1例DEB的基因突变研究

目的检测1例营养不良型大疱性表皮松解症(DEB)家系的基因突变位点。方法对1例DEB患者及其家属成员采用聚合酶链反应及DNA直接测序方法进行COL7A1基因突变检测。结果患者存在COL7A1上第6240位鸟嘌呤G被腺嘌呤A代替(G→A),使得2043位的甘氨酸被精氨酸替代(G2043R),其父母、妹妹及健康人未见此突变。结论COL7A1基因的G2043R突变可能是引起本例临床表现的原因,且是一个denovo突变。     

痒疹样营养不良型大疱性表皮松解症一家系的基因突变

目的 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.方法 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号外显子被剪切,Ⅶ型胶原的胶原区合成后缺少了23个氨基酸.健康对照不存在此突变.结论 COL7A1基因剪接位点的突变是引起该家系临床症状的特异突变,而非多态性改变.     

一遗传性对称性色素异常症家系ADAR基因突变检测

目的 探讨遗传性对称性色素异常症(DSH)一家系ADAR基因突变情况。方法 收集1个遗传性对称性色素异常症家系的外周血标本,采取PCR结合DNA直接测序的方法,检测了该家系中4例患者及3例表型正常者和150例无亲缘关系健康个体的ADAR基因突变情况。结果 该家系中患者存在ADAR基因上第2879位碱基腺嘌呤(A)转换成鸟嘌呤(G),使得ADAR基因的第10号外显子960位密码子由TAT突变成TGT,导致正常的酪氨酸(Tyr)被半胱氨酸(Cys)替代,而该家系的正常人对照及无关健康个体不存在此突变。结论 DSH家系中患者ADAR基因存在错义突变(2879 A→G),这可能是导致DSH发病的分子机制之一。     

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.     

一毛囊角化病家系ATP2A2基因突变的检测

目的 检测一毛囊角化病家系中ATP2A2基因的突变。方法 1例经组织病理结合临床诊断为毛囊角化病,采用聚合酶链反应和DNA测序方法对此家系进行基因突变情况检测。结果 家系中患者在ATP2A2上第1541位腺嘌呤A变为鸟嘌呤G,使编码ATP酶结合域第514位氨基酸由赖氨酸变为精氨酸,家系中未患病者及对照的健康人均不存在此突变。结论 K514R是引起该家系临床病变的一个新的特异突变,不是多态性变化。     

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。     

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。     

表皮松解性角化过度型鱼鳞病二例及其基因突变的研究

目的 研究二例表皮松解性角化过度型鱼鳞病患者基因突变情况。方法 取患者皮损进行组织病理及电镜检查;提取患者外周血DNA,采用聚合酶链反应及DNA直接测序方法,检测患者皮损角蛋白10(K10)及角蛋白1(K1)基因突变;等位基因特异性引物PCR及限制性内切酶片段长度多态性方法筛查正常人群中该等位基因频率。结果 2例患者均存在K1或K10基因的杂合点突变,即K10基因第2140位G→A,K1基因第4226位G→A,分别导致K10第156位的精氨酸变为组氨酸(R156H)及K1第477位氨基酸从谷氨酸变为赖氨酸(E477K),而正常对照无此替代。结论 K10R156H及K1E477K为导致这2例患者临床表型的特异突变。     

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。     

伴丘疹性损害的先天性无毛症一例及其基因突变的研究

目的 研究1例伴丘疹性损害的先天性无毛症患者及其家系中无毛基因的突变情况。方法 取患者皮损进行组织病理检查;提取家系成员的基因组DNA,采用聚合酶链反应扩增无毛基因的全部编码序列并结合DNA直接测序方法,检测患者无毛基因的突变。结果 患者无毛基因存在两处杂合突变:第3外显子的1010位碱基由鸟嘌呤变为腺嘌呤,使第337位氨基酸由甘氨酸突变为天冬氨酸(G337D);第4外显子的1491位碱基由胞嘧啶变为胸腺嘧啶,使第498位氨基酸由谷氨酸突变为终止密码(Q498X)。而其父母及一弟该基因仅存在其中的一处杂合突变。结论 该患者无毛基因中G337D及Q498X两处突变可能使该基因无法编码正常的蛋白,为导致临床表现的特异突变。     

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.     

Three cases of de novo dominant dystrophic epidermolysis bullosa associated with the mutation G2043R in COL7A1

In the absence of a positive family history, it is often difficult to determine whether a single case of mild-to-moderately severe dystrophic epidermolysis bullosa (DEB) represents autosomal recessive or de novo dominant disease. Recent molecular analyses of the type VII collagen gene, COL7A1, have established that the vast majority of such cases are recessive in nature. Nevertheless, a small number of de novo dominant patients have been documented. In this report, we describe three further examples of de novo dominant disease. In each case the COL7A1 mutation comprised the same glycine substitution, G2043R. This mutation has previously been reported in both dominant DEB pedigrees and as a de novo phenomenon and is the most common COL7A1 mutation in dominant DEB throughout the world. These cases emphasize the importance of molecular analysis in providing accurate genetic counselling in this genodermatosis.     

Dystrophic epidermolysis bullosa with one dominant and one recessive mutation of the COL7A1 gene in a child with deafness

Abstract:   Dystrophic epidermolysis bullosa can be inherited in autosomal dominant and recessive forms, the former usually expressed as a milder phenotype, although mild forms of recessive dystrophic epidermolysis bullosa can occur. We present a patient who was found to be a compound heterozygote, inheriting a dominant mutation from his father and a recessive mutation from his mother, resulting in a clinically severe case of dystrophic epidermolysis bullosa. Mutations in the gene for collagen VII ( COL7A1 ) have been documented in both types of dystrophic epidermolysis bullosa. Our patient has also been diagnosed with bilateral auditory neuropathy, a disorder coincidentally also mapped to a nearby gene on chromosome 3p21 (the transmembrane inner ear expressed gene, TMIE ).     

A recurrent glycine substitution mutation, G2043R, in the type VII collagen gene (COL7A1) in dominant dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). Nearly all cases of dominant DEB are caused by glycine substitution mutations occurring within the triple helical region of type VII collagen, and most of the mutations are unique to individual families. In this study, we identified a patient of Hispanic-Mexican origin with a mild form of DEB, which resulted from a de novo dominant glycine substitution, G2043R, in exon 73 of COL7A1. We also investigated a Scottish family with a three-generation pedigree of dominant DEB, in whom the same glycine to arginine substitution mutation was demonstrated. This particular mutation has also been detected previously in three other families with dominant DEB: one Italian, one Hungarian and one Norwegian. Given the widespread geographical distribution of this mutation and the demonstration of its occurrence as a de novo event, G2043R therefore represents the first example of a mutational hotspot in dominant DEB. Interestingly, although both the Mexican and Scottish families we studied had some clinical features in keeping with the Pasini form of the disorder, there was considerable interfamilial variability as well as intrafamilial diversity in the affected individuals.     

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。     

Review of collagen VII sequence variants found in Australasian patients with dystrophic epidermolysis bullosa reveals nine novel COL7A1 variants

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is an inherited skin fragility disorder where blistering occurs in the sub-lamina densa zone at the level of anchoring fibrils (AFs) of the dermo-epidermal junction. Both autosomal dominant (DDEB) and recessive (RDEB) result from mutations in the type VII collagen gene (COL7A1). OBJECTIVE: The purpose of this study was to understand the genotype-phenotype correlation in Australian patients with DEB. METHODS: Skin biopsies from patients were processed for immunofluorescence mapping, the COL7A1 gene was screened for sequence variants. RESULTS: We report 14 Australian families with different forms of dystrophic epidermolysis bullosa (DEB) with 23 different COL7A1 allelic variants, nine of which were novel. Four cases of RDEB-HS combined two premature termination codon (PTC) variants and three other cases of RDEB-HS with combined PTC and spice-site or glycine substitution variants. G2043R, a de novo dominant variant, was also identified in this study. Four "silent" glycine substitutions were found in this study, G2775S, G1673R, G1338V and G2719A. EB17, with combined R2791W and G2210V variants, had a DDEB-Pasini phenotype, in contrast to two family members who had severe DDEB pruriginosa, with the same genotype. CONCLUSION: In this study, the RDEB variants included nonsense variants, splice site variants, internal deletions or insertions, "silent" glycine substitutions within the triple helix or N or C terminal ends of the triple helix and non-glycine missense variants within the triple helix domain. DDEB usually involves glycine substitutions within the triple helix of COL7A1 although other missense variants or splice-site alterations may underlie some cases.