本文選題：單純型大皰性表皮松解癥 + 營(yíng)養(yǎng)不良型大皰性表皮松解癥��； 參考：《山東大學(xué)》2011年博士論文

【摘要】：目的：遺傳性皮膚病大皰性表皮松解癥在電子顯微鏡下根據(jù)水皰形成的位置可分為三類(lèi),真皮-表皮連接區(qū)編碼不同蛋白的基因突變可形成不同的臨床亞型。其中單純型(EBS)水皰形成在表皮的基底細(xì)胞層,而表達(dá)于此水平的角蛋白KRT5和KRT14基因突變均可導(dǎo)致此亞型；營(yíng)養(yǎng)不良型(DEB)水皰形成在錨原纖維水平,到目前為止只有COL7A1基因的突變才能導(dǎo)致此亞型；交界型(JEB)水皰形成在基底膜帶水平的透明板處,電鏡示半橋粒錨絲形成的復(fù)合體異常,研究發(fā)現(xiàn)除編碼層粘連蛋白5的3個(gè)多肽α3、β3和γ2(LAMA3、LAMB3和LAMC2)外、編碼α6β4整合素(ITGB4和ITGA6)和編碼180kDa的大皰性類(lèi)天皰瘡抗原BPAG2(COL17A1)的基因突變也可引起此型。本研究目的主要是了解各型EB基因型與臨床表現(xiàn)型的關(guān)系；豐富基因突變的資料；進(jìn)一步了解各型EB的發(fā)病機(jī)制,對(duì)以后的臨床診斷、產(chǎn)前診斷、基因治療等提供有效的依據(jù)。 方法：收集不同類(lèi)型的大皰性表皮松解癥家系,皮膚活檢進(jìn)行常規(guī)組織病理、免疫熒光及電鏡檢查,采集外周血、提取基因組DNA進(jìn)行基因突變篩查。免疫熒光篩查可以檢查皮膚分層的位置和基底膜中缺少的蛋白(抗原),單克隆抗體可以與基底膜中特定的抗原(蛋白)結(jié)合。當(dāng)特定的抗原蛋白缺失時(shí),就會(huì)缺少相應(yīng)的著色,可以據(jù)此判斷蛋白的缺失和診斷EB子型。透射電子顯微鏡是使用高分辨率顯微鏡研究皮膚樣本來(lái)確認(rèn)皮膚的結(jié)構(gòu)性缺陷。確認(rèn)出EB子型后,可以通過(guò)分子級(jí)的研究(DNA分析)查找基因突變并確認(rèn)遺傳方式(隱性還是顯性)。 結(jié)果：EBS家系：我們?cè)贓BS-K中發(fā)現(xiàn)了兩個(gè)突變KRTS-E168D和KRT14-A413T,其中KRT5-E168D是新發(fā)突變。E168D位于KRT5頭部H1區(qū)的最后一個(gè)氨基酸。頭段的H1區(qū)、桿狀區(qū)的1A和2B以及連接區(qū)L1-2在角蛋白寡聚物的聚合作用中是重要的接觸位點(diǎn),在這4個(gè)區(qū)域的氨基酸通常都很保守。KRT14-A413T發(fā)生于螺旋區(qū)的末端-2B區(qū),這是另一個(gè)病理基因的聚集區(qū)域。在Ⅰ型角蛋白和其他類(lèi)型的錨絲中,第413個(gè)氨基酸殘基不總是丙氨酸,有趣的是其它幾個(gè)替代的氨基酸都是極性的,而且比丙氨酸大,這說(shuō)明由KRT14-A413T所引起的突變可能沒(méi)有如前認(rèn)為的那么重要,那這就能解釋先證者母親和弟弟雖然攜帶這個(gè)突變但是表型正常,當(dāng)KRTl4-A413T單獨(dú)存在時(shí),臨床是沉默的,具有不完全的外顯率。另一例EBS-R具有嚴(yán)重的表型包括泛發(fā)性水皰、粘膜損害、EB痣、掌跖角化過(guò)度和發(fā)育停滯等。免疫熒光示K14染色陰性,而K6和16染色陽(yáng)性。KRT14篩查發(fā)現(xiàn)外顯子3上純和的Y204X突變。我們的患者出生時(shí)表型較重,隨著年齡的增長(zhǎng)而緩解,可能是K5代償了K14突變所引起的后果。非常有趣的是,我們的病例表達(dá)了K6和K16,這兩個(gè)角蛋白的表達(dá)常見(jiàn)于增殖過(guò)快的皮膚比如銀屑病和愈合的傷口。表達(dá)于干細(xì)胞的K15在我們先證者的基底層也發(fā)現(xiàn)有表達(dá)。EBS的臨床表現(xiàn)與突變基因所發(fā)生的位置有關(guān),如EBS-DM臨床癥狀比較嚴(yán)重,多是由較保守的1A、2B桿狀亞區(qū)內(nèi)的基因突變所導(dǎo)致；而EBS-K及EBS-WC的臨床癥狀相對(duì)較輕,基因突變的位置則多發(fā)生于L1-2的連接區(qū)。 DEB家系：我們?cè)?4例DEB患者中發(fā)現(xiàn)了9例新發(fā)COL7A1基因突變,4例RDEB-HS結(jié)合了兩個(gè)PTC突變,另外3例結(jié)合一個(gè)提前終止密碼子(PTC)和剪切位點(diǎn)或甘氨酸置換突變。本研究還發(fā)現(xiàn)4個(gè)沉默的甘氨酸置換突變包括G2775S,G1673R,G1338V和G2791A。先證者2是白色丘疹性DDEB患者,在同一個(gè)等位基因上發(fā)現(xiàn)兩個(gè)突變R2791W和G2210V,而且這個(gè)家系的臨床表型嚴(yán)重程度變化很大。通過(guò)研究發(fā)現(xiàn)除了少數(shù)DDEB病例可由COL7A1基因的缺失突變、錯(cuò)義突變或剪切突變所導(dǎo)致,絕大部分病例還是由甘氨酸的置換突變所導(dǎo)致,且發(fā)生置換的位置多在重復(fù)序列Gl-X-Y的結(jié)構(gòu)區(qū)域之內(nèi)；RDEB的突變形式多樣,復(fù)合了包含甘氨酸置換突變、錯(cuò)義突變、無(wú)義突變、插入或缺失突變及剪切位點(diǎn)突變。 JEB家系：JEB-H具有廣泛的皮膚-粘膜損害,層粘連蛋白染色陰性,分子篩查發(fā)現(xiàn)純和的LAMC2基因IVS21-1GA突變,其父母都是雜合子且攜帶這個(gè)突變。IVS21-1GA可以引起下游PTC,所以可以解釋患者免疫熒光染色陰性及致死型表型的結(jié)果；另一例JEB-nH家庭包括兩個(gè)10歲以上的男孩,伴隨著局限性水皰、趾甲缺失、牙釉質(zhì)凹陷及角膜損害,相比較JEB-H,這個(gè)家系熒光染色減弱而非缺失,發(fā)生于外顯子9最后16個(gè)和內(nèi)含子9頭23個(gè)核苷酸的缺失突變(1359de139)是第一次被發(fā)現(xiàn),這個(gè)突變可能導(dǎo)致框架漂移；我們?cè)?個(gè)JEB-PA家系中一共發(fā)現(xiàn)3個(gè)新發(fā)ITGB4基因突變。2例致死型JEB-PA都是結(jié)合了一個(gè)PTC和一個(gè)錯(cuò)義突變(658de1C/R252C和3903dupC/G273D),而這兩個(gè)致死型病例一例伴有幽門(mén)梗阻而另一例沒(méi)有。非常有趣的是,第3個(gè)非致死型JEB-PA家系中兩個(gè)患者分子篩查都發(fā)現(xiàn)了262GA/3111-1GA突變,但是弟弟伴有幽門(mén)閉鎖而姐姐沒(méi)有,可能一些外力和環(huán)境因素影響了幽門(mén)梗阻的發(fā)生。近來(lái)網(wǎng)蛋白在JEB-PA發(fā)病機(jī)制中的作用被重視起來(lái),擾亂網(wǎng)蛋白和α。β4整合素的相互作用可能是發(fā)生幽門(mén)閉鎖的一個(gè)病因。大多數(shù)JEB-H層粘連蛋白5的兩個(gè)等位基因都有PTC突變,導(dǎo)致mRNA的加速衰減或合成的多肽鏈太短不能與其它二條正常的亞基裝配成層粘連蛋白5,而JEB-nH通常在一個(gè)或兩個(gè)等位基因發(fā)生錯(cuò)義突變。致死型JEB-PA通常在ITGB4等位基因純和或雜合兩個(gè)PTC突變,導(dǎo)致α6β4整合素完全缺乏；錯(cuò)義或者剪切位點(diǎn)突變常發(fā)生于非致死型的JEB-PA患者中。 結(jié)論：一般致死型的表型純合或雜合兩個(gè)PTC突變,導(dǎo)致mRNA加速降解或無(wú)功能的蛋白產(chǎn)生；而非致死型的表型則由錯(cuò)義、無(wú)義、剪切位點(diǎn)、插入或缺失突變所引起。不僅是突變的特性,突變的位置及突變?cè)诘鞍捉Y(jié)構(gòu)和功能中的所有作用也影響臨床表型。雖然研究提示基因型-表型的關(guān)系缺乏絕對(duì)的一致性,但不同的基因突變能預(yù)測(cè)疾病的臨床表型。環(huán)境因素如金屬蛋白酶抑制劑水平的升高或降低作為修飾因素也影響了臨床表型。 意義：通過(guò)探討EB的基因型和臨床表型的關(guān)系提高了我們對(duì)角蛋白、膠原和皮膚細(xì)胞外基質(zhì)分子生物學(xué)的了解,不僅能進(jìn)一步加深對(duì)基底膜在正常健康人及疾病中作用的認(rèn)識(shí),還為下一步尋求新的治療手段奠定堅(jiān)實(shí)的基礎(chǔ)。在今后的研究中對(duì)于EB的治療進(jìn)展將基于基底膜帶的超微結(jié)構(gòu)、多聚體的分子生物學(xué)機(jī)制以及對(duì)于每個(gè)分子所特有性質(zhì)的深入了解上,而通過(guò)本研究將有助于了解遺傳性皮膚病的多樣性及復(fù)雜性,也有助于進(jìn)一步了解其他的獲得性和遺傳性疾病。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R758.59

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 趙曉芳;孟程程;李招發(fā);;層粘連蛋白與疾病相關(guān)性的研究進(jìn)展[J];生物技術(shù)通訊;2010年01期

相關(guān)碩士學(xué)位論文 前1條

1 常小麗;癢疹樣營(yíng)養(yǎng)不良型大皰性表皮松解癥兩家系基因突變分析[D];安徽醫(yī)科大學(xué);2007年

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本文編號(hào)：2003532