单纯型和显性营养不良型大疱性表皮松解症基因突变研究
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摘要
研究背景
人类基因组计划(human genome project, HGP)1990年10月正式启动到2003年工作完成,该计划完成了人类基因组草图的绘制,解开了蕴含人类生命的遗传信息,丰富了遗传标记的数量,促进了基因组技术的发展,并且计划研究人类的全部基因特别是与疾病相关基因的结构、功能以及各种基因突变与疾病的关系,从而阐明人类遗传病的发病机理,并最终实现遗传病的基因诊断和基因治疗。
近年来,我国皮肤遗传病学家在此领域内取得了可喜的成绩。相应的科研成果发表在国际顶尖杂志上,引起了世界遗传学界的高度重视,为争取具有我国自主知识产权的基因研究成果奠定了有力的科学基础。由于我国地域广阔,人口众多,民族多样,遗传资源丰富,这为研究遗传性疾病提供了有利条件。因此,收集、保存和充分利用丰富的遗传资源,在中国开展遗传性皮肤病的研究,不仅有利于提高国内对遗传性疾病的认识和筛查水平,更有利于丰富国际性遗传突变基因图谱。
遗传性大疱性表皮松解症(epidermolysis bullosa,EB)是一组单基因遗传性大疱性皮肤病,呈常染色体显性(AD)或隐性(AR)遗传,其特征为皮肤或粘膜受到轻微损伤即可引起皮肤松解、水疱及大疱,有时也称为“机械性大疱性皮肤病”。2008年6月正式发布新的分类标准,根据透射电镜超微结构以水疱在皮肤中的位置为标准,EB分为单纯型(epidermolysis bullosa simplex,EBS)、交界型(junctional epidermolysis bullosa,JEB)、营养不良型(dystrophic epidermolysis bullosa,DEB)和Kindler综合征四个临床类型,各型又包括不同的亚型。EBS裂隙发生在基底的角质形成细胞内,大部分EBS是由角蛋白5(Kertin5, K5,KRT5)或角蛋白14(Kertinl4, K14, KRT14)基因突变所导致,少数由网蛋白、桥粒斑蛋白、斑菲素蛋白1和α6β4整合素基因突变所引起；JEB裂隙主要发生在透明板,是板层素332,a6β4整合素或ⅩⅦ型胶原异常所致；DEB裂隙发生在致密板下锚丝纤维水平,是由Ⅶ型胶原(Type VII collagen, COL7A1)基因突变所致；Kindler综合征皮肤分离可以发生在表皮、交界部位或致密板下层,因此无法将其归类于EBS、JEB或DEB中的任何一类。其致病基因系粘着斑相关蛋白Fermitin家族同源物1蛋白。
单纯型大疱性表皮松解症(EBS)是第一个在分子水平阐明的人类遗传性水疱性疾病,也是第一个被发现的人类角蛋白(K)疾病,是EB中最常见的临床类型,发病率约为1：30000-1：50000。根据皮肤分离发生的部位将EBS分为基底层上亚型和基底层亚型,各亚型又分别包括多个不同的病种。其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)是EBS中最常见的一型,呈常染色体显性遗传,有完全外显率,发生率为1/500000,曾称为Koebner型(EBS-K)。临床特征为水疱较为广泛,手部关节、肘部、膝部、足部和其他易反复损伤的部位发生水疱、大疱及粟丘疹。尼氏征阴性。很少累及甲与黏膜。儿童通常在出生后不久患病,几个月内有所改善,但在开始爬行时和儿童期可能复发,部分患者的水疱可较长期存在和泛发。夏季严重,冬季有所缓解。损害稀少,不会引起严重萎缩。随病程发展可消退,患者一般发育和健康状况正常。致病基因是角蛋白5(K5)和角蛋白14(K14)。K5基因是Ⅱ型角蛋白家族(GenBank no:NM000424)成员,染色体位于12q11-q13,包含9个外显子；K14基因是Ⅰ型角蛋白家族(GenBank no:NM000526)成员,染色体位于17q12-q21,包含8个外显子。近年来,在EBS各亚型患者中共发现约214种K5和223种K14基因突变(http://www.interfil.org)。分析文献发现多数EBS患者K5和K14基因突变主要是在分子头段的H1区(仅见于K5基因)、杆状区的1A及2B两段和连接区的L1-2段,而且发现基因突变的位置不同导致的临床表现也不同从而引起不同的临床亚型。许多研究证实K5和K14突变位点与临床表现密切相关,提出角蛋白有两个突变“热点”区,在1A起始区及2B结尾区有两个高度保守的α螺旋杆状区,称为螺旋起始区(HIP)及螺旋结尾区(HTP),分别与头区和尾区相连,是角蛋白中间丝装配延长的重要结构,其序列改变可损害蛋白功能,导致严重的EBS-DM。发生在非保守的连接区L1-2的基因突变可导致临床比较轻的其他泛发型EBS (EBS, gen-nonDM)和局限型EBS (EBS-loc)。
营养不良型大疱性表皮松解症(DEB)是一种会形成瘢痕的EB,水疱位于皮肤的深层组织,真皮上层的致密板下。将DEB分为显性遗传(DDEB)和隐性遗传(RDEB)两大亚型。胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)是DEB中非常罕见的临床亚型。1946年Kuske报道1例发生于胫前区的瘙痒性水疱、萎缩及瘢痕的DEB,称为胫前DEB。出生时即可发生,亦可在婴儿期或儿童期发病。主要发生于胫前、前臂,少数亦可发生于躯干。瘙痒明显,可见瘢痕、粟丘疹及甲营养不良。成人期损害可呈苔藓样斑块。病情呈慢性进行性发展,尤其进入青春期后加重。DDEB-Pt致病基因Ⅶ型胶原(COL7A1)。Ⅶ型胶原是构成锚原纤维的主要成分,与锚斑结合,并与真皮纤维交织在一起,在维持真表皮连接结构稳定性中具有十分重要的作用。Ⅶ型胶原是三个相同的α1链构成的同源三聚体,每一α1多肽链含有中央胶原性三螺旋区,两侧为非胶原性氨基端和梭基端。胶原性结构区由特征性Gly-X-Y重复序列构成三螺旋结构域。Ⅶ型胶原基因(COL7A1)定位于3p21.1,由118个外显子组成(Genbank No:L23982),是迄今报道的外显子最多基因。据统计到目前为止报道的COL7A1基因突变点总共有730多个(http://www.col7.info)。绝大多数DDEB的基因突变均为甘氨酸替换突变(某一碱基的点突变导致甘氨酸被其它氨基酸替换)。而RDEB通常表现为两条同源染色体均发生突变,它的突变位点变化较大,通常为以下几种方式：PTC(premature termination codon,提前终止密码)/PTC,错义突变/PTC,错义突变／错义突变,PTC/剪切位点突变等。
本研究对中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者进行详细临床资料调查并对基因突变进行检测,分别检测到1个K14基因错义突变c.280G>A (p.Ala94Thr)和1个COL7A1基因甘氨酸替换突变c.G6109A (p.Gly2037Arg)。并证实这2个突变为致病性突变,系国内外首次报道。DDEB-Pt散发患者是否存在de novo突变,有待进一步深入研究和探讨。本研究扩充了中国汉族人单纯型大疱性表皮松解症和营养不良型大疱性表皮松解症的遗传及临床数据库,为患者的遗传咨询、产前诊断及基因治疗等奠定了基础。
目的
检测中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者的基因突变,了解EBS和DEB基因型与临床表型的关系,丰富基因突变的资料；进一步了解发病机制,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
收集中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者及其家庭成员和100名健康对照的外周血标本,利用改良盐析法提取外周静脉血中的基因组DNA,分别设计覆盖K14基因所有外显子编码区及其外显子-内含子交界序列6对引物和COL7A1基因118个外显子编码区及其外显子-内含子交界序列的72对引物,通过PCR反应扩增和测序进行序列分析。
结果
本研究检测了1例其他泛发型单纯型大疱性表皮松解症(EBS,gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者。病例1：其他泛发型EBS (EBS, gen-nonDM)患儿为5岁女性,出生时全身出现广泛的表皮松解,在搔抓、创伤或压力下形成大小不一的水疱,疱内容物清、易破溃、易愈合。全身皮肤摩擦部位反复出现水疱、大疱,大小不等,自行破溃后均可愈合,愈合后无瘢痕。病情反复发作。该患儿无类似疾病家族史,其父母非近亲结婚。组织病理学示：表皮角化过度,表皮下水疱,炎症细胞浸润。病例2：DDEB-Pt患儿为11岁男性,出生时右侧食指第二关节腹侧可见一蚕豆大小水疱,疱壁破裂后残留瘢痕,随后于双侧踝关节出现蚕豆大小丘疹、水疱,水疱破溃后局部皮肤出现增生性瘢痕。曾于多家医院就诊,给予维生素E、中药(成分不详)口服及激素软膏外用治疗,皮疹未能控制,并扩展至头皮、躯干及四肢,瘢痕增大、肥厚,皮疹尤以双小腿伸侧及肘关节部位为重。自2011年以来患儿出现慢性荨麻疹,皮疹处瘙痒更剧烈,搔抓或碰撞、摩擦后出现水疱,且瘢痕增厚,扩展,质地变韧。该患儿无类似疾病家族史,其父母非近亲结婚。组织病理学示：表皮角化过度,多个角栓形成,棘层不规则增生,可见表皮下大疱形成,个别棘细胞松解脱落至疱内,真皮乳头水肿,真皮浅层毛细血管增生扩张,疱内及血管周围可见炎细胞浸润,包括淋巴细胞、嗜酸性粒细胞及嗜中性粒细胞等,真皮胶原纤维增生。分别以两患儿的基因组DNA为模板,所有设计扩增KRT14及COL7A1基因在各自的条件下分别扩增出各自的产物,所有产物纯化后在ABI3730XL测序仪进行序列分析,测序结果与Ensembl网站所公布的KRT14和COL7A1基因序列进行对照,并与NCBI-SNP数据库进行比对。发现病例1的K14基因1号外显子第280位碱基鸟嘌呤(G)被腺嘌呤(A)取代(c.280G>A),使得头区第94位密码子由GCT变成ACT,导致K14蛋白头部区域丙氨酸(Ala)突变为苏氨酸(Thr),即c.280G>A错义突变(p.Ala94Thr),相同的突变在患儿父母和家系外100名正常对照中未发现；病例2的COL7A1基因73号外显子的第6109位碱基鸟嘌呤(G)转化为腺嘌呤(A),使得三螺旋区第2037位密码子由GCT变成ACT,编码氨基酸由甘氨酸(Gly)变为精氨酸(Arg),即c.G6109A (p.Gly2037Arg)甘氨酸替换突变,相同的突变在患儿父母和家系外100名正常对照中未发现。
结论
运用提取基因组DNA,PCR扩增和直接测序等分子生物学技术对中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者进行详细临床资料调查并对基因突变进行检测,共检测到1个K14基因和1个COL7A1基因突变,分别是K14基因错义突变c.280G>A (p.Ala94Thr)和COL7A1基因甘氨酸替换突变c.G6109A (p.Gly2037Arg)。证实这2个突变为致病性突变,其中K14基因错义突变c.280G>A (p.Ala94Thr)和COL7A1基因甘氨酸替换突变c.G6109A(p.Gly2037Arg)系国内外首次报道。DDEB-Pt散发患者是否存在de novo突变,有待进一步深入研究和探讨。本研究扩充了中国汉族人单纯型大疱性表皮松解症和营养不良型大疱性表皮松解症的遗传及临床数据库,为患者的遗传咨询、产前诊断及基因治疗等奠定了基础。
Background
The Human Genome Project (HGP) was officially started in October1990to2003, which finished mapping the human genome draft, discovered the genetic information that contained to the human life, enriched the number of genetic markers, promoted the development of genomic technology, and plans to continuous study on the all genes that owned by human beings, especially in the genes structure and function that related to the diseases, and the relationship between variety gene mutations and diseases, further clarify the pathogenesis of human genetic diseases, and eventually realize the gene diagnosis and gene therapy of the genetic diseases.
In recent years, most experts have made a great achievements in the skin genetic diseases. The scientific research results were published in the top international journals, the conclusions has attracted a great attention to the genetics community in the world, which provides the scientific basis for gene research results with independent intellectual property rights in our country. For the vast territory, large population, ethnic diversity and rich genetic resources, which provides favorable conditions for the study of hereditary skin diseases. Therefore, the collection, preservation and utilization to the rich genetic resources, carrying out the genetic skin disease research in China, which is not only beneficial to improve the domestic understanding and screening level of genetic diseases, but also advantageous to rich the genetic mutant gene mapping in the world.
Epidermolysis Bullosa is a group of hereditary bullosa skin disease, express in autosomal dominant (AD) or autosomal recessive (AR) genetic, characterized by damage on the skin or mucous membrane by minor trauma can cause blistering, sometimes referred to as "mechanical bullosa skin diseases". In June,2008, the new classification standard was released officially, according to the standard of the blisters' position in the skin that identified by transmission electron microscope, EB is divided into Epidermolysis Bullosa Simplex (EBS), Junctional Epidermolysis Bullosa (JEB), Dystrophic Epidermolysis Bullosa (DEB) and Kindler syndrome clinical types, each type also includes different subtypes. EBS cleft occurred in the basal keratinocytes, most of the EBS are caused by the gene mutations of keratin5(Kertin5, K5, KRT5) or keratin14(Kertin14, K14, KRT14), a few of them are caused by gene mutation of net protein, desmoplakin, plakophilin1and a6β4integrin; JEB cleft occurred mainly in the transparent plate, is caused by abnormality of lamina332, α6β4integrin or type XVII collagen; DEB, with separation under the lamina densa, is caused by the defect in collagen VII (Type VII collagen, COL7A1). Kindler syndrome skin separation can occur in the epidermis, junction or sub-layer of densa board, consequently, it cannot be classified as EBS, JEB or DEB in any types. The pathogenic gene is caused by mutation in Fermitin family homologous Protein1.
EBS is the first human genetic vesicular disease that described from molecular level, it is also the first human keratin (K) disease, is a most common clinical type of EB, the morbidity is about1:30000-1:50000. According to the position of the skin separation occurs, the EBS can be divided into the basal layer upper side subtype and basal layer subtype, different subtype also includes a lot varieties diseases. The other Sporadic type of EBS is the most common type, express in the autosomal dominant inheritance, with complete penetrance, morbidity of1/500000, called Koebner (EBS-K) type. The clinical features were blister widespread and occurred in hand joint, elbow, knee, foot and other easy to repeated position, characterized of Nissl negative, the nails and mucosa are insensitive to it. Infants usually infected after birth a few days, but it would be better a few months later, however, it maybe relapse at the beginning of crawling and childhood, a parts of patients would have the blister at long term and widespread occurrence. It will be severed during the summer, and eased a little during the winter, it have a few damage, and does not cause serious adermotrophia. It also will be dissipated during the treatment, the patients who have this disease have the general development and normal health status. The pathogenic gene is keratin5and keratin14. K5gene is belongs to a type II keratin family (GenBank no:NM000424), chromosome is located in12q11-ql3, contains9exons; K14gene is belongs to a type I keratin family (GenBank no:NM000526), chromosome is located in the17q12-q21, contains8exons. In recent years, about214of K5and223of K14gene mutations were found in different EBS patients (http://www.interfil.org). According to the literature review, most K5and K14gene mutation with EBS patients are occurred in H1region of the molecular head segment (only in K5gene),1A and2B region of the rod district, and the L1-2segment of1A and2B connecting region, we also found that the clinical manifestations differences caused by different gene mutation region, which lead to the different clinical subtypes.
Many studies have confirmed there is a good relationship between K5and K14mutation region and the clinical manifestations, proposed two mutation "hot" areas for the keratin, they are located in the1A starting area and2B end area of two highly conserved α helical rod district, called helical initiation position (HIP) and helical terminal position (HTP), and they are connected with head position and tail position, respectively, which is an important structure of keratin intermediate filament assembly extend. Any sequence changes of this structure can damage the functions of the proteins, leading to severe EBS-DM. The gene mutation occurs in non conservative connected region L1-2can lead to other general EBS and EBS-loc.
DEB is one kind of EB with a scar, blisters were located in deep tissue of the skin, under the density board of the upper dermis. DEB can be divided into dominant (DDEB) and recessive (RDEB) types. DDEB-Pt is a rare clinical DEB subtype. Kuske (1946) reported a DEB case with the itching blisters, atrophy and scar in the shins anterior position, called as the shins anterior DEB. It can occurs at birth, may also be in infancy or childhood, mainly occurs in the shins anterior, forearm, a few occurs on the trunk. The patients with this disease have a obviously itching, visible scar, milia and nail dystrophy. Its damage for the adults showed lichenoid patch. The disease is a chronic progressive, and will be worse after the puberty.
The DDEB-Pt pathopoiesis gene is a type Ⅶ collagen (COL7A1). Type Ⅶ collagen is the component of anchoring fibrils, it combined with the anchor spot, and interweave with the leather fiber, it plays a very important role in the structure stability maintenance of the dermal epidermal junction. Collagen type Ⅶ is a homotrimer consist of three identical αl chains, each α1polypeptide chain containing central collagenous Tri-helix region, the non collagenous amino terminal and carboxyl terminal were located in its two sides. The repetitive Gly-X-Y sequence consists a Tri-helix structure position in the collagen structural region. Type Ⅶ collagen gene (COL7A1) located in3p21.1, consisting of118exons (Genbank No:L23982), is a gene with most exon reported until now. According to the statistics, more than730COL7A1gene mutations point were reported until now (http://www.col7.info). The most of DDEB mutations are glycine substitution mutation (a base point mutation caused glycine substitution by other amino acids). While RDEB is usually mutated with both two homologous chromosomes mutations, its mutation point changes greatly, usually in the following ways:PTC (Premature Termination Codon)/PTC, missense mutation/PTC, missense mutation/missense mutations, PTC/splice site mutations.
This study focused on the one Chinese Epidermolysis Bullosa Simplex patient (EBS, gen-nonDM) and one shins anterior Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) patient, surveyed all of clinical information and detected the gene mutations of two patients, one K14gene and one COL7A1gene were mutated, the results showed that K14gene missense mutation of c.280G>A(p.Ala94Thr), while COL7A1genes glycine substitution mutation of c.G6109A(p.Gly2037Arg). We also confirmed that these2mutations are pathogenic mutations, this result is first report in domestic and abroad by us. Whether Sporadic patients with DDEB-Pt are de novo mutation or not, it's need further study and discussion in the future. The study extends the genetic and clinical database of Epidermolysis Bullosa Simplex and Sporadic Dystrophic Epidermolysis Bullosa for Chinese person, laid the foundation for genetic counseling, prenatal diagnosis and gene therapy.
Objective
Determine the gene mutation in the patients with Epidermolysis Bullosa Simplex(EBS, gen-nonDM) and shins anterior Sporadic Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt), understand the relationship between the clinical phenotype and genotypes of EBS and DEB, enrich the gene mutation data; further understanding the pathogenesis of EB,provide an effective information for the future clinical diagnosis, prenatal diagnosis, gene disease therapy.
Methods
Collected the blood samples of Epidermolysis Bullosa Simplex (EBS, gen-nonDM) patient,shins anterior Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) Sporadic patient and their two families' members and100healthy controls person, extracted the genomic DNA in peripheral venous blood using modified salting out method, conducted the PCR amplification sequence analysis for all exons of the gene coding region and exon intron junction sequences of6pairs primers of K5and118exons of the gene coding region and exon intron junction sequences of72pairs primers located in COL7A1gene.
Results
This study examined one Epidermolysis Bullosa Simplex (EBS, gen-nonDM) patient and one shins anterior Sporadic Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) patient. The patient of cases one is a5year old female, the body appears widely Epidermolysis Bullosa at her born, form a lot of different sizes blister after scratching, trauma or stress, the blister with some clear contents inside are easy to break, and also easy to healing. The friction parts of the skin recurrent blister and bullae, unequal in size, these blister and bullae can own broken, and also can self healing, no scars after healing. The disease repeated again and again. The patient has no similar family history of the disease, the parents are not consanguineous marriage. Histopathology showed:epidermal hyperkeratosis, sub-epidermal blistering, inflammatory cell infiltration. The patient of case2is a11years old male, with the bean size blister in the second joint ventral of right index finger at his born, there is scar left after blister wall rupture, subsequently, bean size papula and blister appears in the bilateral ankle, appears the hypertrophic scar in parts of the skin after blister burst. The patient was treated in many hospitals, given vitamin E, traditional Chinese medicine (composition is unknown) oral and hormone ointment for the treatment, the rash did not control, and extended to the scalp, trunk and limbs, the scar increased, hypertrophy, rash especially in the shank part side and the elbow are very heavy. Since2011, the patient have the chronic urticaria, the rash itching is more severe, the blisters were occurred after scratching, collision or friction, and scar thickening, extension, texture tougher day by day. The patient has no similar family history of the disease, the parents are not consanguineous marriage. Histopathology showed:epidermis hyperkeratosis, formed several multiple angle plug, irregular acanthosis hyperplasia, the bullous formed under the visible epidermal, individual acantholysis shedding to the blister inside, dermal papilla edema, superficial dermal capillary hyperplasia and dilatation, blister and perivascular inflammatory cell infiltration, including lymphocytes, eosinophilic granulocyte and neutrophils, dermal collagen fiber hyperplasia. Using the genomic DNA of two patients as template, all18pairs primers in case one and72pairs primers in case two were amplified in different condition, all amplify products were purified and sequenced, then compared with K14and COL7A1gene sequence that published in the Ensembl website, the results showed, in case one, guanine (G) in280loci of K14gene exon1replaced by adenine (A)(c.280G>A), which resulted in the94th codon in the head position changed from GCT to ACT, and leads to the alanine (Ala) in the head position of K14gene mutated to the threonine (Thr), namely c.280G>A(p.Ala94Thr) missense mutation, the same mutation was not found in the her parents and100normal control person; in case two, guanine (G) in6109loci of COL7A1gene exon73replaced by adenine (A), which resulted in the2037th codon of Tri-helix region changed from GCT to ACT, encoding amino acid changed from glycine (Gly) to arginine (Arg), namely c.G6109A(p.Gly2037Arg) glycine substitution mutation, the same mutation was not found in the his parents and100normal control person.
Conclusion
Using the molecular biological techniques, such as the genomic DNA extraction, PCR amplification and direct sequencing, detected the gene mutation of the patients who are one of Epidermolysis Bullosa Simplex (EBS, gen-nonDM) and another of Sporadic Dystrophic Epidermolysis Bullosa (DDEB-Pt), the results showed that one is K14genes missense mutation c.280G>A (p.Ala94Thr) and another is COL7A1genes glycine substitution mutation c.G6109A (p.Gly2037Arg). We are also confirmed that all these2mutations are pathogenic mutation, this result is a first report in domestic and abroad by us. Whether Sporadic patients with DDEB-Pt are de novo mutation or not, it's need further study and discussion in the future. The study extends the genetic and clinical database of Epidermolysis Bullosa Simplex and Dystrophic Epidermolysis Bullosa for Chinese person, laid the foundation for genetic counseling, prenatal diagnosis and gene therapy.
人类基因组计划(human genome project, HGP)1990年10月正式启动到2003年工作完成,该计划完成了人类基因组草图的绘制,解开了蕴含人类生命的遗传信息,丰富了遗传标记的数量,促进了基因组技术的发展,并且计划研究人类的全部基因特别是与疾病相关基因的结构、功能以及各种基因突变与疾病的关系,从而阐明人类遗传病的发病机理,并最终实现遗传病的基因诊断和基因治疗。
近年来,我国皮肤遗传病学家在此领域内取得了可喜的成绩。相应的科研成果发表在国际顶尖杂志上,引起了世界遗传学界的高度重视,为争取具有我国自主知识产权的基因研究成果奠定了有力的科学基础。由于我国地域广阔,人口众多,民族多样,遗传资源丰富,这为研究遗传性疾病提供了有利条件。因此,收集、保存和充分利用丰富的遗传资源,在中国开展遗传性皮肤病的研究,不仅有利于提高国内对遗传性疾病的认识和筛查水平,更有利于丰富国际性遗传突变基因图谱。
遗传性大疱性表皮松解症(epidermolysis bullosa,EB)是一组单基因遗传性大疱性皮肤病,呈常染色体显性(AD)或隐性(AR)遗传,其特征为皮肤或粘膜受到轻微损伤即可引起皮肤松解、水疱及大疱,有时也称为“机械性大疱性皮肤病”。2008年6月正式发布新的分类标准,根据透射电镜超微结构以水疱在皮肤中的位置为标准,EB分为单纯型(epidermolysis bullosa simplex,EBS)、交界型(junctional epidermolysis bullosa,JEB)、营养不良型(dystrophic epidermolysis bullosa,DEB)和Kindler综合征四个临床类型,各型又包括不同的亚型。EBS裂隙发生在基底的角质形成细胞内,大部分EBS是由角蛋白5(Kertin5, K5,KRT5)或角蛋白14(Kertinl4, K14, KRT14)基因突变所导致,少数由网蛋白、桥粒斑蛋白、斑菲素蛋白1和α6β4整合素基因突变所引起；JEB裂隙主要发生在透明板,是板层素332,a6β4整合素或ⅩⅦ型胶原异常所致；DEB裂隙发生在致密板下锚丝纤维水平,是由Ⅶ型胶原(Type VII collagen, COL7A1)基因突变所致；Kindler综合征皮肤分离可以发生在表皮、交界部位或致密板下层,因此无法将其归类于EBS、JEB或DEB中的任何一类。其致病基因系粘着斑相关蛋白Fermitin家族同源物1蛋白。
单纯型大疱性表皮松解症(EBS)是第一个在分子水平阐明的人类遗传性水疱性疾病,也是第一个被发现的人类角蛋白(K)疾病,是EB中最常见的临床类型,发病率约为1：30000-1：50000。根据皮肤分离发生的部位将EBS分为基底层上亚型和基底层亚型,各亚型又分别包括多个不同的病种。其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)是EBS中最常见的一型,呈常染色体显性遗传,有完全外显率,发生率为1/500000,曾称为Koebner型(EBS-K)。临床特征为水疱较为广泛,手部关节、肘部、膝部、足部和其他易反复损伤的部位发生水疱、大疱及粟丘疹。尼氏征阴性。很少累及甲与黏膜。儿童通常在出生后不久患病,几个月内有所改善,但在开始爬行时和儿童期可能复发,部分患者的水疱可较长期存在和泛发。夏季严重,冬季有所缓解。损害稀少,不会引起严重萎缩。随病程发展可消退,患者一般发育和健康状况正常。致病基因是角蛋白5(K5)和角蛋白14(K14)。K5基因是Ⅱ型角蛋白家族(GenBank no:NM000424)成员,染色体位于12q11-q13,包含9个外显子；K14基因是Ⅰ型角蛋白家族(GenBank no:NM000526)成员,染色体位于17q12-q21,包含8个外显子。近年来,在EBS各亚型患者中共发现约214种K5和223种K14基因突变(http://www.interfil.org)。分析文献发现多数EBS患者K5和K14基因突变主要是在分子头段的H1区(仅见于K5基因)、杆状区的1A及2B两段和连接区的L1-2段,而且发现基因突变的位置不同导致的临床表现也不同从而引起不同的临床亚型。许多研究证实K5和K14突变位点与临床表现密切相关,提出角蛋白有两个突变“热点”区,在1A起始区及2B结尾区有两个高度保守的α螺旋杆状区,称为螺旋起始区(HIP)及螺旋结尾区(HTP),分别与头区和尾区相连,是角蛋白中间丝装配延长的重要结构,其序列改变可损害蛋白功能,导致严重的EBS-DM。发生在非保守的连接区L1-2的基因突变可导致临床比较轻的其他泛发型EBS (EBS, gen-nonDM)和局限型EBS (EBS-loc)。
营养不良型大疱性表皮松解症(DEB)是一种会形成瘢痕的EB,水疱位于皮肤的深层组织,真皮上层的致密板下。将DEB分为显性遗传(DDEB)和隐性遗传(RDEB)两大亚型。胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)是DEB中非常罕见的临床亚型。1946年Kuske报道1例发生于胫前区的瘙痒性水疱、萎缩及瘢痕的DEB,称为胫前DEB。出生时即可发生,亦可在婴儿期或儿童期发病。主要发生于胫前、前臂,少数亦可发生于躯干。瘙痒明显,可见瘢痕、粟丘疹及甲营养不良。成人期损害可呈苔藓样斑块。病情呈慢性进行性发展,尤其进入青春期后加重。DDEB-Pt致病基因Ⅶ型胶原(COL7A1)。Ⅶ型胶原是构成锚原纤维的主要成分,与锚斑结合,并与真皮纤维交织在一起,在维持真表皮连接结构稳定性中具有十分重要的作用。Ⅶ型胶原是三个相同的α1链构成的同源三聚体,每一α1多肽链含有中央胶原性三螺旋区,两侧为非胶原性氨基端和梭基端。胶原性结构区由特征性Gly-X-Y重复序列构成三螺旋结构域。Ⅶ型胶原基因(COL7A1)定位于3p21.1,由118个外显子组成(Genbank No:L23982),是迄今报道的外显子最多基因。据统计到目前为止报道的COL7A1基因突变点总共有730多个(http://www.col7.info)。绝大多数DDEB的基因突变均为甘氨酸替换突变(某一碱基的点突变导致甘氨酸被其它氨基酸替换)。而RDEB通常表现为两条同源染色体均发生突变,它的突变位点变化较大,通常为以下几种方式：PTC(premature termination codon,提前终止密码)/PTC,错义突变/PTC,错义突变／错义突变,PTC/剪切位点突变等。
本研究对中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者进行详细临床资料调查并对基因突变进行检测,分别检测到1个K14基因错义突变c.280G>A (p.Ala94Thr)和1个COL7A1基因甘氨酸替换突变c.G6109A (p.Gly2037Arg)。并证实这2个突变为致病性突变,系国内外首次报道。DDEB-Pt散发患者是否存在de novo突变,有待进一步深入研究和探讨。本研究扩充了中国汉族人单纯型大疱性表皮松解症和营养不良型大疱性表皮松解症的遗传及临床数据库,为患者的遗传咨询、产前诊断及基因治疗等奠定了基础。
目的
检测中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者的基因突变,了解EBS和DEB基因型与临床表型的关系,丰富基因突变的资料；进一步了解发病机制,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
收集中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者及其家庭成员和100名健康对照的外周血标本,利用改良盐析法提取外周静脉血中的基因组DNA,分别设计覆盖K14基因所有外显子编码区及其外显子-内含子交界序列6对引物和COL7A1基因118个外显子编码区及其外显子-内含子交界序列的72对引物,通过PCR反应扩增和测序进行序列分析。
结果
本研究检测了1例其他泛发型单纯型大疱性表皮松解症(EBS,gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者。病例1：其他泛发型EBS (EBS, gen-nonDM)患儿为5岁女性,出生时全身出现广泛的表皮松解,在搔抓、创伤或压力下形成大小不一的水疱,疱内容物清、易破溃、易愈合。全身皮肤摩擦部位反复出现水疱、大疱,大小不等,自行破溃后均可愈合,愈合后无瘢痕。病情反复发作。该患儿无类似疾病家族史,其父母非近亲结婚。组织病理学示：表皮角化过度,表皮下水疱,炎症细胞浸润。病例2：DDEB-Pt患儿为11岁男性,出生时右侧食指第二关节腹侧可见一蚕豆大小水疱,疱壁破裂后残留瘢痕,随后于双侧踝关节出现蚕豆大小丘疹、水疱,水疱破溃后局部皮肤出现增生性瘢痕。曾于多家医院就诊,给予维生素E、中药(成分不详)口服及激素软膏外用治疗,皮疹未能控制,并扩展至头皮、躯干及四肢,瘢痕增大、肥厚,皮疹尤以双小腿伸侧及肘关节部位为重。自2011年以来患儿出现慢性荨麻疹,皮疹处瘙痒更剧烈,搔抓或碰撞、摩擦后出现水疱,且瘢痕增厚,扩展,质地变韧。该患儿无类似疾病家族史,其父母非近亲结婚。组织病理学示：表皮角化过度,多个角栓形成,棘层不规则增生,可见表皮下大疱形成,个别棘细胞松解脱落至疱内,真皮乳头水肿,真皮浅层毛细血管增生扩张,疱内及血管周围可见炎细胞浸润,包括淋巴细胞、嗜酸性粒细胞及嗜中性粒细胞等,真皮胶原纤维增生。分别以两患儿的基因组DNA为模板,所有设计扩增KRT14及COL7A1基因在各自的条件下分别扩增出各自的产物,所有产物纯化后在ABI3730XL测序仪进行序列分析,测序结果与Ensembl网站所公布的KRT14和COL7A1基因序列进行对照,并与NCBI-SNP数据库进行比对。发现病例1的K14基因1号外显子第280位碱基鸟嘌呤(G)被腺嘌呤(A)取代(c.280G>A),使得头区第94位密码子由GCT变成ACT,导致K14蛋白头部区域丙氨酸(Ala)突变为苏氨酸(Thr),即c.280G>A错义突变(p.Ala94Thr),相同的突变在患儿父母和家系外100名正常对照中未发现；病例2的COL7A1基因73号外显子的第6109位碱基鸟嘌呤(G)转化为腺嘌呤(A),使得三螺旋区第2037位密码子由GCT变成ACT,编码氨基酸由甘氨酸(Gly)变为精氨酸(Arg),即c.G6109A (p.Gly2037Arg)甘氨酸替换突变,相同的突变在患儿父母和家系外100名正常对照中未发现。
结论
运用提取基因组DNA,PCR扩增和直接测序等分子生物学技术对中国汉族1例其他泛发型单纯型大疱性表皮松解症(EBS, gen-nonDM)和1例胫前显性遗传型营养不良型大疱性表皮松解症(DDEB-Pt)散发患者进行详细临床资料调查并对基因突变进行检测,共检测到1个K14基因和1个COL7A1基因突变,分别是K14基因错义突变c.280G>A (p.Ala94Thr)和COL7A1基因甘氨酸替换突变c.G6109A (p.Gly2037Arg)。证实这2个突变为致病性突变,其中K14基因错义突变c.280G>A (p.Ala94Thr)和COL7A1基因甘氨酸替换突变c.G6109A(p.Gly2037Arg)系国内外首次报道。DDEB-Pt散发患者是否存在de novo突变,有待进一步深入研究和探讨。本研究扩充了中国汉族人单纯型大疱性表皮松解症和营养不良型大疱性表皮松解症的遗传及临床数据库,为患者的遗传咨询、产前诊断及基因治疗等奠定了基础。
Background
The Human Genome Project (HGP) was officially started in October1990to2003, which finished mapping the human genome draft, discovered the genetic information that contained to the human life, enriched the number of genetic markers, promoted the development of genomic technology, and plans to continuous study on the all genes that owned by human beings, especially in the genes structure and function that related to the diseases, and the relationship between variety gene mutations and diseases, further clarify the pathogenesis of human genetic diseases, and eventually realize the gene diagnosis and gene therapy of the genetic diseases.
In recent years, most experts have made a great achievements in the skin genetic diseases. The scientific research results were published in the top international journals, the conclusions has attracted a great attention to the genetics community in the world, which provides the scientific basis for gene research results with independent intellectual property rights in our country. For the vast territory, large population, ethnic diversity and rich genetic resources, which provides favorable conditions for the study of hereditary skin diseases. Therefore, the collection, preservation and utilization to the rich genetic resources, carrying out the genetic skin disease research in China, which is not only beneficial to improve the domestic understanding and screening level of genetic diseases, but also advantageous to rich the genetic mutant gene mapping in the world.
Epidermolysis Bullosa is a group of hereditary bullosa skin disease, express in autosomal dominant (AD) or autosomal recessive (AR) genetic, characterized by damage on the skin or mucous membrane by minor trauma can cause blistering, sometimes referred to as "mechanical bullosa skin diseases". In June,2008, the new classification standard was released officially, according to the standard of the blisters' position in the skin that identified by transmission electron microscope, EB is divided into Epidermolysis Bullosa Simplex (EBS), Junctional Epidermolysis Bullosa (JEB), Dystrophic Epidermolysis Bullosa (DEB) and Kindler syndrome clinical types, each type also includes different subtypes. EBS cleft occurred in the basal keratinocytes, most of the EBS are caused by the gene mutations of keratin5(Kertin5, K5, KRT5) or keratin14(Kertin14, K14, KRT14), a few of them are caused by gene mutation of net protein, desmoplakin, plakophilin1and a6β4integrin; JEB cleft occurred mainly in the transparent plate, is caused by abnormality of lamina332, α6β4integrin or type XVII collagen; DEB, with separation under the lamina densa, is caused by the defect in collagen VII (Type VII collagen, COL7A1). Kindler syndrome skin separation can occur in the epidermis, junction or sub-layer of densa board, consequently, it cannot be classified as EBS, JEB or DEB in any types. The pathogenic gene is caused by mutation in Fermitin family homologous Protein1.
EBS is the first human genetic vesicular disease that described from molecular level, it is also the first human keratin (K) disease, is a most common clinical type of EB, the morbidity is about1:30000-1:50000. According to the position of the skin separation occurs, the EBS can be divided into the basal layer upper side subtype and basal layer subtype, different subtype also includes a lot varieties diseases. The other Sporadic type of EBS is the most common type, express in the autosomal dominant inheritance, with complete penetrance, morbidity of1/500000, called Koebner (EBS-K) type. The clinical features were blister widespread and occurred in hand joint, elbow, knee, foot and other easy to repeated position, characterized of Nissl negative, the nails and mucosa are insensitive to it. Infants usually infected after birth a few days, but it would be better a few months later, however, it maybe relapse at the beginning of crawling and childhood, a parts of patients would have the blister at long term and widespread occurrence. It will be severed during the summer, and eased a little during the winter, it have a few damage, and does not cause serious adermotrophia. It also will be dissipated during the treatment, the patients who have this disease have the general development and normal health status. The pathogenic gene is keratin5and keratin14. K5gene is belongs to a type II keratin family (GenBank no:NM000424), chromosome is located in12q11-ql3, contains9exons; K14gene is belongs to a type I keratin family (GenBank no:NM000526), chromosome is located in the17q12-q21, contains8exons. In recent years, about214of K5and223of K14gene mutations were found in different EBS patients (http://www.interfil.org). According to the literature review, most K5and K14gene mutation with EBS patients are occurred in H1region of the molecular head segment (only in K5gene),1A and2B region of the rod district, and the L1-2segment of1A and2B connecting region, we also found that the clinical manifestations differences caused by different gene mutation region, which lead to the different clinical subtypes.
Many studies have confirmed there is a good relationship between K5and K14mutation region and the clinical manifestations, proposed two mutation "hot" areas for the keratin, they are located in the1A starting area and2B end area of two highly conserved α helical rod district, called helical initiation position (HIP) and helical terminal position (HTP), and they are connected with head position and tail position, respectively, which is an important structure of keratin intermediate filament assembly extend. Any sequence changes of this structure can damage the functions of the proteins, leading to severe EBS-DM. The gene mutation occurs in non conservative connected region L1-2can lead to other general EBS and EBS-loc.
DEB is one kind of EB with a scar, blisters were located in deep tissue of the skin, under the density board of the upper dermis. DEB can be divided into dominant (DDEB) and recessive (RDEB) types. DDEB-Pt is a rare clinical DEB subtype. Kuske (1946) reported a DEB case with the itching blisters, atrophy and scar in the shins anterior position, called as the shins anterior DEB. It can occurs at birth, may also be in infancy or childhood, mainly occurs in the shins anterior, forearm, a few occurs on the trunk. The patients with this disease have a obviously itching, visible scar, milia and nail dystrophy. Its damage for the adults showed lichenoid patch. The disease is a chronic progressive, and will be worse after the puberty.
The DDEB-Pt pathopoiesis gene is a type Ⅶ collagen (COL7A1). Type Ⅶ collagen is the component of anchoring fibrils, it combined with the anchor spot, and interweave with the leather fiber, it plays a very important role in the structure stability maintenance of the dermal epidermal junction. Collagen type Ⅶ is a homotrimer consist of three identical αl chains, each α1polypeptide chain containing central collagenous Tri-helix region, the non collagenous amino terminal and carboxyl terminal were located in its two sides. The repetitive Gly-X-Y sequence consists a Tri-helix structure position in the collagen structural region. Type Ⅶ collagen gene (COL7A1) located in3p21.1, consisting of118exons (Genbank No:L23982), is a gene with most exon reported until now. According to the statistics, more than730COL7A1gene mutations point were reported until now (http://www.col7.info). The most of DDEB mutations are glycine substitution mutation (a base point mutation caused glycine substitution by other amino acids). While RDEB is usually mutated with both two homologous chromosomes mutations, its mutation point changes greatly, usually in the following ways:PTC (Premature Termination Codon)/PTC, missense mutation/PTC, missense mutation/missense mutations, PTC/splice site mutations.
This study focused on the one Chinese Epidermolysis Bullosa Simplex patient (EBS, gen-nonDM) and one shins anterior Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) patient, surveyed all of clinical information and detected the gene mutations of two patients, one K14gene and one COL7A1gene were mutated, the results showed that K14gene missense mutation of c.280G>A(p.Ala94Thr), while COL7A1genes glycine substitution mutation of c.G6109A(p.Gly2037Arg). We also confirmed that these2mutations are pathogenic mutations, this result is first report in domestic and abroad by us. Whether Sporadic patients with DDEB-Pt are de novo mutation or not, it's need further study and discussion in the future. The study extends the genetic and clinical database of Epidermolysis Bullosa Simplex and Sporadic Dystrophic Epidermolysis Bullosa for Chinese person, laid the foundation for genetic counseling, prenatal diagnosis and gene therapy.
Objective
Determine the gene mutation in the patients with Epidermolysis Bullosa Simplex(EBS, gen-nonDM) and shins anterior Sporadic Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt), understand the relationship between the clinical phenotype and genotypes of EBS and DEB, enrich the gene mutation data; further understanding the pathogenesis of EB,provide an effective information for the future clinical diagnosis, prenatal diagnosis, gene disease therapy.
Methods
Collected the blood samples of Epidermolysis Bullosa Simplex (EBS, gen-nonDM) patient,shins anterior Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) Sporadic patient and their two families' members and100healthy controls person, extracted the genomic DNA in peripheral venous blood using modified salting out method, conducted the PCR amplification sequence analysis for all exons of the gene coding region and exon intron junction sequences of6pairs primers of K5and118exons of the gene coding region and exon intron junction sequences of72pairs primers located in COL7A1gene.
Results
This study examined one Epidermolysis Bullosa Simplex (EBS, gen-nonDM) patient and one shins anterior Sporadic Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) patient. The patient of cases one is a5year old female, the body appears widely Epidermolysis Bullosa at her born, form a lot of different sizes blister after scratching, trauma or stress, the blister with some clear contents inside are easy to break, and also easy to healing. The friction parts of the skin recurrent blister and bullae, unequal in size, these blister and bullae can own broken, and also can self healing, no scars after healing. The disease repeated again and again. The patient has no similar family history of the disease, the parents are not consanguineous marriage. Histopathology showed:epidermal hyperkeratosis, sub-epidermal blistering, inflammatory cell infiltration. The patient of case2is a11years old male, with the bean size blister in the second joint ventral of right index finger at his born, there is scar left after blister wall rupture, subsequently, bean size papula and blister appears in the bilateral ankle, appears the hypertrophic scar in parts of the skin after blister burst. The patient was treated in many hospitals, given vitamin E, traditional Chinese medicine (composition is unknown) oral and hormone ointment for the treatment, the rash did not control, and extended to the scalp, trunk and limbs, the scar increased, hypertrophy, rash especially in the shank part side and the elbow are very heavy. Since2011, the patient have the chronic urticaria, the rash itching is more severe, the blisters were occurred after scratching, collision or friction, and scar thickening, extension, texture tougher day by day. The patient has no similar family history of the disease, the parents are not consanguineous marriage. Histopathology showed:epidermis hyperkeratosis, formed several multiple angle plug, irregular acanthosis hyperplasia, the bullous formed under the visible epidermal, individual acantholysis shedding to the blister inside, dermal papilla edema, superficial dermal capillary hyperplasia and dilatation, blister and perivascular inflammatory cell infiltration, including lymphocytes, eosinophilic granulocyte and neutrophils, dermal collagen fiber hyperplasia. Using the genomic DNA of two patients as template, all18pairs primers in case one and72pairs primers in case two were amplified in different condition, all amplify products were purified and sequenced, then compared with K14and COL7A1gene sequence that published in the Ensembl website, the results showed, in case one, guanine (G) in280loci of K14gene exon1replaced by adenine (A)(c.280G>A), which resulted in the94th codon in the head position changed from GCT to ACT, and leads to the alanine (Ala) in the head position of K14gene mutated to the threonine (Thr), namely c.280G>A(p.Ala94Thr) missense mutation, the same mutation was not found in the her parents and100normal control person; in case two, guanine (G) in6109loci of COL7A1gene exon73replaced by adenine (A), which resulted in the2037th codon of Tri-helix region changed from GCT to ACT, encoding amino acid changed from glycine (Gly) to arginine (Arg), namely c.G6109A(p.Gly2037Arg) glycine substitution mutation, the same mutation was not found in the his parents and100normal control person.
Conclusion
Using the molecular biological techniques, such as the genomic DNA extraction, PCR amplification and direct sequencing, detected the gene mutation of the patients who are one of Epidermolysis Bullosa Simplex (EBS, gen-nonDM) and another of Sporadic Dystrophic Epidermolysis Bullosa (DDEB-Pt), the results showed that one is K14genes missense mutation c.280G>A (p.Ala94Thr) and another is COL7A1genes glycine substitution mutation c.G6109A (p.Gly2037Arg). We are also confirmed that all these2mutations are pathogenic mutation, this result is a first report in domestic and abroad by us. Whether Sporadic patients with DDEB-Pt are de novo mutation or not, it's need further study and discussion in the future. The study extends the genetic and clinical database of Epidermolysis Bullosa Simplex and Dystrophic Epidermolysis Bullosa for Chinese person, laid the foundation for genetic counseling, prenatal diagnosis and gene therapy.
引文
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