【作者】 汤占利；

【导师】 李春阳；

【作者基本信息】 山东大学 ， 皮肤病与性病学， 2011， 博士

【摘要】 研究背景大疱性表皮松解症(epidermolysis bullosa, EB)是表皮与真皮分离的在临床上有多种表现的一组遗传性皮肤病的总称,根据超微结构显示组织分裂的准确位置,EB可分为四种主要类型：单纯型EB(epidermolysis bullosa simplex, EBS);交界型EB(junctional epidermolysis bullosa, JEB);营养不良型EB(dystrophic epidermolysis bullosa, DEB)和Kindler综合征(Kindler syndrome)。EBS:裂隙发生在基底的角质形成细胞内,是角蛋白5、14或plectin蛋白异常所致；JEB：裂隙主要发生在透明板,是lamina-332,整联蛋白a 6β4或ⅩⅦ型胶原异常所致；DEB：裂隙发生在致密板下锚丝纤维水平,是Ⅶ型胶原异常所致；而Kindler综合征患者的裂隙可以发生于表真皮交界处的任何结构部位,主要由于粘着斑相关蛋白Fermitin家族同源物1蛋白异常所导致。DEB的表皮松解部位发生于致密板下带,根据遗传方式的不同可分为常染色体显性(DDEB)和常染色体隐性遗传(RDEB)两种类型。近年来随着分子生物学技术的发展研究表明DEB是由位于基底膜致密板下带锚丝纤维的主要组成部分---Ⅶ型胶原基因COL7A1突变导致。痒疹样营养不良型大疱性性表皮松解症(dystrophic epidermolysis bullosa pruriginosa, DEBP)是DEB的一种少见临床亚型。DEBP患者可于出生后早期表现为DEB的临床表型,并随着年龄增长,伴随着水疱或大疱性损害减少,逐渐出现痒疹样损害；患者也可以出生后无任何临床表现,逐渐于双胫前缓慢出现痒疹样改变,伴有轻度的甲营养不良性改变;少数患者可在成年后才出现典型的DEBP皮损而无任何其他DEB的临床表现；极少数DEBP患者还可以在典型临床表现的基础上出现白色丘疹样皮疹。DEBP可呈常染色体显性遗传或隐性遗传,但多数病例呈常染色体显性遗传。目前,已报导引起DEBP的突变包括错义突变、移码突变以及剪切位点突变。其中,引起COL7A1基因编码的Ⅶ型胶原三螺旋区的甘氨酸替代的杂合错义突变为DEBP最常见的基因突变类型。这是由于Ⅶ型胶原蛋白结构特征是Gly-Xaa-Yaa连续重复,由于甘氨酸是最小的氨基酸,对于胶原蛋白的结构和功能至关重要,当甘氨酸被其他种类的氨基酸替代后,可能干扰Ⅶ型胶原的三螺旋结构域的形成,妨碍其结构的完整性和稳定性,使皮肤基底膜致密板下带锚纤维(其主要成分为Ⅶ型胶原)不能维持正常的功能,导致DEBP发生。但引起DEBP患者剧烈瘙痒并最终在反复搔抓刺激下出现痒疹样改变的具体原因尚不清楚。本研究拟对收集到的3例临床表型差异较大的DEBP病例进行COL7A1基因突变的研究,进一步探讨DEBP患者临床表型与基因型之间可能的关联,丰富DEBP突变的数据库,为患者家系的产前诊断奠定基础。目的检测三例痒疹样营养不良型大疱性表皮松解症患者的COL7A1基因突变情况。方法1、收集三例患者临床资料,病例1为17岁男性,出生后反复于手、足等摩擦部位出现水疱及大疱,消退后留有轻度增生性瘢痕。随年龄增大,患者摩擦性水疱逐渐减轻,但缓慢于双胫前、背部、上肢伸侧出现扁平丘疹及结节,伴有剧烈瘙痒,家族中其母亲及多位亲属有类似情况,否认父母近亲结婚。病例2和病例3均为成年女性,青春期后发病,主要表现双胫前、背部逐渐增多的结节,伴有剧烈瘙痒。病例2有家族史,病例3为散发病人。查体：三例患者均可见到双胫前、背部多发的淡粉红色或褐色扁平丘疹及结节,表皮剥蚀,部分表面伴有明显角化,其中病例1皮疹较为广泛多发,并且伴有腹部多发肤色扁平肤色或者白色丘疹及增生性瘢痕。三例患者均可见到甲变薄,远端萎缩甚至缺如。三例患者的皮肤组织病理均提示：角化过度,棘层肥厚,表皮下轻度裂隙,真皮浅层血管周围少量淋巴细胞浸润。2、电镜检查标本制备：环钻钻取病例1及病例3胫前结节处皮损一块(3mm),立即用5%戊二醛溶液固定,送至电镜室行透射电镜检测。3、DNA的提取取患者及其亲属外周血5ml,2%EDTA抗凝,以低渗溶血以及酚—氯仿抽提法提取DNA。4.PCR扩增及DNA测序根据COL7A1基因序列设计72对特异性引物扩增3例患者全部外显子,同时扩增患者亲属及150例无关正常人对照的相应突变外显子。扩增条件：94℃预变性5min；94℃变性45s,56℃-62℃退火1 min,72℃延伸1min,共35个循环；72℃后延伸10min。1.5%的琼脂糖凝胶电泳检查PCR产物；所有PCR产物经纯化后送至北京天一辉远测序公司测序。结果病例1及2有家族史,病例3为散发患者。病例1和3皮损透射电镜显示部分区域锚纤维丝轻度减少,病例1可见致密板下裂隙。以三例患者的基因组DNA为模板,所有72对引物在各自的条件下分别扩增出各自的产物,包括所有118个外显子的编码序列以及侧翼序列。所有产物纯化后测序结果与Ensembl网站所公布的COL7A1基因序列进行对照,发现病例1的COL7A1基因编码外显子的第6734位核苷酸发生G→T杂合突变,使位于85号外显子2245位密码子发生GGT→GTT突变,导致Gly2245Val杂合突变,其母亲及家族中其他患者带有该突变,其父亲未检测到该突变；病例2的COL 7A1基因编码外显子的第6859位核苷酸发生G→A杂合突变,使位于87号外显子2287位密码子发生GGA→AGA突变,导致Gly2287Arg杂合突变,家族中其他患者带有该突变；病例3的CIL7A1基因编码外显子的第5318位核苷酸发生G→T杂合突变,使位于42号外显子1773位密码子发生GGT→GTT突变,导致Gly1773Val杂合突变,其未患病的父母未检测到该突变,提示其为新发突变。突变在病例1和2家族中的患者均呈现共分离现象。所有突变均由反向测序得到验证。150例无关正常人对照均未发现这三种突变。结论COL 7A1的p.G2245V、p.G2287R和p.G1773V甘氨酸替代突变,可能是引起这三例患者临床表型的病因,进一步证实甘氨酸替代突变在DEBP中的重要致病意义；其中p. G2245V、p.G1773V为两种未报道过的新突变,这丰富了DEBP的临床表型与基因型之间的关系研究,并为此三例患者家族的产前诊断奠定了基础。更多还原

【Abstract】 BackgroundEpidermolysis bullosa (EB) is a group of disease with dermal-epidermal separation in the different level of basement membrane zone. According to the ultrastructure and the precise split location, EB can be divided into four main types: epidermolysis bullosa simplex (EBS), junctional epidermolysis bullosa (JEB), dystrophic epidermolysis bullosa (DEB) and Kindler syndrome. EBS, with cleft within basal keratinocytes, is caused by mutations in KRT5, KRT14 or plectin protein. JEB, of which the separation occurs predominantly in the lamina lucida, can be due to the defects of lamina-332, integrinα6β4 and collagen XVII. DEB, with separation under the lamina densa, is caused by the defect in collagen VII. Finally, Kindler syndrome, without definitely locations of the cleft, is caused by mutation in Fermitin family homologous protein 1.Dystrophic epidermolysis bullosa (DEB) is a subtype of inherited epidermolysis bullosa characterized by clefts under the lamina densa. DEB can be inherited by autosomal dominant or autosomal recessive pattern, based on the mutation mode. DEB is caused by mutations in type VII collagen gene which encodes one of the important components of the anchoring fibrils.Dystrophic epidermolysis bullosa pruriginosa (DEBP) is a rare type of dystrophic epidermolysis bullosa (DEB). Patients with DEBP may show clinical features of DEB early after birth, with vesicles and blisters, which were diminished with age. Gradually patients developed pruriginosa-like lesions, mainly on the shins. Patients can also have no clinical manifestations until adult age, when pruriginosa lesions gradually appeared on the shins, with slightly nail destrophy. Albopapuloid lesions may be found in some patients with DEBP.Although DEBP can be inherited either in an autosomal dominant or in an autosomal recessive pattern, most of them are autosomal dominant. Disease-causing mutations in COL7A1 gene of DEBP includes missense mutations, frameshift mutations and splice mutation. Glycine substitution missense mutations in COL7A1 is the most common one in DEBP, which affects the characterized structural Gly-Xaa-Yaa in collagen proteins. Because glycine is the smallest amino acid and plays an important role in the function and structure of VII collagen. The Tri-helix structural formation of VII collagen was interfered as Glycine substituted by other amino acids, affecting its stability and integrity. As a result, functions of anchoring fibrils under lamina densa in bsasement, which is mainly composed by collagen VII, may be altered. The mechanism of severe pruritus in DEBP and the cause of the prurigo-like lesions are still unclear. Our research focused on the relationship between clinical phenotypes and genotypes in three cases with DEBP. It may further rich DEBP mutations database and prepare for the prenatal diagnosis in the families of these three cases.ObjectiveTo determine mutations of COL7A1 gene in three cases with dystrophic epidermolysis bullosa pruriginosa.Methods1. The first patient was a 17 y old male who developed vesicle and bulla on the friction site of hand and foot after brith.The frictional vesicle and bulla diminished gradully with age, but papules and nodules were noted on his shins,dorsa,and extremities with severe pruritus.The same condition was formed in this mother and relatives.with denial parent consanguineous marriage. The second and third cases were adult female. The first onset was occurred after juvenile and manifested by gradually increased nodule on the shins and dorsa with severe pruritus.The second case has family history and the third was sporadic.Physical examination revealed multiple purple red or black papules and nodula,with epidermal excoriations, hyperkeratosis on shins and trunk.Nail involvement including dystrophy,atrophy was found in three cases.The lesion in first case were severe,with multiple papule and scare on dorsa. The pathology revealed hyperkeration,acanthosis,fissure formation under epidermis, and lymphocyte infiltration around blood vessel in upper dermis.2. Electromicroscope examination:The skin lesion was taken from the shins of the patient 1 and patient 3, fixed in 5% glutaraldehyde solutions, and observed by transmission electromicroscope.3. Genomic DAN extraction:5ml peripheral blood was taken from the patients and their relatives. DNA was isolated by phenol-chloroform.4. PCR amplification and DNA sequencing:72 pairs of primers were designed according to COL7A1 gene sequence. PCR were peformed and all the amplicons were subjected to the direct sequencing.150 normal individuals were also sequenced as controls. The purified PCR products were sequenced by Beijing Tianyihuyuan company.ResultsPatient 1 and 2 are familial cases, whereas patient 3 is sporadic. Transmission electron microscopy examination showed cleft under lamina densa and slightly decreased anchoring fibrils in part of the regions. Genomic DNA was subjected to mutation detection by PCR methods. All the exons, including the flanking intronic sequences, were amplified and the products were purified and send for direct sequencing. By comparison with COL7A1 sequences published by Ensembl, we detect a heterozygous G-T substitution affecting nucleotide 6734, exons of 85 leading to Gly2245Val heterozygous mutation in case 1. In case 2, sequencing of the PCR product revealed a heterozygous G-A mutation at the nucleotide 6859, resulting in Gly2287Arg. These mutations were also found in their affected relatives. In case 3, heterozygous G-T mutation at the nucleotide 5318, leading to Gly1773Val was detected, but the same mutation was not found in her unaffected parents. All mutations was confirmed by reverse sequencing. The novel mutations, Gly2245Val and Gly1773Val, were not detected in 150 normal controls. ConclusionMutations of p.G2245V、p.G2287R and p.G1773V of COL7Al may be the causes of the clinical phenotype of the three patients. Further proved glycine replace mutation the important pathogenic significance in DEBP.Among them,2245V p.G p.G 1773V for two not reported new mutations. This riched DEBP genotype phenotype and the clinical research, and had laid the foundation of prenatal diagnosis.更多还原