一残毁性掌跖角皮症家系致病基因突变检测

发布时间：2018-08-29 10:47

【摘要】：研究背景残毁性掌跖角皮症(Mutilanting Palmoplantar Keratoderma)又称残毁性遗传性角质瘤(keratoma hereditaria mutilans),于1929年由德国学者Vohwinkel首先描述并命名,因此该病又被称为沃温克耳综合症(Vohwinkel syndrome, VS, OMIM124500)。它是一种罕见的常染色体显性遗传病,多呈家族性发病,同时也有散发病例的报道。该病好发于白种人女性,多于婴幼儿起病,病程呈进行性发展。其典型的临床表现包括：对称性弥漫性掌跖角化,掌跖表面呈蜂窝状小凹馅；手足背部及肘膝关节伸侧等可呈典型的海星状或条状角化过度斑块；远端指(趾)间关节呈环形缩窄并逐渐断离(假阿洪病样改变)。目前已定位克隆鉴定出两个与残毁性掌跖角化病有关的致病基因：分别是定位在13号染色体13q11-12上编码缝隙连接蛋白Cx26的GJB2基因和定位在1号染色体1q21上编码兜甲蛋白的LOR基因。由GJB2基因突变导致的残毁性掌跖角化病常伴有听力异常,被称为典型的残毁性掌跖角化病；由LOR基因突变导致的残毁性掌跖角化病常伴有鱼鳞病,又被称为变异型残毁性掌跖角化病。临床上我们收集到一残毁性掌跖角化病伴有感音神经性耳聋的家系,并对该家系进行了致病基因的研究。据我们所知,本研究为国内首次对该病进行致病基因突变检测。目的对一个中国汉族残毁性掌跖角化病家系进行致病基因突变检测,以明确其致病基因及突变类型,以期进一步明确临床表型与基因型的关系。方法收集该家系5例患者、12例正常人和100例健康正常对照外周血标本和家系患者临床资料。用外周血DNA提取试剂盒提取外周血基因组DNA,采用聚合酶链反应(PCR)扩增候选基因GJB2基因的整个编码序列和侧翼序列在内的1015bp,扩增产物纯化后应用ABI PRISM3730XL自动测序仪直接双向测序,Sequencher4.10.1Demo软件与基因组序列进行比对分析,以明确具体的突变位置和突变方式。结果PCR产物测序结果显示：该家系所有患者GJB2基因均存在一个杂合错义突变196G→C,导致第一细胞外区域(E1)第66位天冬氨酸被组氨酸替代(即D66H),而家系中12例正常人和100例非家系成员正常对照的DNA测序结果均未发现此突变。反向测序结果亦证实该突变。结论该家系所有患者均存在GJB2基因突变(D66H),此基因为已知的残毁性掌跖角皮症致病基因,实验数据表明GJB2基因中D66H错义突变也是中国汉族人群中残毁性掌跖角化伴耳聋的致病原因之一,可以通过基因诊断和产前诊断阻止致病基因的传递。
[Abstract]:Background residual palmoplantar keratoderma (Mutilanting Palmoplantar Keratoderma), also known as residual hereditary keratinoma (keratoma hereditaria mutilans), was first described and named by German scholar Vohwinkel in 1929, so it is also known as Wawenkel syndrome (Vohwinkel syndrome, VS, OMIM124500). It is a rare autosomal dominant hereditary disease with familial and sporadic cases. The disease is more common in Caucasian women than in infants. The course of disease is progressive. The typical clinical manifestations include symmetrical diffuse palmar metatarsal keratosis with honeycombed concave filling on the palmar metatarsal surface typical starfish or striped hyperkeratosis on the back of the hand and foot and extension of the elbow and knee joint etc. The distal interphalangeal joint was circumferential constricted and gradually disconnected (pseudahun disease). Two pathogenetic genes related to residual palmoplantar keratosis have been identified by location-specific cloning: the GJB2 gene which encodes gap junction protein Cx26 on chromosome 13 13q11-12 and the LOR gene which is located on chromosome 1 1q21. The residual palmoplantar keratosis caused by GJB2 gene mutation is often accompanied by hearing abnormality, which is called typical residual palmoplantar keratosis, and the residual palmoplantar keratosis caused by LOR gene mutation is often accompanied by ichthyosis. It is also called variant residual palmoplantar keratosis. A family with residual palmoplantar keratosis associated with sensorineural hearing loss was collected and studied. As far as we know, this study is the first time to detect the pathogenic gene mutation in China. Objective to detect the pathogenetic gene mutation in a Chinese Han Chinese family with residual palmoplantar keratosis in order to clarify the relationship between clinical phenotype and genotype. Methods the clinical data of peripheral blood samples of 5 patients and 100 healthy controls were collected. The genomic DNA, of peripheral blood was extracted by peripheral blood DNA extraction kit. The whole coding sequence and flanking sequence of candidate gene GJB2 gene were amplified by polymerase chain reaction (PCR). The amplified products were purified by ABI PRISM3730XL automatic sequencing apparatus. Sequencher 4.10.1 Demo software was used to analyze the genome sequence. In order to identify the specific mutation location and mutation mode. Results the sequencing of PCR products showed that the GJB2 gene of all patients in this family had a heterozygous mutation 196G / C, leading to the replacement of the 66th position aspartic acid (D 66H) in the first extracellular region (E1) by histidine (D 66H), while 12 cases in the family were normal. This mutation was not found in the DNA sequencing of human and 100 non-pedigree normal controls. Reverse sequencing also confirmed the mutation. Conclusion GJB2 gene mutation (D66H) is present in all patients in this family, which is due to the known pathogenetic gene of residual palmoplantar keratoderma. Experimental data show that D66H missense mutation in GJB2 gene is also one of the causes of deafness in Chinese Han population. It can prevent the transmission of pathogenic gene through gene diagnosis and prenatal diagnosis.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R758.5

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