外胚层发育不良患者基因突变分析及功能验证

目的 寻找先天性外胚层发育不良（ectodermal dysplasia, ED）可能致病的基因突变并进行功能验证。方法 收集8例ED患者血液样本并进行基因组抽提,利用全外显子测序（whole-exome sequencing,WES）对患者基因组DNA进行高通量测序。对测序结果进行质量控制,确保质检合格后,从中筛选可能的致病基因突变。利用软件对筛选的基因突变致病性进行预测,利用免疫荧光实验及双荧光素酶实验对基因突变致病性进行功能验证。结果 所有样本全外显子测序有效率（effective rate）均为97.5%以上,错误率（error rate）均小于0.03%,Q20所占比例大于97.0%,目标区域的平均测序深度均为90×以上,提示测序数据质量良好。经筛选后,共发现3个可能致病的EDA基因点突变：c.959A>G,c.1073A>G,c.1001G>A,数据库比对显示其均为罕见变异,软件预测均为致病性突变。免疫荧光实验发现3个EDA突变可导致p65蛋白核移位减少,双荧光素酶实验进一步表明3个EDA突变可导致NF-κB通路活性降低。结论 本研究利用全外显子测序在 ED患者基因组中发现了EDA突变,并对突变致病基因进行了功能验证,为进一步理解ED的发病机制提供了依据。     

外胚层发育不良的新分类及相关基因(附病例报告)

外胚层发育不良是一类病因复杂,临床表现多样且具有遗传性的疾病。目前,从分子基础和生物学功能角度对本病提出了新的临床-遗传学分类方法。本文对外胚层发育不良的分类方法及其相关的致病基因作了综述并附1例病例报告。     

少汗型外胚层发育不良症患者全唾液分析

目的研究少汗型外胚层发育不良症患者唾液流速和成分变化。方法测量HED男性患者、女性携带者、以及男性、女性正常对照组唾液流速和成分,对结果进行统计学分析。结果HED男性患者（18例）、女性携带者（15例）较正常对照组（男性30例、女性30例）唾液流速低,唾液无机成分和蛋白含量升高,淀粉酶活性降低。结论唾液流速减低,使HED患者更容易患口腔疾病,如龋病、念珠菌感染等;同时,唾液成分的改变也有助于HED的临床诊断。     

少汗型外胚层发育不良症eda-A1基因突变分析及其真核表达载体的构建

目的克隆少汗型外胚层发育不良症（HED）eda-A1基因并进行突变分析,同时构建eda-A1基因编码序列突变型（M）和野生型（W）的真核表达载体pcDNA3.1（-）-eda-A1-M/W,为进一步明析eda基因的功能奠定基础。方法设计含有BamHⅠ和HindⅢ的酶切位点的引物, 从HED患者和正常人外周血淋巴细胞中提取总mRNA,利用RTPCR法克隆eda-A1（M/W）基因cDNA序列;并运用BamHⅠ和HindⅢ双酶切pcDNA3.1（-）载体和eda-A1基因片段,将eda-A1 （M/W） 插入到pcDNA3.1 （-） 载体中,从而构建新的真核表达载体,命名为pcDNA3.1 （-）-eda-A1-M和pcDNA3.1（-）-eda-A1-W。结果成功克隆少汗型外胚层发育不良症eda-A1基因,并发现未见报道过的907位A→C错义突变,导致306位编码氨基酸由谷氨酰胺变异为脯氨酸;经PCR法、重组载体双酶切法、DNA测序验证,成功构建了pcDNA3.1（-）-eda-A1-W/M的真核表达载体。结论成功构建少汗型外胚层发育不良症eda-A1基因（突变型和野生型）真核表达载体pcDNA3.1（-）-eda-A1-M/W,为进一步研究该基因在牙齿发育中的生物学作用奠定了实验基础。     

EDA基因在4个少汗型外胚层发育不全家系中的检测及分析

目的 对4个少汗型外胚层发育不全家系的EDA基因进行测序分析,研究突变的位置、类型,为临床诊断提供遗传学依据.方法 提取先证者及其亲属的基因组DNA,其中患者5人,无症状者12人,另外抽取100名无先天缺牙家族史的正常成人外周血,提取基因组DNA作为对照,设计EDA基因8个外显子的引物,通过聚合酶链反应和DNA测序的方法与正常序列比对.结果 4个家系的患者均存在EDA基因不同位点的突变,分别为c.466C>T、c.663-697缺失、c.587-615缺失、c.878T>G,携带者存在杂合突变,正常对照不存在以上突变.结论 EDA基因的c.466C>T、c.663-697缺失、c.587-615缺失、c.878T>G突变是导致家系先证者及患者出现少汗型外胚层发育不全的病因.其中,EDA基因的c.663-697缺失、c.587-615缺失、c.878T>G是未报道的新突变.     

A novel splicing mutation of ectodysplasin A gene responsible for hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) is characterized by hypohidrosis, hypodontia, sparse hair, and characteristic facial features. This condition is caused by an ectodysplasin A (EDA) gene mutation. In this study, we examined two HED pedigrees and investigated the molecular genetics of the defect. Direct sequencing analysis revealed a previously unidentified mutation in the EDA splice donor site (c.526 + 1G>A). The function of the mutant EDA gene was predicted through online investigations and subsequently confirmed by splicing analysis in vitro. The mutation resulted in the production of a truncated EDA‐A1 protein caused by complete omission of exon 3. This novel functional skipping–splicing EDA mutation was considered to be the cause of HED in the two pedigrees reported here. Our findings, combined with those reported elsewhere, provide an improved understanding of the pathogenic mechanism of HED as well as important information for a genetic diagnosis.     

X连锁无汗性外胚叶发育不全家系ED1基因的突变检测

目的探讨国内X连锁无汗性外胚叶发育不全家系中ED1基因的突变情况，为该病的遗传咨询、产前诊断、确诊携带者提供依据。方法收集2个X连锁无汗性外胚叶发育不全家系及1个散发患者的外周血样本，盐析法提取基因组DNA，采用聚合酶链反应（polymerase chain reaction，PCR）和直接测序对ED1基因进行突变检测。结果家系一患者ED1基因第9外显子发生错义突变（1045G〉A），家系二和散发患者ED1基因第3外显子发生错义突变，分别为467G〉A和466C〉T。结论ED1基因的错义突变可导致X连锁无汗性外胚叶发育不全。这3个突变与国外学者的报道一致。     

少汗型外胚层发育不全患者咬合重建1例

少汗型外胚层发育不全（HED）患者常伴有颌间距离丧失、牙槽骨发育不良以及先天缺牙等,修复治疗较难。本文报道1例20岁的HED患者,为其设计上颌固定、下颌套筒冠义齿修复进行咬合重建。1年后复查,患者的义齿使用良好,咬合功能恢复正常。     

Anthropometric and cephalometric measurements in X-linked hypohidrotic ectodermal dysplasia

OBJECTIVE: To describe the somatic development and craniofacial morphology in males affected with hypohidrotic ectodermal dysplasia (HED) and female carriers and to find clinical markers for early clinical diagnosis of possible female carriers. DESIGN: A clinical and radiographic examination of the affected males and the female carriers. SETTING AND SAMPLE POPULATION: Twenty-four affected males and 43 female carriers with a known mutation in the ED1 gene were examined in a dental clinic in either Copenhagen or Aarhus, Denmark. EXPERIMENTAL VARIABLES: Height, body mass index (BMI) and head circumference. Cephalometric analysis of the craniofacial morphology. OUTCOME MEASURE: Data on the somatic and craniofacial development in the affected males and female carriers. RESULTS: No difference was observed regarding body height in the affected males and female carriers, BMI values were lower than the mean in most affected boys and adolescence and head circumference was somewhat decreased in both groups compared to normative data. The cephalometric analysis showed a reduced maxilla length and prognathism, a normal size and shape of the mandible and a reduced sagittal jaw relationship in both HED groups. Furthermore, affected males had a retroclined nasal bone and a more anteriorly inclined maxilla. A short nose, protruding lips, reduced facial convexity and facial height, characterized the soft tissue profile of the affected males. In female carriers, the lips were significantly retruded when compared with controls. CONCLUSION: No specific somatic or cephalometric markers could be observed, in the female carrier group.     

外胚层发育不全无汗综合征患者基因突变的检测

目的：检测外胚层发育不全无汗综合征患者的EDA基因的突变。方法：提取外胚层发育不全无汗综合征患者的基因组DNA，采用PCR方法扩增EDA基因的第5、9外显子，直接将PCR产物送测序。结果：确证该患者是X染色体隐性遗传的外胚层发育不全无汗综合征，患者EDA基因的第5外显子存在突变位点：918位碱基C突变为A，致使226位线氨酸变为终止码，结论：该患者是X染色隐性遗传的外胚层发育不全无汗综合征，其EDA基因5外显子存在突变。