Hallopeau-Siemens型营养不良型大疱性表皮松解症一例产前诊断

目的 鉴定一常染色体隐性遗传营养不良型大疱性表皮松解症家系的突变后,对患者的下一代开展产前诊断.方法 首先对患者和患者妻子进行COL7A1基因全部118个外显子的扩增和直接测序.然后从孕15周患者妻子的羊水中提取胎儿的DNA,应用聚合酶链反应(PCR)、DNA直接测序和限制性片段长度多态性(RFLP)的方法来检测突变位点,从而进一步确定该胎儿是否患病.结果 发现该患者COL7A1基因的1条等位基因第2号外显子上存在S48P的错义突变,而另1条等位基因第27号外显子上存在3625del11缺失突变,造成编码区阅读框架的移位,最终导致蛋白终止密码(PTC)的产生.患者妻子该基因全序列完全正常.胎儿COL7A1基因的1条等位基因第27号外显子上存在3625del11缺失突变,而另1个第2号外显子序列正常.因此证实该胎儿为携带者,胎儿出生后临床表型正常.结论 完成我国首例常染色体隐性遗传的营养不良型大疱性表皮松解症的DNA基础的产前诊断.     

遗传性皮肤病

20 0 3 1 2 97　痒疹样营养不良型大疱性表皮松解症一家系的基因突变 /姜薇 (北京大学第一医院皮肤科 )…∥中华皮肤科杂志 . 2 0 0 2 ,3 5 ( 6) . 442～444鉴定一痒疹样营养不良型大疱性表皮松解症家系的基因突变 ,为进一步开展基因诊断和基因治疗奠定基础。应用聚合酶链反应 (PCR )、DNA直接测序明确突变位点 ,根据突变位点设计等位基因特异性引物 ,用PCR来检测突变位点以及采用逆转录 聚合酶链反应 (RT PCR)和克隆测序进一步确定该家系的致病原因。结果 :该家系中患者COL7A1基因的 87号外显子存在剪接位点突变 ,导致 87号对显子…     

2个痒疹样营养不良型大疱性表皮松解症家系基因突变分析

目的:检测2个痒疹样营养不良型大疱性表皮松解症家系的基因突变。方法: 应用聚合酶链反应(PCR)、RT-PCR、DNA直接测序明确突变位点。结果:发现家系1中先证者COL7A1基因的61号外显子5217位发生GC的改变,造成甘氨酸(GGT)被精氨酸(CGT)取代;家系2中先证者87号内含子c.6900+1GC的剪接突变,导致87号外显子被剪切,86号外显子与88号外显子直接拼接,Ⅶ型胶原的胶原区缺少了23个氨基酸;健康对照不存在此两种突变。结论: 2个家系发现的突变均为导致该疾病发生的特异性突变。     

一例显性营养不良型大疱性表皮松解症的基因突变检测

目的 研究1例营养不良型大疱性表皮松解症家系中的基因突变情况.方法 经组织病理、电镜及免疫荧光方法结合临床诊断为显性营养不良型大疱性表皮松解症1例,采用聚合酶链反应(PCR)DNA直接测序,限制性内切酶反应及应用D3S1359、D20S161、D5S818、D17S1293、CSFIPO五个座位微卫星DNA多态标志的方法对此例患者家系进行基因突变情况检测.结果 家系中患者存在COL7A1上第6240位鸟嘌呤G被腺嘌呤A替代突变导致Ⅶ胶原第2043位的甘氨酸被精氨酸替代,而其父母及对照的健康人均不存在此突变.结论 G2043R是引起该家系临床病变的特异突变,不是多态性变化,且此突变为一个denovo突变.     

X性连锁少汗性外胚层发育不良家系ED1基因突变检测

目的 探讨X性连锁少汗性外胚层发育不良(XLHED)家系中ED1基因突变。方法 收集2个X性连锁少汗性外胚层发育不良家系外周血标本;采用聚合酶链反应(PCR)结合DNA直接双向测序的方法。结果 家系1中ED1基因的第8个外显子下游与内含子8交界处存在一个新的剪接点缺失突变(IVS8+5 del G)。家系2中第9个外显子处存在一个错义突变(A959G)。这些突变未在两个家系的正常人及188例无关正常对照者中出现。结论 中国人ED1基因突变可引起XLHED,且IVS8+5del G为一个新的突变。     

Hallopeau-Siemens型隐性营养不良型大疱性表皮松解症一例的基因突变研究

目的鉴定一常染色体隐性遗传营养不良型大疱性表皮松解症家系的基因突变。方法应用PCR、DNA直接测序明确突变位点,根据突变位点设计特异性引物,用PCR检测突变位点从而进一步确定该家系的致病原因。结果发现该患者COL7A1基因的一条等位基因第2号外显子上存在S48P的错义突变,而另一条等位基因第27号外显子上存在3625del11缺失突变,造成编码区阅读框架的移位,最终导致蛋白终止密码(PTC)的产生。隐性营养不良型大疱性表皮松解症患者这种两个突变的组合在国际上为首次报道。结论 COL7A1基因的缺失突变和错义突变引起该患者临床症状的特异突变。     

X连锁无汗性外胚叶发育不良家系的基因突变检测

目的 鉴定X连锁无汗性外胚叶发育不良(EDA)家系的基因突变及其突变类型,为建立对该病的基因诊断与遗传咨询提供依据。方法 应用聚合酶链反应-单链构象多态性(PCR-SSCP)分析法,结合DNA测序,检测了汉族人X连锁EDA一家系的基因突变位点与突变方式。结果 EDA致病基因(EDA1基因)外显子1的PCR产物经SSCP分析显示,患者及其携带者母亲出现异常单链条带。DNA测序表明,先证者该基因外显子1的第404位碱基胞嘧啶C被鸟嘌呤G颠换,致使EDA蛋白跨膜区第54位组氨酸突变成谷胺酰胺(H54Q),其母亲同一位置碱基呈现C～G杂合双峰。结论 本EDA家系中患者EDA1基因外显子1存在错义突变(404C→G),这可能是导致EDA发病的分子机制之一。     

Hallopeau-Siemens型常染色体隐性遗传大疱性表皮松解症一家系的基因突变研究

目的鉴定一Hallopeau—Siemens型常染色体隐性遗传真皮型大疱性表皮松解症家系的基因突变，为进一步开展产前诊断奠定基础。方法提取患者及其父母的基因组DNA，应用聚合酶链反应、DNA直接测序明确突变位点，并使用限制性片段长度多态性分析进一步确定该家系的致病原因。结果发现患者COL7A1基因存在2个突变：①第12号外显子上第4326位碱基由胞嘧啶突变为胸腺嘧啶，使第525位氨基酸由精氨酸（G）突变为终止密码（R525X）；②第105号外显子上第27716位碱基由胞嘧啶突变为胸腺嘧啶，使第2510位氨基酸由精氨酸（G）突变为终止密码（R2510X）。其母为R525X突变杂合子，其父为R2510X突变杂合子。结论COL7A1基因的R525X无义突变和R2510X无义突变是引起该患者临床症状的特异突变。     

一个遗传性对称性色素异常症家系DSRAD基因剪接位点突变的鉴定

目的 鉴定遗传性对称性色素异常症家系DSRAD基因的突变。方法 收集遗传性对称性色素异常症1个家系成员的血样，提取基因组DNA，用PCR扩增结合直接测序的方法进行DSRAD基因的检测。在内含子区域检测到一个碱基替换后，进一步提取患者外周血RNA，行RT-PCR，直接测序后分析其异常剪接方式。结果 在该家系患者的11号内含子区域检测到1个非经典的剪接位点突变，（c.3021-2G > A），RT-PCR结果发现，12号外显子缺失，在13号外显子处发生移码突变。结论 DSRAD基因11号内含子区域剪接位点突变（c.3021-2G > A）造成mRNA的异常剪接，导致邻近的12号外显子缺失和13号外显子移码突变，从而引起该家系患者发病。     

遗传性对称性色素异常症一家系致病基因的定位和突变研究

目的 探讨遗传性对称性色素异常症家系的致病基因。方法 明确先证者的临床诊断后,收集该家系成员的血样抽提基因组DNA,应用基因分型和连锁分析的方法进行基因定位,并对该定位区域内DSRAD基因直接测序,分析其突变位点。结果 基因分型和连锁分析将该家系的致病基因定位于1号染色体,和已知报道的区域一致。突变研究发现该家系所有患者的DSRAD基因2号外显子均携带CAA→TAA的突变,使得517位氨基酸由谷氨酰胺变成中止密码子。结论 该遗传性对称性色素异常症家系中的患者存在DSRAD基因的无义突变。     

A novel splice site mutation in collagen type VII gene in a Chinese family with dominant dystrophic epidermolysis bullosa pruriginosa

Dystrophic epidermolysis bullosa pruriginosa, a subtype of epidermolysis bullosa dystrophica and a heterogeneous inherited disease, is characterized by pruritus, excoriated nodular prurigo-like lesions, skin fragility, altered anchoring fibrils and loss of dermal-epidermal adhesion. Mutation in type VII collagen gene (COL7A1) is thought to be implicated in the underlying change for dystrophic epidermolysis bullosa pruriginosa. We report here a large family of dominant dystrophic epidermolysis bullosa pruriginosa. Mutation analysis using polymerase chain reaction and direct sequencing demonstrated a novel nucleotide substitution of 6899A-->G in exon 87 in one COL7A1 allele of the proband and 18 affected family members. This substitution was not found in 100 normal alleles. Polymerase chain reaction and sequencing of the cDNA, reverse transcribed from the proband's peripheral lymphocyte RNA, suggest that this mutation causes aberrant COL7A1 mRNA splicing of exon 87 skipping. Clinical features and pedigree analysis suggest that 6899A-->G substitution is a mutation with full penetrance and variable expressivity.     

Allelic heterogeneity of dominant and recessive COL7A1 mutations underlying epidermolysis bullosa pruriginosa.

The inherited mechanobullous disease, dystrophic epidermolysis bullosa, is caused by type VII collagen gene (COL7A1) mutations. We studied six unrelated patients with a distinct clinical subtype of this disease, epidermolysis bullosa pruriginosa, characterized by pruritus, excoriated prurigo nodules, and skin fragility. Mutation analysis using polymerase chain reaction amplification of genomic DNA, heteroduplex analysis and direct nucleotide sequencing demonstrated pathogenetic COL7A1 mutations in each case. Four patients had a glycine substitution mutation on one COL7A1 allele (G1791E, G2242R, G2369S, and G2713R), a fifth was a compound heterozygote for a splice site mutation (5532 + 1G-to-A) and a single base pair deletion (7786delG), and a sixth patient was heterozygous for an out-of-frame deletion mutation (6863del16). This study shows that the molecular pathology in patients with the distinctive clinical features of epidermolysis bullosa pruriginosa is heterogeneous and suggests that other factors, in addition to the inherent COL7A1 mutation(s), may be responsible for an epidermolysis bullosa pruriginosa phenotype.     

Genotype-phenotype correlation in Chinese patients with dystrophic epidermolysis bullosa pruriginosa

Dystrophic epidermolysis bullosa pruriginosa (DEB-Pr) is a rare variant of dystrophic epidermolysis bullosa (DEB) due to dominant or recessive mutations in the COL7A1 gene. More than 40 mutations in COL7A1 have been described in DEB-Pr. The aim of this study was to understand the genotype-phenotype correlation in Chinese patients with DEB-Pr. Three Chinese families with typical clinical features of DEB-Pr were studied. The results were analysed in association with the eight Chinese DEB-Pr patients reported in the literature. In the three Chinese families with DEB-Pr, we found two dominant cases with G1773R and c.6900+1G>C mutations, and one case with heterozygous G2701W mutation of uncertain inheritance mode. In the 10 Chinese patients with dominant type of DEB-Pr, 7 glycine substitutions and three splicing site mutations of exon 87 skipping were identified. Glycine substitution mutations in the triple helix region and exon 87 skipping, leading to the in-frame deletion of 23 amino acid residues in the triple-helix, are often seen in Chinese patients with dominant DEB-Pr, although the glycine substitutions are also frequently present in dominant DEB.     

Exon 87 skipping of the COL7A1 gene in dominant dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare, inherited, blistering disorder resulting from mutations in the COL7A1 gene, which encodes the anchoring fibrils, type VII collagen. We herein describe a further Japanese girl diagnosed with dominant DEB (DDEB). She had blisters sporadically and erosions healed with mild scarring and milia on the knees and pretibial regions. Severe pruritus was present at this time. Direct nucleotide sequencing of genomic DNA disclosed a heterozygous same splice-site mutation c.6900G>A in the COL7A1, which causes in-frame exon 87 skipping. So far, five different COL7A1 mutations leading to exon 87 skipping have been identified in rare forms of DEB: four DDEB pruriginosa and one pretibial DDEB. Therefore, a recent study suggested that exon 87 skipping in COL7A1 was related to the phenotype of DDEB pruriginosa. When she was 18 years old, however, the blister formation and pruritus markedly decreased. Therefore, her clinical symptoms were consistent to very mild DDEB but not to DDEB pruriginosa. Taken together, in-frame exon 87 skipping through c.6900G>A mutation may account for the mild skin features, rather than DDEB pruriginosa, in the present case.     

COL7A1与PLEC双基因突变致大疱性表皮松解症一例国内首报

【摘要】 目的 对1例营养不良型大疱性表皮松解症患儿家系进行基因突变分析。方法 收集1例营养不良型大疱性表皮松解症患儿临床资料,提取患儿及其父母外周血DNA进行全基因组外显子测序,将测序结果与既往报道的大疱性表皮松解症基因进行比对,比对结果采用Sanger测序方法进行验证并预测生物学信息,在100例健康对照中验证该位点。结果 患儿存在复合杂合突变,共携带3个致病突变,即COL7A1基因c.3625_3635 del11、c.6270delT突变和PLEC基因c.12772G＞A突变。其中COL7A1基因c.6270delT突变和PLEC基因c.12772G＞A突变皆为新发突变。COL7A1基因c.3625_3635 del11及c.6270delT突变来自父亲,导致肽链合成提前终止,产生截短蛋白;PLEC基因c.12772G＞A突变来自母亲,导致网蛋白第4258位谷氨酸被赖氨酸替代（p.Glu4258Lys）。结论 该患儿是由COL7A1与PLEC双基因突变所致的常染色体隐性遗传营养不良型大疱性表皮松解症。     

A founder synonymous COL7A1 mutation in three Danish families with dominant dystrophic epidermolysis bullosa pruriginosa identifies exonic regulatory sequences required for exon 87 splicing

Dystrophic epidermolysis bullosa pruriginosa (DEB-Pr) (OMIM 604129) represents a distinct variant within the DEB clinical spectrum. It is characterized by intense pruritus and distinctive nodular prurigo-like and/or hypertrophic lichenoid lesions mainly localized on the arms, legs and upper shoulders. DEB-Pr is caused by either dominant (DDEB-Pr) or recessive mutations in the COL7A1 gene encoding type VII collagen (COLVII). The full spectrum of COL7A1 mutations in DEB-Pr remains elusive and the genotype-phenotype correlation is largely incomplete. Here, we report and functionally characterize a previously unrecognized translationally silent exonic COL7A1 mutation that results in skipping of exon 87 and is associated with DDEB-Pr phenotypes in several members of three apparently unrelated Danish families. A haplotype segregation study suggested a common ancestor in these kindred. Functional splicing analysis of the mutant exon by a COL7A1 minigene construct and computational prediction for splicing regulatory cis-sequences prove that the mutation alters the activity of an exonic splicing enhancer (ESE) critical for exon inclusion. These findings substantiate for the first time the involvement of an ESE mutation in the pathogenesis of DEB and have implications for genetic counselling of Danish families with DDEB.     

A novel missense mutation in the COL7A1 gene causes epidermolysis bullosa pruriginosa

Epidermolysis bullosa (EB) pruriginosa, characterized by severe itching and the presence of nodular prurigo‐like or lichenoid lesions, is a rare clinical type of dystrophic EB. Mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils, have been implicated in the pathogenesis of the disorder. In the present study, we screened a Chinese family with EB pruriginosa for COL7A1 mutations by PCR amplification of genomic sequences and direct nucleotide sequencing. The mutation consists of a G→T substitution at nucleotide 6724 in exon 85, which leads to the substitution of glycine by tryptophan at codon 2242. This report adds new variants to the known COL7A1 mutations underlying EB pruriginosa, and provides the basis for genetic counselling and prenatal diagnosis for affected families.