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。     

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.     

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。     

营养不良型大疱性表皮松解症的诊治进展

营养不良型大疱性表皮松解症是遗传性大疱性表皮松解症中较为严重的类型，目前已发现，Ⅶ型胶原基因COL7A1是其致病的相关基因，呈现不同的临床表型与COL7A1等位基因突变的位点和形式不同相关。除目前临床应用的有创性羊膜腔穿刺产前诊断技术，超声、母体血胎儿有核红细胞分离以及游离胎儿DNA提取技术的发展将推动无创性产前诊断技术的提高。近年来，从分子缺陷水平对于营养不良型大疱性表皮松解症的治疗又有新的尝试。     

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.     

两个隐性营养不良型大疱性表皮松解症家系COL7A1基因突变分析及产前诊断

目的 检测2个隐性营养不良型大疱性表皮松解症家系COL7A1基因突变情况,并在患儿母亲再次妊娠时进行胎儿产前诊断.方法 收集2例隐性营养不良型大疱性表皮松解症患儿临床资料,提取患儿及其父母的外周血DNA,应用PCR扩增COL7A1基因的全部118个外显子并测序;用100例无血缘关系的健康人外周血作对照.确定致病突变后,在患儿母亲再次妊娠时,羊膜腔穿刺术获取羊水细胞.分别自直接抽提的羊水细胞以及培养后的羊水细胞中提取基因组DNA进行扩增和测序,检测COL7A1基因突变,同患儿的检测结果进行比较,行产前诊断.胎儿娩出后抽取静脉血,检测COL7A1基因突变.所有测序结果都经过双向测序验证.结果 2例患儿均发现COL7A1基因复合杂合突变.例1COL7A1基因存在c.5453G＞A及c.6781C＞T复合杂合突变,分别导致编码氨基酸发生p.G1818D突变和产生提前终止密码p.R2261Efs*25,其中c.5453G＞A来自父亲,c.6781C＞T来自母亲.例2COL7A1基因存在c.6205C＞T及c.8272_8272delG复合杂合突变,导致编码蛋白发生p.R2069C及p.V2758Sfs*28复合杂合突变,其中c.6205C＞T来自父亲,c.8272_8272delG来自母亲.2例患儿的母亲再次怀孕时所取羊水细胞及羊水培养细胞均未测到患儿所携带的COL7A1基因突变.胎儿出生后皮肤黏膜正常,无水疱,基因检测亦未发现患儿所携带的COL7A1基因突变.结论 2例营养不良型大疱性表皮松解症患儿均存在COL7A1基因复合杂合突变,并成功在2例患儿的母亲再次妊娠时进行了产前诊断.     

隐性营养不良型大疱性表皮松解症家系调查1例及COL7A1基因突变分析

目的:分析1例隐性营养不良型大疱性表皮松解症患者的家系遗传及COL7A1基因突变情况。方法:收集临床资料,提取患儿及其父母外周血DNA,利用GenCap目标基因技术对患儿全基因组外显子区域DNA捕获并富集,再利用IlluminaHiSeq 2 000第二代测序仪进行测序,与正常人进行数据比对分析,采用Sanger测序法进行验证。结果:患儿COL7A1基因第104号外显子出现7769delG纯合突变,该突变分别遗传自父母,父母该位点有杂合变异,父母及其家系中其他成员表型均正常。结论:该例患儿诊断为严重泛发型隐性营养不良型大疱性表皮松解症,致病机制为COL7A1基因出现纯合突变,产生提早终止密码。     

隐性营养不良型大疱性表皮松解症4例COL7A1基因突变分析

【摘要】 目的 分析4例隐性营养不良型大疱性表皮松解症（RDEB）患儿基因突变与临床表型情况。方法 收集4例RDEB患儿临床资料,提取患儿及其父母外周血DNA,使用先天性大疱性表皮松解症多基因芯片进行高通量测序,确定致病基因位点后,采用Sanger测序法进行双向验证。结果 4例患儿基因检测均显示COL7A1基因复合杂合突变,共8个突变位点。例1为中度泛发型RDEB,存在父源c.7828C>T和母源c.448G>A突变;例2为中度泛发型,存在父源c.3625_3635del和母源 c.2726_2728del突变;例3为局限型,存在父源突变c.682+1G>A和等位基因突变c.5532+1G>A,其父母外周血DNA未发现c.5532+1G>A突变;例4为重度泛发型,存在父源c.5196delC和母源c.500_501insAGGG突变。其中c.2726_2728del和c.5196delC突变既往未见报道。结论 4例RDEB患儿均存在COL7A1基因复合杂合突变,可能为其致病原因。其中,双等位基因移码突变携带者的表型重于双等位基因剪切位点、错义和无义、移码和氨基酸缺失及插入组成的复合杂合突变携带者表型。     

隐性营养不良型大疱性表皮松解症3例患者的COL7A1基因突变检测

目的检测3例隐性营养不良型大疱性表皮松解症患者COL7A1基因突变位点。方法提取3例患者及其相关亲属外周血DNA,采用PCR扩增COL7A1基因编码区的全部外显子及其侧翼序列并测序。结果基因检测发现3例患者共有5个突变位点,包括2个错义突变(p.P2232L与p.G1531E),2个无义突变(p.R2492*与p.R2777*)和1个剪切位点突变(ds IVS6+2TC),其中p.P2232L,p.G1531E,ds IVS6+2TC这三个突变位点未曾报道。结论上述突变可能参与了隐性营养不良型大疱性表皮松解症的致病机制。     

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。     

A novel homozygous splice site mutation in COL7A1 in a Chinese patient with severe recessive dystrophic epidermolysis bullosa and squamous cell carcinoma

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils in the dermo–epidermal junction. The development of cutaneous squamous cell carcinoma (SCC) is one of the most serious complications of this disease. We report herein a Chinese patient with the severe generalized subtype of RDEB (RDEB‐sev gen) complicated by SCC. Methods Skin biopsies were examined for histology, basement membrane ultrastructure, and type VII collagen expression. Genomic DNA was extracted from the peripheral blood samples and subjected to polymerase chain reaction amplification and direct automated DNA sequencing. Results Histopathological examination of the patient’s skin revealed an undetectable expression of type VII collagen polypeptides in the basement membrane zone. Mutation analysis identified a novel splice site mutation in intron 64 (IVS64+5g‐>a) of COL7A1 gene, which resulted in an in‐frame deletion of exon 64 in both alleles. Conclusions This report contributes to the expanding database of COL7A1 mutations and emphasizes the need to elucidate the underlying genetic mechanisms associated with the increased incidence of SCC in RDEB patients.     

Two cases of recessive dystrophic epidermolysis bullosa diagnosed as severe generalized

Recessive dystrophic epidermolysis bullosa (RDEB) is a congenital bullous disease resulting from defective anchoring fibrils at the dermal-epidermal junction and mutations in the type VII collagen gene. In this report, we describe two patients with severe generalized RDEB. Patient 1 was a 24-day-old male infant, and patient 2 was a 1-day-old female infant. Immunofluorescence microscopy demonstrated absence of type VII collagen labeling in a skin sample of patient 1, and reduced staining in patient 2. Electron microscopy revealed absence of anchoring fibrils below the lamina densa in patient 1, and reduced or rudimentary anchoring fibrils in patient 2. Mutation analyses of COL7A1 in these patients revealed heteroallelic recessive mutations which resulted in premature termination codons (PTC): 6573+1G>C in intron81 and 886del6ins14 in exon 7 in patient 1, and 6573+1G>C in intron81 and 4535insC in exon 44 in patient 2. Heteroallelic combinations of PTC mutation generally result in the severe generalized type. Patient 2 has developed a digital fusion at age 2, which is a typical manifestation of severe generalized RDEB. The RDEB subtype is considered to be determined based on comprehensive information, including analysis of alleles, protein expression, ultrastructure and clinical symptoms after growth. However, mutation analyses of COL7A1 can provide valuable information estimating a diagnosis in early infancy.     

Recurrent molecular abnormalities in type VII collagen in southern Italian patients with recessive dystrophic epidermolysis bullosa

In this study we searched for mutations in the type VII collagen gene (COL7A1) in 10 families from Southern Italy with severe generalised recessive dystrophic epidermolysis bullosa using PCR amplification of genomic DNA, heteroduplex analysis and direct nucleotide sequencing. Our principal aim was to identify any recurrent mutations in COL7A1 that might facilitate future mutation detection strategies in this population. Three recurrent COL7A1 mutations were delineated in six of the 10 families: a frameshift mutation in exon 4, 497insA, was detected in three affected individuals from three families, a deletion mutation at the acceptor splice site of intron 114/exon 115, 8441-14del21, was found in five patients in three of the families, and an intron 49 acceptor splice site mutation, 4783-1 G-to-A, was identified in three subjects in two families (GenBank accession no, L02870). Haplotype analyses showed evidence for propagation of common ancestral mutant COL7A1 alleles for each of these recurrent mutations. These results contribute significantly to understanding the nature of COL7A1 pathology in patients from Southern Italy and in designing future approaches to mutation detection.