一个表皮松解性掌跖角化病家系的KRT9基因突变分析

目的明确一个伴随有类似关节指垫样病损和指甲病变的表皮松解性掌跖角化病的中国家系中角蛋白9(keratin9,KRT9)基因突变情况。方法用聚合酶链反应技术扩增家系成员及家系外50名正常人KRT9基因的编码区及外显子与内含子交界处,DNA序列分析寻找突变位点,然后经限制性内切酶Dde分析验证。结果患者KRT9基因第1外显子第160位密码子发生AAT→AGT的突变(N160S),而家系正常成员及家系外50个正常人中均不存在此突变。结论KRT9基因的第1外显子第160位密码子发生AAT→AGT突变(N160S)导致该家系患者发生表皮松解性掌跖角化病。     

先天性大疱性鱼鳞病样红皮病基因突变检测

目的 研究1例先天性大疱性鱼鳞病样红皮病散发患者的基因突变情况,探讨基因型与表现型的相关性.方法 应用聚合酶链反应扩增KRT1和KRT10基因的热点突变区,通过DNA直接测序的方法,对先天性大疱性鱼鳞病样红皮病患者、家系中的正常成员和50名无亲缘关系的正常个体的KRT1和KRT10基因进行突变检测.结果 在患者KRT10基因的第1外显子上发现了1个错义突变(467G→A),导致角蛋白10(KRT10)1A区的精氨酸由组氨酸替代(R156H),而家系正常成员和无亲缘关系的50名正常对照中均未发现该突变.结论 KRT10基因第1外显子突变(467G→A)在该例先天性大疱性鱼鳞病样红皮病患病的分子机制中发挥重要作用.     

一个表皮松解性掌跖角化病维吾尔家系致病基因研究

目的 对一个维吾尔族表皮松解性掌跖角化病(epidermolytic palmoplantar keratoderma,EPPK)家系角蛋白9基因(keratin 9 gene,KRT9)进行测序,以检测其是否为该病的致病基因.方法 提取新疆地区一个维吾尔族EPPK家系外周血基因组DNA,针对已知候选基因KRT9和KRT1,分别对其所在染色体位置17q12-q21和12q13选取遗传标记进行连锁分析研究,确定连锁区域后,对区域内KRT9基因所有外显子进行测序分析.结果 分别得到48个家庭成员遗传标记的基因型和单倍型,经Linkage软件计算分析,发现标记D17S1787在=0时Lod值达到8.65,并最终将该病候选区域定位于遗传标记17/TG/36620115-D17S846之间约1 Mb范围内.排除该病与位于染色体12q13上的遗传标记DI2S96(θ=0时Lod=-∞)连锁.未发现KRT9基因存在致病性突变.结论 提示该表皮松解性掌跖角化病家系的致病基因位于染色体17q21.2上(chr17:36620083-37146934)约1 Mb区域内,且突变位点不位于KRT9基因编码区.     

一个弥漫性表皮松解性掌跖角化病家系角蛋白9基因R162W突变

目的 研究1个弥漫性表皮松解性掌跖角化病(epidermolytic plamoplantar keratoderma,EPPK)家系中的基因突变情况.方法 收集1个弥漫性表皮松解性掌跖角化病家系的外周血标本,采取聚合酶链反应结合DNA直接测序的方法,检测了该家系中4例患者及3名表型正常者和100名无亲缘关系健康个体的KRT9基因突变情况.结果 该家系中患者存在KR79基因上第484位C突变成T,使得KRTY基因的第1外显子162位密码子由CGG突变成TGG,导致正常精氨酸被色氨酸所取代,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 EPPK家系中患者KRT9基因存在错义突变(484C→T),这可能是导致EPPK发病的分子机制之一.     

表皮松解性掌跖角化症的分子遗传学研究进展

阐述了角蛋白分子的结构、功能以及由角蛋白突变所引起的一些遗传性角蛋白病 ,总结了迄今所发现的导致表皮松解性掌跖角化症的 17种不同的角蛋白 9基因突变类型 ,以及在各个种族或民族中开展的有关角蛋白基因突变的研究情况。     

一个表皮松解性掌跖角化病家系的角蛋白9基因突变研究

目的 确定一个中国汉族表皮松解性掌跖角化症(epidermolytic palmoplantar keratoderma,EPPK)家系角蛋白9基因(keration 9,KRT9)的突变情况,并分析基因型与表型的对应关系.方法 收集该家系12例患者、13名健康者和100名无亲缘关系的正常人的外周血,提取基因组DNA.采用PCR扩增KRT9基因的第1和第6外显子,对PCR产物进行双向测序以检测基因突变.结果 在所有患者中均检测到KRT9基因第1外显子中的杂合型488G→A突变,导致第163位的精氨酸被谷氨酰胺取代(R163Q).在家系中13名正常人和家系外100名正常人未检测到同样的突变.结论 KRT9基因的错义突变488G→A是该家系发生表皮松解性掌跖角化症的主要原因.     

A mutational hot spot in keratin 10 (KRT 10) in patients with epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis (EHK), (bullous congenital ichthyosiformerythroderma), is an autosomal dominant human skin disorder.Recently, we and others have described mutations in keratins1 and 10 (K1 and K10) in patients with this disease. Structure-functionmodels predict that these mutations would impair normal filamentassembly and function. We have extended our earlier studiesto include 8 more incidences of EHK. In half of these families,we were unable to locate a mutation within the rod domains ofeither K1 or K10. However, polymorphic restriction site andsequence analysis of the other families revealed a mutationalhot spot within the 1A alpha-helical segment of K10. These involveArginine to Histidine, Arginine to Cysteine and Arginine toLeucine substitutions at residue 10 of the rod domain. Interestingly,mutations in the corresponding Arginine residue in keratin K14have been identified in patients with epidermolysis bullosasimplex. The large number of mutations found at this positionin both keratins K10 and K14 suggests that other epithelia celldisorders will be discovered that are caused by the correspondingmutation in related type I keratin genes.     

中国南方斑驳病一家系的一种新的KIT基因突变

目的研究1个斑驳病家系的基因突变情况。方法经组织病理、电镜检查结合典型的临床特征确立斑驳病的诊断。采用聚合酶链反应及DNA直接测序的方法对此家系进行基因突变检测。结果家系中先证者存在K／T基因第2528位G→A（或C→T），使密码子AGT〉AAT，导致A850N。100名健康对照组不存在此突变。结论S850N可能是引起该家系临床表型的原因。     

A recurrent 1992delCT mutation of the type X collagen gene in a Japanese patient with Schmid metaphyseal chondrodysplasia

Summary We report here a recurrent frameshift mutation within the carboxyl-terminal noncollagenous domain coding region of the type X collagen gene (COL10A1) in a Japanese patient with Schmid metaphyseal chondrodysplasia. The mutation involves deletion of a CT dinucleotide from position 1992 (1992delCT), and produces a frameshift which creates a premature termination codon close to the site of the deletion. The predicted length of the mutant polypeptide is 664 amino acids, which is shorter than the wild type polypeptide (680 amino acids). A 1992delCT mutation ofCOL10A1 has been previously reported in one family. The independent occurrence ofde novo mutation of this specific dinucleotide repeat suggests that this region is a possible mutational hot spot onCOL10A1.     

局灶性真皮发育不全患者PORCN基因突变研究

目的 检测局灶性真皮发育不全(focal dermal hypoplasia,FDH)患者PORCN基因突变情况,探讨基因型与表型的相关性.方法 收集1例局灶性真皮发育不全患者及其家系3名正常成员及50名无血缘关系的健康对照者的外周血,采用PCR扩增PORCN基因所有外显子及其邻近的剪切位点进行直接测序.结果 患者PORCN基因核苷酸序列第2外显子上第149位碱基由G变为C,导致第38位氨基酸丙氨酸被脯氨酸代替(A38P),该患者家属及健康对照无此突变.患者临床表现中度.结论 该患者存在PORCN基因A38P突变,为一新的错义突变,可能是导致FDH发病机制之一.G149C基因型与中度的表型之间的关系与A38P错义突变对蛋白的影响有关,与以往结论不同.     

一例Apert综合征患者的FGFR2基因突变分析

目的 确定1例Apert综合征患者是否存在成纤维细胞生长因子受体2(fibroblast growth factor receptor 2,FGFR2)基因突变.方法 收集患者及其父母的外周血,提取基因组DNA.采用PCR扩增FGFR2基因第7和第9外显子,对PCR产物进行双向测序检测基因突变.结果 发现患者FGFR2基因第7外显子的934 C→G突变,导致了FGFR2蛋白第252位丝氨酸被色氨酸取代(S252W),与国外报道的致病性突变一致.结论 FGFR2基因第7外显子的P34 C→G突变是该例Apert综合征的致病原因.     

中国汉族人念珠状发患者hHB6基因突变检测

目的 对同患念珠状发的母女俩患者进行分子遗传学分析,确定其致病原因.方法 调查患者家系系谱,取患者和正常对照的头发进行常规的显微观察,获取临床信息.抽提所有参与实验的家系成员的全基因组DNA.PCR扩增人类毛发碱性角蛋白6基因(human hair basic keratin 6 gene,hHB6)的所有外显子和外显子-内含子交界区.采用直接测序法检测突变.用限制性片段长度多态性分析验证突变和在该家系内突变是否与疾病共分离,以及该突变是否在正常人群中存在.结果 在其中一个患者中检测到了hHB6基因的一个杂合突变c.1204G＞A(p.E402K).限制性片段长度多态性分析确证了母女俩患者均携带该突变,而家系中其他人(表型均正常)和150名不相关的正常中国汉族人不携带该突变.结论 在中国汉族人念珠状发患者中检测到了hHB6基因c.1204G＞A(P.E402K)突变,由母亲遗传给其女儿.结果表明毛发角蛋白hHB6在念珠状发发病机制中起关键的作用,初步显示hHB6基因常见c.1204G＞A(P.E402K)突变也是导致中国人患念珠状发的原因.     

A novel nonsense mutation of the PEPD gene in a Japanese patient with prolidase deficiency

A nonsense mutation at amino acid residue 184 in the human peptidase D (PEPD) gene caused the production of a truncated polypeptide. Characterizing molecular defects in patients provides clues to elucidate the relationship between the phenotype and the genotype. Received: November 8, 1999 / Accepted: November 24, 1999     

Characterization of a novel mutation in exon 10 of the adrenoleukodystrophy gene

We have detected a novel mutation in the adrenoleukodystrophy (ALD) gene in skin fibroblasts in primary culture derived from a patient suffering from the adrenocortical insufficiency-only-phenotype of ALD. This nonsense mutation (C2400T/Q672X) is the only mutation reported to date affecting exon 10. It leads to a translation product lacking the 74 C-terminal amino acids. As a consequence of the loss of this region, which immediately follows the putative nucleotide binding domain, the ALD protein (ALDP) was not detectable at all by ALDP-specific monoclonal antibodies. Since ALDP-specific mRNA was readily detected in these fibroblasts, the loss of protein is probably not attributable to RNA instability but may be explained by protein instability. If the Q672X mutation leads in fact to an unstable translation product this would be consistent with the hypothesis that the C-terminus is crucial for ALDP stability.     

一个近亲婚配家系中的一种P基因新突变

目的在DNA水平上对1个有2例患者的姨表兄妹近亲婚配家系中的眼皮肤白化病患者进行分型诊断。方法用PCR扩增先证者P基因及TYR基因各外显子、外显子-内含子交界区及3＇端和5＇端非翻译区，以DNA序列测定技术检测基因突变，以DNA测序技术和限制性内切酶酶切法检测该家系其他成员及群体中正常人的相应基因位点。结果先证者和其白化病妹妹为P基因A787T突变纯合子，其父母和表型正常的弟弟均为A787T突变杂合子。先证者TYR基因未见突变。群体中102名表型正常者中无A787T突变等位基因。结论在基因水平确定我国存在眼皮肤白化病Ⅱ型，同时发现了一种P基因病理性新突变。     

先天性厚甲症Ⅱ型一家系KRT17基因突变研究

目的 研究1个先天性厚甲症Ⅱ型家系的基因突变情况.方法 收集该家系的详细临床资料,外周血提取基因组DNA,PCR扩增KRT17热点突变区,通过PCR扩增产物直接测序方法对该家系患者、正常成员和100名无亲缘关系的正常人进行KRT17基因突变检测.结果 在该家系患者KRT17基因的第1外显子上发现了1个错义突变(296 T→C),导致KRT17的1A区亮氨酸由脯氨酸替代(L99P),而家系中正常成员和家系外100名正常对照中均未能发现该突变.结论 该家系患者的临床表现为KRT17发生突变(L99P)所致,结合文献复习证实部分PC-Ⅱ家系基因型与表现型之间存在一定相关性.

Abstract：

Objective To investigate the keratin 17 gene (KRT17)mutation in a pedigree with pachyonychia congenita type 2 (PC-Ⅱ ). Methods DNA was extracted from the blood samples of the patients, unaffected members of the pedigree, and 100 unrelated healthy controls. PCR was performed to amplify the hot spots in KRT17 gene. PCR products were directly sequenced to detect mutation. Results A heterozygous 296T--C mutation was found in all the affected members of this family, which resulted in the substitution of leucine by proline in codon 99 (L99P) in the 1A domain of the KRT17, but not in the healthy individuals from the family and the 100 unrelated controls. Conclusion The mutation of KRT17 may play a major role in the pathogenesis of this pedigree with pachyonychia congenita type 2.     

Crouzon综合征基因突变检测

目的 研究1个Crouzon综合征家系及1例散发的Crouzon综合征患者的成纤维生长因子受体2(fibroblast growth factors receptor 2,FGFR2)基因突变情况.方法 在1个Crouzon综合征家系的10名成员,和另一例散发者的外周血提取基因组DNA,PCR扩增FGFR2基因的第8和10外显子(部分家族成员仅扩增第8外显子),产物纯化后直接进行DNA测序检测突变.结果 家系中3名成员及另1例散发者FGFR2基因第8外显子的833位核苷酸发生G→T的转换突变,该突变为错义突变,使该位点所编码的氨基酸由半胱氨酸变为苯丙氨酸(C278F).该突变为杂合子突变.结论 FGFR2基因突变是Crouzon综合征致病原因.     

斑驳病的一种新的KIT基因突变

目的 研究1个斑驳病家系先证者及其母亲的基因突变情况。方法 经组织病理、电镜检查结合典型的临床特征确立斑驳病的诊断。采用聚合酶链反应及DNA直接测序的方法对1个斑驳病家系进行基因突变检测。结果 家系中先证者存在KIT基因第1833位G〉A，导致V6041。100名健康对照者及其它家庭成员不存在此突变。结论 V6041是引起该家系临床表型的原因。     

Aberrant heterodimerization of keratin 16 with keratin 6A in HaCaT keratinocytes results in diminished cellular migration

Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments. In keratinocytes, type II keratin 6 (K6) interacts with type I keratin 16 (K16). We previously showed that the intermediate filament protein K16 is up-regulated in aged human skin. Here, we report that there is an obvious imbalance of K16 to K6 mRNA in in vivo and in vitro aging, which possibly leads to cellular effects. To unveil a possible biological function of K16 overexpression we investigated the migration potential of keratinocytes having up-regulated K16 expression in vitro. Two cell lines were established by transfection of human keratinocytes (HaCaT cells) with K16 or control vectors and subsequent fluorescence-activated cell sorting. By performing migration assays we were able to show a 90% reduction in the migration ability of the K16-overexpressing keratinocytes. In addition, a delay in wound closure associated with K16-overexpressing cells was shown by scratch assays. Transient overexpression of K6A in K16-overexpressing keratinocytes partially corrected the cell-migration defect. By real-time PCR we excluded co-regulation of the annotated interaction partner, K6, in the K16 cell line. Finally, we observed a decreased level of tyrosine phosphorylation in K16-overexpressing cells. Taken together, these data highlight the possibility of a physiological role for K6/K16 heterodimers in keratinocyte cell migration, in addition to the heterodimer's known functions in cell differentiation and mechanical resilience.