大疱性表皮松解症基因型—表型的研究

发布时间：2018-06-10 14:26

本文选题：单纯型大疱性表皮松解症 + 营养不良型大疱性表皮松解症　；参考：《山东大学》2011年博士论文

【摘要】：目的：遗传性皮肤病大疱性表皮松解症在电子显微镜下根据水疱形成的位置可分为三类,真皮-表皮连接区编码不同蛋白的基因突变可形成不同的临床亚型。其中单纯型(EBS)水疱形成在表皮的基底细胞层,而表达于此水平的角蛋白KRT5和KRT14基因突变均可导致此亚型；营养不良型(DEB)水疱形成在锚原纤维水平,到目前为止只有COL7A1基因的突变才能导致此亚型；交界型(JEB)水疱形成在基底膜带水平的透明板处,电镜示半桥粒锚丝形成的复合体异常,研究发现除编码层粘连蛋白5的3个多肽α3、β3和γ2(LAMA3、LAMB3和LAMC2)外、编码α6β4整合素(ITGB4和ITGA6)和编码180kDa的大疱性类天疱疮抗原BPAG2(COL17A1)的基因突变也可引起此型。本研究目的主要是了解各型EB基因型与临床表现型的关系；丰富基因突变的资料；进一步了解各型EB的发病机制,对以后的临床诊断、产前诊断、基因治疗等提供有效的依据。方法：收集不同类型的大疱性表皮松解症家系,皮肤活检进行常规组织病理、免疫荧光及电镜检查,采集外周血、提取基因组DNA进行基因突变筛查。免疫荧光筛查可以检查皮肤分层的位置和基底膜中缺少的蛋白(抗原),单克隆抗体可以与基底膜中特定的抗原(蛋白)结合。当特定的抗原蛋白缺失时,就会缺少相应的着色,可以据此判断蛋白的缺失和诊断EB子型。透射电子显微镜是使用高分辨率显微镜研究皮肤样本来确认皮肤的结构性缺陷。确认出EB子型后,可以通过分子级的研究(DNA分析)查找基因突变并确认遗传方式(隐性还是显性)。结果：EBS家系：我们在EBS-K中发现了两个突变KRTS-E168D和KRT14-A413T,其中KRT5-E168D是新发突变。E168D位于KRT5头部H1区的最后一个氨基酸。头段的H1区、杆状区的1A和2B以及连接区L1-2在角蛋白寡聚物的聚合作用中是重要的接触位点,在这4个区域的氨基酸通常都很保守。KRT14-A413T发生于螺旋区的末端-2B区,这是另一个病理基因的聚集区域。在Ⅰ型角蛋白和其他类型的锚丝中,第413个氨基酸残基不总是丙氨酸,有趣的是其它几个替代的氨基酸都是极性的,而且比丙氨酸大,这说明由KRT14-A413T所引起的突变可能没有如前认为的那么重要,那这就能解释先证者母亲和弟弟虽然携带这个突变但是表型正常,当KRTl4-A413T单独存在时,临床是沉默的,具有不完全的外显率。另一例EBS-R具有严重的表型包括泛发性水疱、粘膜损害、EB痣、掌跖角化过度和发育停滞等。免疫荧光示K14染色阴性,而K6和16染色阳性。KRT14筛查发现外显子3上纯和的Y204X突变。我们的患者出生时表型较重,随着年龄的增长而缓解,可能是K5代偿了K14突变所引起的后果。非常有趣的是,我们的病例表达了K6和K16,这两个角蛋白的表达常见于增殖过快的皮肤比如银屑病和愈合的伤口。表达于干细胞的K15在我们先证者的基底层也发现有表达。EBS的临床表现与突变基因所发生的位置有关,如EBS-DM临床症状比较严重,多是由较保守的1A、2B杆状亚区内的基因突变所导致；而EBS-K及EBS-WC的临床症状相对较轻,基因突变的位置则多发生于L1-2的连接区。 DEB家系：我们在14例DEB患者中发现了9例新发COL7A1基因突变,4例RDEB-HS结合了两个PTC突变,另外3例结合一个提前终止密码子(PTC)和剪切位点或甘氨酸置换突变。本研究还发现4个沉默的甘氨酸置换突变包括G2775S,G1673R,G1338V和G2791A。先证者2是白色丘疹性DDEB患者,在同一个等位基因上发现两个突变R2791W和G2210V,而且这个家系的临床表型严重程度变化很大。通过研究发现除了少数DDEB病例可由COL7A1基因的缺失突变、错义突变或剪切突变所导致,绝大部分病例还是由甘氨酸的置换突变所导致,且发生置换的位置多在重复序列Gl-X-Y的结构区域之内；RDEB的突变形式多样,复合了包含甘氨酸置换突变、错义突变、无义突变、插入或缺失突变及剪切位点突变。 JEB家系：JEB-H具有广泛的皮肤-粘膜损害,层粘连蛋白染色阴性,分子筛查发现纯和的LAMC2基因IVS21-1GA突变,其父母都是杂合子且携带这个突变。IVS21-1GA可以引起下游PTC,所以可以解释患者免疫荧光染色阴性及致死型表型的结果；另一例JEB-nH家庭包括两个10岁以上的男孩,伴随着局限性水疱、趾甲缺失、牙釉质凹陷及角膜损害,相比较JEB-H,这个家系荧光染色减弱而非缺失,发生于外显子9最后16个和内含子9头23个核苷酸的缺失突变(1359de139)是第一次被发现,这个突变可能导致框架漂移；我们在3个JEB-PA家系中一共发现3个新发ITGB4基因突变。2例致死型JEB-PA都是结合了一个PTC和一个错义突变(658de1C/R252C和3903dupC/G273D),而这两个致死型病例一例伴有幽门梗阻而另一例没有。非常有趣的是,第3个非致死型JEB-PA家系中两个患者分子筛查都发现了262GA/3111-1GA突变,但是弟弟伴有幽门闭锁而姐姐没有,可能一些外力和环境因素影响了幽门梗阻的发生。近来网蛋白在JEB-PA发病机制中的作用被重视起来,扰乱网蛋白和α。β4整合素的相互作用可能是发生幽门闭锁的一个病因。大多数JEB-H层粘连蛋白5的两个等位基因都有PTC突变,导致mRNA的加速衰减或合成的多肽链太短不能与其它二条正常的亚基装配成层粘连蛋白5,而JEB-nH通常在一个或两个等位基因发生错义突变。致死型JEB-PA通常在ITGB4等位基因纯和或杂合两个PTC突变,导致α6β4整合素完全缺乏；错义或者剪切位点突变常发生于非致死型的JEB-PA患者中。结论：一般致死型的表型纯合或杂合两个PTC突变,导致mRNA加速降解或无功能的蛋白产生；而非致死型的表型则由错义、无义、剪切位点、插入或缺失突变所引起。不仅是突变的特性,突变的位置及突变在蛋白结构和功能中的所有作用也影响临床表型。虽然研究提示基因型-表型的关系缺乏绝对的一致性,但不同的基因突变能预测疾病的临床表型。环境因素如金属蛋白酶抑制剂水平的升高或降低作为修饰因素也影响了临床表型。意义：通过探讨EB的基因型和临床表型的关系提高了我们对角蛋白、胶原和皮肤细胞外基质分子生物学的了解,不仅能进一步加深对基底膜在正常健康人及疾病中作用的认识,还为下一步寻求新的治疗手段奠定坚实的基础。在今后的研究中对于EB的治疗进展将基于基底膜带的超微结构、多聚体的分子生物学机制以及对于每个分子所特有性质的深入了解上,而通过本研究将有助于了解遗传性皮肤病的多样性及复杂性,也有助于进一步了解其他的获得性和遗传性疾病。
[Abstract]:Objective: hereditary dermatosis bullous epidermolysis can be divided into three types under the electron microscope according to the position of the blister. The gene mutation of different proteins encoded by the dermal epidermal junction area can form different clinical subtypes. The simple type (EBS) blister is formed at the basal cell layer of the epidermis, and the expression of the keratin KRT5 at this level The mutation of the KRT14 gene can lead to this subtype; the blisters of the malnutrition (DEB) form at the level of the anchorage fiber, so far only the mutation of the COL7A1 gene can lead to this subtype; the boundary type (JEB) blister is formed at the level of the basal membrane zone at the transparent plate, and the electron microscope shows the abnormal complex of the complex of the half bridge. The gene mutations that encode alpha 6 beta 4 integrin (ITGB4 and ITGA6) and 180kDa's bullous pemphigoid antigen BPAG2 (COL17A1) can also be caused by the 3 peptides of laminin 5, beta 3 and 2 (LAMA3, LAMB3 and LAMC2). The aim of this study is to understand the relationship between the genotype and the clinical phenotype of each type, and to enrich the genetic mutation. Objective: to further understand the pathogenesis of various types of EB, and provide an effective basis for future clinical diagnosis, prenatal diagnosis and gene therapy.
Methods: to collect different types of epidermolysis bullosa family, skin biopsy was carried out by routine histopathology, immunofluorescence and electron microscopy, peripheral blood was collected, genomic DNA was collected to screen the gene mutation. Immunofluorescence screening could examine the location of skin layer and the lack of protein in the basement membrane (Kang Yuan). Monoclonal antibodies can be used. A specific antigen (protein) binding in the basement membrane. When a specific antigen protein is missing, the corresponding coloring is lacking, which can be used to determine the deletion of the protein and the diagnosis of the EB subtype. Transmission electron microscopy is used to study the skin samples using a high-resolution microscope to identify the structural defects of the skin. After the identification of the EB subtype, it can be passed through the molecules. Study (DNA analysis) looked for gene mutations and identified genetic patterns (recessive or dominant).
Results: EBS family: we found two mutations KRTS-E168D and KRT14-A413T in EBS-K, in which KRT5-E168D is the last amino acid in the H1 region of the KRT5 head. The H1 region of the head, the 1A and 2B in the rod-shaped region, and the connection zone L1-2 are important contact sites in the polymerization of the keratin oligomers, in these 4 regions. The amino acids in the domain are usually very conservative that.KRT14-A413T occurs at the end of the -2B region of the spiral region, which is an aggregation area for another pathological gene. In the type I keratin and other types of anchors, the 413rd amino acid residues are not always alanine. Interestingly, the other substitutes are polar and are larger than alanine. The mutation caused by KRT14-A413T may not be as important as previously thought, which explains that the mother and brother of the precursor, though carrying the mutation but have normal phenotypes, are silent and have incomplete exotoxicity when KRTl4-A413T exists alone. Another case of EBS-R has a severe phenotype including generalized blister, sticky Membrane damage, EB nevus, hyperkeratoplantaris of palmar and plantaris and stagnation of development. Immunofluorescence K14 staining was negative, while K6 and 16 staining positive.KRT14 screening found a pure and Y204X mutation in exon 3. Our patients were born with heavier phenotypes and were relieved with age growth. It is possible that K5 can compensate for the consequences of K14 mutation. It is very interesting that we The cases expressed K6 and K16, the expression of these two keratin is common in fast proliferating skin, such as psoriasis and wound healing. The K15 expressed in the stem cells in the basal layer of our precursor also found that the clinical manifestation of the expression of.EBS was related to the location of the mutant gene, for example, the clinical symptoms of EBS-DM were more serious, and most of them were compared. The 1A, 2B baculovirus subregion was caused by genetic mutation, and the clinical symptoms of EBS-K and EBS-WC were relatively mild, and the location of the gene mutation occurred more in the connection area of L1-2.
DEB family: we found 9 new COL7A1 mutations in 14 patients with DEB, 4 cases of RDEB-HS combined with two PTC mutations, and 3 combined with an early termination codon (PTC) and shear site or glycine replacement mutation. This study also found that 4 silent glycine replacement mutations included G2775S, G1673R, G1338V, and G2791A. precursor 2. It was a white papular DDEB patient with two mutations R2791W and G2210V on the same allele, and the clinical phenotypic severity of the family changed greatly. In addition, a few cases of DDEB were found to be caused by missing mutations, missense or shear mutations of the COL7A1 gene, and most of the cases were also caused by glycine. The replacement mutation is caused by the mutation, and the location of the replacement is mostly within the structural area of the repeat sequence Gl-X-Y; the mutation of RDEB is diverse and complex, including the mutation of the glycine replacement, the missense mutation, the nonsense mutation, the insertion or deletion mutation and the shear site mutation.
JEB family: JEB-H has extensive skin mucosal lesion, negative laminin staining. Molecular screening found a pure and LAMC2 gene IVS21-1GA mutation. The parents are heterozygotes and carry the mutation.IVS21-1GA to cause the downstream PTC, so it can explain the result of the negative immunofluorescence staining and the fatal phenotype of the patient; another case of J. The EB-nH family includes two boys over 10 years old, accompanied by localized blisters, missing toenails, dental enamel depression, and corneal damage. Compared with JEB-H, the family's fluorescence staining is weakened but not missing, which occurs first in the exon 9 last 16 and in the intron 9 23 nucleotides (1359de139). This mutation may be the first time. It led to frame drift; we found 3 new ITGB4 mutations in 3 JEB-PA families and.2 cases of fatal JEB-PA were combined with one PTC and one missense mutation (658de1C/R252C and 3903dupC/G273D), and one of the two fatal cases was accompanied by pyloric obstruction and the other was not. It was very interesting that third non lethal JEB-P. Two patients in the A family have found a 262GA/3111-1GA mutation, but the younger brother is associated with pyloric atresia while sister does not, and some external and environmental factors may affect the occurrence of pyloric obstruction. Recently, the role of the protein in the pathogenesis of JEB-PA has been emphasized, and the interaction of the protein and the alpha 4 integrin may be the interaction of the net protein and the alpha. One of the causes of pyloric atresia. Most of the two alleles of JEB-H laminin 5 have PTC mutations, resulting in accelerated attenuation of mRNA or synthesis of polypeptide chains that are too short to be assembled with two other normal subunits to form laminin 5, while JEB-nH usually occurs in one or two alleles with a missense mutation. Fatal JEB-PA pass. Two PTC mutations are often found in the pure and or heterozygous of the ITGB4 allele, leading to the complete deficiency of the alpha 6 beta 4 integrin, and the missense or shear site mutation often occurs in the non lethal JEB-PA patients.
Conclusion: two PTC mutations of phenotypic homozygous homozygous or heterozygosity of the general lethal type lead to the accelerated degradation of mRNA or the production of non functional proteins; and the non lethal phenotype is caused by missense, nonsense, shear site, insertion or deletion mutation. It is not only the characteristic of mutation, the location of the mutation and all the effects of the mutation in the structure and function of the protein. The clinical phenotype is affected. Although studies suggest that the genotype phenotype relationship is not absolutely consistent, different gene mutations can predict the clinical phenotype of the disease. Environmental factors such as the increase or decrease of the level of metalloproteinase inhibitors also affect the clinical phenotype.
Significance: by exploring the relationship between genotypes and clinical phenotypes of EB, our understanding of the molecular biology of keratin, collagen and skin extracellular matrix can not only further deepen the understanding of the role of the basement membrane in normal healthy people and diseases, but also lay a solid foundation for the next step of seeking new treatments. The progress in the treatment of EB in the study will be based on the ultrastructure of the basal membrane band, the molecular biological mechanism of the polymer and the in-depth understanding of the specific properties of each molecule. This study will help to understand the diversity and complexity of hereditary dermatosis and help to further understand other acquired and hereditary diseases. Disease.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.59

【共引文献】