析结果显示, IL4R基因中的SNP18685711、SNP18686951、SNP18686994、SNP18687043、SNP18687051、SNP18687259六个位点与疾病存在显著相关性(P<0.05),连锁不平衡分析它们处在同一单倍型结构域中Block(D’=1),其中三个SNP位点改变氨基酸序列,对其进一步进行蛋白二级结构预测显示这些位点一定程度上影响了蛋白的二级结构从而导致功能的改变,本实验首次在中国人群中系统性的对染色体5q31-33区域的细胞因子IL13,IL4,IL5,IL9,IL3,CD14,IL12B基因及IL4R基因的SNP位点与哮喘的相关性进行了检验,证明了IL4R基因中的六个SNP位点与哮喘易感性具有相关性,这六个位点通过改变蛋白序列影响蛋白结构和功能,从而增加了哮喘的易感性。石骨症是遗传性破骨细胞功能障碍引起的骨放射密度显著增高的骨代谢疾病。石骨症遗传异质性明显,在不同家族之间,甚至同一家族的不同成员之间表型可以有显著差异。临床上石骨症分为三型:常染色体隐性遗传的恶性婴儿型石骨症,患儿通常在5岁以前死亡;常染色体隐性遗传的中间型石骨症,通常在10岁以前发病,所有患儿都可以存活至成年;常染色体显性遗传的成人型石骨症,发病年龄不限,对生命无威胁。本研究报道的家系是陕西一个遗传了4代的石骨症家系,属常染色体显性遗传II型,但稍具有不同于以往国内外报道的临床表型。我们经过对该家系进行连锁分析,将致病基因定位到遗传标记D16SA和D16SD之间,这个位点与Benichou通过全基因组扫描的一个位点即16P13.3重合。最近,Cleiren等在12个ADOP-II型家系中找到了ClCN7的突变,而ClCN7基因在我们定位范围之内,因此我们对ClCN7基因进行了突变检测,但是没有发现与疾病一致的突变,由于石骨症遗传异质性非常复杂,而且该家系患者有不同于以往国内外报道的临床表型,因此很可能是该区域其他基因的突变导致了该类型常染色体显性石骨症发生.瘢痕疙瘩是皮肤损伤后引发的胶原异常积聚导致的瘢痕样病变。该疾病的遗传方式及遗传因素比较复杂,而且遗传性大家系非常罕见。近年来,研究者确认遗传性瘢痕疙瘩症的遗传模式符合常染色体显性遗传,并伴有不完全显性和表型差异, Alexander等通过对两个瘢痕疙瘩家系进行全基因组扫描定位研究,分别将两个家系定位于D2S410和D2S1353之间40cm的区域和D7S678和D7S494之间16cM的区域内,这是迄今为止国际上对遗传性瘢痕疙瘩唯一的定位报道.我们利用一个采集于内蒙古赤峰的家系对这两个致病位点进行了连锁分析,结果显示该家系的致病基因与2q23和7p11位点上的微卫星标记不具有紧密连锁关系,说明对于遗传性瘢痕疙瘩还存在不同于国外报道的第三个致病位点,同时对遗传性瘢痕疙瘩症符合常染色体显性遗传以及具有遗传异质性提供了更为丰富的证据,我们需要收集其他家系或扩大所研究家系的样本以进行进一步的研究,通过克隆瘢痕疙瘩症的致病基因最终阐明瘢痕疙瘩形成的分子机制.更多还原

【Abstract】 Asthma has become the most common chronic childhood disease in developed nations, affecting more than 155 million individuals. Both, gene–gene as well as gene–environment interactions contribute to its overall phenotype. The development of asthma appears to be determined by the interaction between host susceptibility (genetics) and a variety of environmental exposures. Numerous genetic studies have mapped an asthma susceptibility genes to a region on chromosome 5q31-q33 in several populations. This region contains a cluster of proinflammatory cytokines important in immune regulation including the genes encoding the T helper 2-type cytokines (the interleukin genes IL3, IL4, IL5, IL9, and IL13),Two members of this cluster, IL4 and IL13, have been both genetically and functionally implicated in the pathogenesis of asthma. The increasing knowledge of the human genome and the large number of SNPs that are becoming available as well as improved technology for genotyping make large scale association studies possible. In an effort to discover additional polymorphisms in genes whose variants have been implicated in asthma, we scrutinized the genetic polymorphisms in IL13, IL4, IL4R,IL3, IL9, CD14, IL5, IL12B genes to evaluate it as potent candidate genes for asthma host genetic study. We performed extensive screening of these genes by direct sequencing to detect polymorphisms and statistical analysis to examine the genetic effects on asthma.Here, we present thirty genetic polymorphisms found in these genes and the results of an association study in Chinese population. Our genetic association analysis of polymorphisms revealed that six polymorphisms in IL4R gene, three of which resulted in an amino-acid change, showed significant association with the risk of asthma (P=0.0002). In Chinese population, these six polymorphisms segregated in strong linkage disequilibrium. This information about the genetic association of important genes with asthma might provide valuable insights into strategies for the pathogenesis of asthma.Osteopetrosis is an inherited skeletal condition characterized by increased bone radiodensity. There are three clinical groups: infantile-malignant autosomal recessive, fatal within the first few years of life; intermediate autosomal recessive, appears during the first decade of life but does not follow a malignant course; and autosomal dominant, with full-life expectancy but many orthopaedic problems.Here, we identified and analysed a four-generation family affected with ADO type II. We used linkage analysis strategy to map the disease locus and found a linkage between markers on 16q13 and the ADO type II phenotype. According to previous reports, Benichou used a whole genome scan strategy to map the disease locus on the same region 16q13-22. Following the assignment of the gene causing ADO type II to chromosome 16p13.3, Wim Van Hul report seven different mutations in the gene encoding the CICN7 chloride channel in all 12 ADO type II families analysed. For excluding the possibility that the mutation segregating in this family was in the CICN7 gene we have directly sequence the CICN7 gene in two affected members of this family, no mutation were identified. These results suggested there is other gene located on this region responsible for this disorder in this family.Keloid formation is a common scarring disorder that can occur with an autosomal dominant inheritance pattern but incomplete clinical penetrance and variable expression.in some families. A.lexander performed a genome-wide linkage search for genes predisposing to keloid formation in two large families, and identified keloid susceptibility loci on chromosomes 2q23 and 7p11.This is a only report of gene locus for familial keloid formation so far.We have collected one family with a high occurrence of keloids, excluded these two keloids susceptibility loci on chromosomes 2q23 and 7p11 by linkage analysis and haplotype analysis. Based on the high occurrence of keloid in the African -American population and the observed variation in severity of keloid formation, it is likely that additional loci for keloids exist in Chinese population. Despite detailed histological and biochemical analyses of keloid tissue and keloid fibroblasts in culture, the causes for keloid formation remain unknown. Identifying these genes will lead to a better understanding of the biological mechanisms that regulate scarring.更多还原