KRT6a基因N172del新发突变导致I型先天性厚甲症

目的：检测1例I型先天性厚甲症患者KRT6a基因突变。方法：提取该患者及其父母和100名正常对照外周血白细胞基因组DNA,设计针对KRT6a和KRT16基因的特异性引物,PCR扩增KRT6a和KRT16基因的全部外显子,并进行直接测序。结果：PCR扩增结合DNA测序发现该患者KRT6a基因第1外显子存在异常,第514—516位的3个核苷酸AAC缺失,导致第172位氨基酸一天冬酰胺（N）缺失。患者父母及100名正常对照均未发现此突变。未发现KRT16基因突变。结论：KRT6a基因N172del突变可能是导致本例I型先天性厚甲症的致病突变,该突变非遗传自父母,为新发突变。     

KRT6a基因N172del新发突变导致Ⅰ型先天性厚甲症

目的:检测1例Ⅰ型先天性厚甲症患者KRT6a基因突变.方法:提取该患者及其父母和100名正常对照外周血白细胞基因组DNA,设计针对KRT6a和KRT16基因的特异性引物,PCR扩增KRT6a和KRT16基因的全部外显子,并进行直接测序.结果:PCR扩增结合DNA测序发现该患者KRT6a基因第1外显子存在异常,第514-516位的3个核苷酸AAC缺失,导致第172位氨基酸-天冬酰胺(N)缺失.患者父母及100名正常对照均未发现此突变.未发现KRT16基因突变.结论:KRT6a 基因N 172del突变可能是导致本例Ⅰ型先天性厚甲症的致病突变,该突变非遗传自父母,为新发突变.     

表皮松解性角化过度型鱼鳞病一家系K10基因突变的研究

目的:检测一表皮松解性角化过度型鱼鳞病家系K10基因突变位点.方法:提取该家系成员的外周血DNA,采用聚合酶链反应(PCR)及DNA直接测序方法,检测患者角蛋白1(K1)及K10的基因突变.结果:该家系2例患者存在K10基因的杂合点突变,即在K10基因第2140位G→A,导致其第156位的精氨酸变为组氨酸(R156H).结论:K10 R156H是导致该家系2例患者临床表型的特异突变,进一步证实K10基因第156位密码子是突变热点.     

先天性厚甲症2家系中角蛋白16和17的基因突变

目的了解先天性厚甲症Ⅰ型(PC-Ⅰ)及Ⅱ型(PC-Ⅱ)患者家系的基因突变。探讨其基因突变和临床表现的关系。方法扩增外周血基因组DNA中角蛋白16基因的第1~6外显子及K17基因的第1外显子,对PCR产物进行序列分析。结果PC-Ⅰ家系(家系1)中患者K16基因第127位密码子由CGC突变CCC,导致K16角蛋白1A区的精氨酸由脯氨酸替代(R127P);PC-Ⅱ家系(家系2)中2例患者K17基因第99位密码子由CTG突变为CCG,导致K17角蛋白1A区的亮氨酸由脯氨酸替代(L99P),而这两个家系中的正常人及与此两家系无关的50例正常人未发现此突变。结论该PC-Ⅰ家系存在角蛋白16的R127P突变,PC-Ⅱ家系存在角蛋白17的L99P突变。3例厚甲症患者检测到的2个角蛋白突变均由K16及K17发生错义突变,导致其编码的相应氨基酸由脯氨酸替代。此类突变可引发较重的临床表现,即呈现典型PC-Ⅰ型或PC-Ⅱ型,不会呈现其他较轻的临床亚型。     

先天性厚甲症Ⅱ型一家系角蛋白17基因突变的研究

目的 研究一先天性厚甲症Ⅱ型(PC-Ⅱ)患者家系基因突变,探讨基因突变和临床表现的关系.方法 PCR扩增外周血基因组DNAK17基因第一外显子，PCR产物进行序列分析。结果 家系中3例患者（2例为迟发型厚甲，分别在4岁和15-16岁发生）K17基因第92位密码子由AAT突变为AGT，导致K17角蛋白1A区N92S突变，而该家系中的2例正常人及与该家系无关的50例正常人未发现此突变。结论 该PC-Ⅱ型家系存在K171A区N92S突变，K171A区突变可呈现为迟发型先天性厚甲。角蛋白基因突变位置可能不是PC-Ⅱ厚甲发生年龄的唯一决定因素，可能还存在其它遗传或环境因素决定PC-Ⅱ厚甲发生的年龄。     

播散性浅表性光化性汗孔角化症MVK基因突变检测

目的：研究2个中国汉族播散性浅表性光化性汗孔角化症（DSAP）家系患者MVK基因突变。方法：提取2个DSAP家系及1 0 0 名无亲缘关系的健康对照外周血DNA,采用PCR扩增患者MVK基因的全部外显子及其侧翼序列,用Sanger测序法对PCR扩增产物直接测序检测基因突变。结果：发现1 个新的剪切位点突变（c.1040-2A>C）和1 个已报道的错义突变（c.1094T>C）。结论：本研究进一步证实MVK基因突变与DSAP发病相关。     

慢性家族性良性天疱疮五家系ATP2C1基因突变分析

目的：检测中国汉族慢性家族性良性天疱疮5家系ATP2C1基因突变情况。方法：提取5家系中12例患者、13名表型正常及100名正常对照外周血基因组DNA，经PCR扩增后进行DNA测序，并使用Chromas软件解析。结果：家系1中3例患者存在ATP2C1基因第18号外显子c.1738A>G（p.I580V）突变，家系2 中2例患者存在ATP2C1基因第25号外显子c.2416C>T(p.R806*)突变，家系3中3例患者存在ATP2C1基因第15号外显子c.1250G>A（p.R417K）突变，家系4和家系5 中ATP2C1基因未发现突变。上述家系内表型正常个体及100名正常对照中均未检测到相应突变。结论：ATP2C1基因突变可能在3例汉族HHD家系内发挥致病作用。     

Ⅱ型先天性厚甲症一家系KRT基因突变检测

目的:检测先天性厚甲症一家系中KRT6b和KRT17基因突变位点。方法:提取先证者、其父母(母亲为患者,父亲正常人)及100名正常对照者外周静脉血DNA,PCR技术扩增KRT6b和KRT17基因编码序列,Sanger测序法对PCR扩增产物进行测序。结果:先症者及其母亲在KRT17基因1号外显子上存在错义突变(c.275AG),KRT6b基因不存在任何突变。先证者父亲及100名正常对照者中未检测到任何突变。结论:此家系患者是由于KRT17基因突变(c.275AG,p.Asn92Ser)所致。     

KRT6A基因N171S新生突变引起Ⅰ型先天性厚甲症一例

目的 探讨Ⅰ型先天性厚甲症患者KRT6A基因的突变与l临床表型的关系.方法 提取1例新疆维吾尔族Ⅰ型先天性厚甲症散发病例及其父母和50例正常对照外周血白细胞基因组DNA,应用特异性引物进行PCR扩增,产物纯化后直接进行测序.结果 PCR扩增结合DNA测序发现该患者KRT6A基因第1外显子存在异常,第512位由腺嘌呤(A)突变为鸟嘌呤(G),导致KRT6A角蛋白1A起始区第171位编码氨基酸由天冬酰胺(N)变为丝氨酸(S),即发生N171S错义突变.患儿父母及与家系无血缘关系的50例正常对照均未发现此突变,说明N171S导致新疆维吾尔族Ⅰ型先天性厚甲症的新生基因突变.结论 KRT6A基因N171S突变是导致本例新疆维吾尔族Ⅰ型先天性厚甲症的新生突变.     

一先天性厚甲家系角蛋白基因突变鉴定

目的 探讨一个先天性厚甲家系角蛋白基因突变。方法 用PCR及Sanger测序技术对先天性厚甲家系先症者KRT17基因所有外显子和KRT6B基因编码螺旋起始和终止区域序列进行突变鉴定,针对发现的可疑位点,Sanger测序检测家系其他成员该位点的变异情况。结果 基因检测结果表明,家系患者KRT17基因错义突变c.263T 〉 C,该突变导致角蛋白17（K17）第88位氨基酸由蛋氨酸变成苏氨酸（p.M88T）,KRT6B基因未见异常。结论 KRT17基因c.263 T 〉 C（p.M88T）突变是该先天性厚甲家系致病基因突变。     

A novel missense mutation L468Q of keratin 6a in pachyonychia congenita type 1

BACKGROUND: Pachyonychia congenita is an autosomal dominant disorder that usually develops in early infancy. The major features of the syndrome are hypertrophic nail dystrophy, palmoplantar keratoderma and oral leucokeratosis, accompanied by other ectodermal defects, according to subtype. OBJECTIVE: To analyse the K6a gene mutation in a sporadic Chinese patient with pachyonychia congenita type 1 (PC-1) and to explore the relationship between the genotype and phenotype of PC-1. METHODS: Genomic DNA was extracted from peripheral blood of the patient with PC-1 and 100 unrelated controls. The whole coding region of K6a gene was amplified using long-range polymerase chain reaction (PCR); nested PCR was then used to amplify the mutation 'hot-spot' of the K6a gene. The PCR products were directly sequenced to detect the mutation. RESULTS: A novel missense mutation L468Q in the helix 2B domain of the K6a polypeptide was identified in the patient but not in the healthy individuals from the family and 100 unrelated control individuals. CONCLUSIONS: We describe this mutation for the first time, and provide further evidence that the helix boundary motif sequences of K6a are a mutation 'hot-spot'.     

A mutation detection strategy for the human keratin 6A gene and novel missense mutations in two cases of pachyonychia congenita type 1

Pachyonychia congenita type 1 (PC-1) is an autosomal dominant ectodermal dysplasia characterized by hypertrophic nail dystrophy, focal non-epidermolytic palmoplantar keratoderma and variable features of oral leukokeratosis and follicular keratosis. Previously, we have shown that this disease can be caused by mutations in type I keratin K16 and one mutation has been reported in its type II keratin expression partner, K6a. Mutation analysis for K6a has been hampered by the presence of multiple copies of the K6 gene in the human genome, of which some are expressed and others are pseudogenes. Here, we describe a mutation detection strategy where the entire KRT6A gene, approximately 7 kb, is specifically amplified by long-range PCR. Using this technique, we have detected two novel mutations in the 1A domain of the K6a polypeptide, N171K and F174S. Mutations were confirmed in the affected individuals and were excluded from 50 unaffected unrelated individuals by restriction enzyme analysis of KRT6A PCR products. Additionally, mutation N171K was confirmed by RT-PCR in mRNA derived from lesional palmoplantar epidermis of an affected individual, confirming the specificity of the genomic PCR for the functional K6a gene. This, together with a similar strategy which we have developed for the K16 gene, provide a robust system for mutation detection and prenatal diagnosis for patients with PC-1.     

弹性假黄色瘤一例ABCC6基因突变分析

目的 报告1例弹性假黄色瘤,并检测ABCC6基因突变情况。方法 分析1例弹性假黄色瘤先证者的临床资料,并收集其父母、儿子及100例无亲缘关系的健康对照的外周血,提取基因组DNA,PCR扩增ABCC6基因编码区31个外显子,并进行DNA直接测序与ABCC6编码蛋白质功能预测。结果 先证者父母及儿子表型均正常。基因突变分析显示,先证者存在ABCC6基因c.373G > A（p.E125K）和c.3703C > T（p.R1235W）的复合杂合突变,先证者母亲为c.3703C > T（p.R1235W）杂合突变携带者,先证者父亲和儿子为c.373G > A（p.E125K）杂合突变携带者,而100例无亲缘关系的健康对照均未检测到该两种突变。物种间序列比对分析发现,ABCC6编码蛋白质第125位谷氨酸和第1235位精氨酸为进化高度保守的序列,SIFT和Polyphen?2软件预测c.373G > A（p.E125K）和c.3703C > T（p.R1235W）突变为有害变异位点。结论 ABCC6基因c.373G > A（p.E125K）和c.3703C > T（p.R1235W）的复合杂合突变可能是该例弹性假黄色瘤患者发病的原因。     

多发性脂囊瘤角蛋白17基因的突变研究

目的:研究多发性脂囊瘤(SM)角蛋白17基因的突变,为进一步开展基因诊断和基因治疗奠定基础。方法:抽取多发性脂囊瘤患者、患者父母以及100例正常人静脉血提取基因组DNA,采用聚合酶链反应(PCR)的方法对角蛋白17外显子1及其侧翼和外显子6进行扩增,并对其PCR产物直接进行双向测序以检测突变。结果:在家系中两例患者第42位密码子由CTG突变为CCG,结果导致角蛋白17 V1区杂合错义突变(L42P),即亮氨酸由脯氨酸替代,而此家系中的正常人和与该家系无关的100例正常人的DNA测序结果未发现此突变。结论:此家系中患者表型可能由角蛋白17基因头区的L42P突变所致。     

家族性良性天疱疮一家系报道及其ATP2C1基因筛查

目的 报道一个家族性良性天疱疮家系并对其致病基因ATP2C1进行突变筛查。方法 对先证者及其家族4代成员进行临床调查。采集每一成员静脉血标本,同时采集50例健康人血液标本作为对照。提取外周血基因组DNA,分别对 ATP2C1基因的所有28个外显子及其侧翼内含子序列进行PCR扩增,再对每一扩增产物进行直接测序,最后将测序结果分别与基因库（NM_014382.2和NC_000003.9）的编码序列和基因组序列进行逐一比对分析。结果 调查该家系4代24个成员,共有8例患者。基因筛查显示先证者和该家族其他患者的ATP2C1基因第17号外显子上发生一单核苷酸碱基置换,即c（1696C→T）;同时该家族中第2代、第3代正常成员和50例健康对照均未检测到这一碱基变化。第4代4个成员中,仅有1个成员,即Ⅳ3,亦检测到这一变化。结论 该家系患者ATP2C1基因发生c（1696C→T）无义突变,可能是家族性良性天疱疮的致病突变;Ⅳ3携带该突变,但到目前为止,其未发生家族性良性天疱疮的相关临床症状,有必要对其进行密切随访。     

Identification of sporadic mutations in the helix initiation motif of keratin 6 in two pachyonychia congenita patients: further evidence for a mutational hot spot

Pachyonychia congenita (PC) is a rare, autosomal dominant, ectodermal dysplasia characterized most distinctly by the presence of symmetric nail hypertrophy. In the Jadassohn-Lewandowsky form, or PC-1, additional cutaneous manifestations may include palmoplantar hyperkeratosis, hyperhidrosis, follicular keratoses, and oral leukokeratosis. Mutations have previously been identified in the 1A helix initiation motif of either keratin 6 or keratin 16 in patients with PC-1. In the current study, we have identified 2 sporadic, heterozygous mutations in the 1A helix region of the K6 isoform (K6a). The first mutation identified was a 3 base pair deletion (K6adelta N171). The second mutation was a C-to-A transversion resulting in an amino acid substitution (K6a N171K). These data, in combination with previous reports, provide further evidence that this location is a mutational hot spot.     

一个表皮松解性掌跖角化病家系的KRT1基因突变分析

目的 探讨一个中国汉族人表皮松解性掌跖角化病(EPPK)家系的角蛋白基因KRT1、KRT9、KRT10突变情况.方法 收集1个EPPK家系的临床资料,提取外周血DNA,通过PCR扩增角蛋白KRT1、KRT9、KRT10基因编码区的全部外显子及其侧翼序列并测序,以表型正常家系成员及50例健康人为正常对照.结果 发现家系内6例患者均存在KRT1基因错义突变c.1436T>C,导致第479位的异亮氨酸被苏氨酸取代(I479T),在家系中6例正常人及50例对照者未发现上述突变.结论 错义突变KRTI的c.1436T>C可能为导致该家系临床表型的主要原因.本例为国内首次发现的KRT1突变引起的EPPK家系.

Abstract：

Objective To analyze the mutations in keratin 1 (KRT1), KRT9 and KRT10 genes in a Chinese family with epidermolytic palmoplantar keratoderma (EPPK). Methods Clinical data were collected from a family with EPPK. Genomic DNA was extracted from the peripheral blood of 12 family members, including 6 patients and 6 unaffected members, as well as from 50 unrelated normal human controls. PCR was performed to amplify all the exons and flanking sequences of KRT1, KRT9 and KRT10 genes followed by DNA sequencing.Results A missense mutation C.1436T > C was found in the highly conserved helix termination motif of KRT1 gene of all the patients, resulting in a substitution of isoleucine by threonine at position 479 of the KRT1 protein. No mutation was found in the unaffected members or unrelated controls. Conclusions The missense mutation C.1436T > C in K.RT1 gene is likely to be the main cause of the phenotype of EPPK in this family.This is the first report of a pedigree with KRT1 gene mutation-induced EPPK in China.