A型着色性干皮病1例XPAC基因的复合无义突变

目的检测1例A型着色性干皮病患者及其父母的XPAC基因的突变情况。方法收集患者及其父母资料,提取外周血DNA,采用PCR扩增XPAC基因编码区的全部外显子,DNA直接测序,明确突变位点,并以50个无关正常人作对照。结果患者XPAC基因分别于第631及第682位发生C→T突变,使第211位及第228位氨基酸均由精氨酸变成终止密码子(R211X,R228X)突变,两突变分别来自其父母。50例健康对照者不存在此两种突变。结论R211X及R228X复合无义突变为引起该患者的A型着色性干皮病的病因。     

板层状鱼鳞病患者转谷氨酰胺酶1活性缺失及其基因突变

目的 检测一板层状鱼鳞病家系中患者转谷氨酰胺酶1的活性及其编码基因的突变。方法 以免疫组化法检测患者转谷氨酰胺酶1的活性,PCR扩增该基因的全部编码序列,并行DNA测序。结果 患者皮肤转谷氨酰胺酶1的活性完全缺失。PCR结合DNA测序发现患者该基因第4外显子存在异常:第604位碱基由胞嘧啶突变为胸腺嘧啶,使第202位氨基酸由谷氨酰胺(Q)变为终止密码(R202X),导致其编码的蛋白缺失了C端的615个氨基酸。其父母皆为杂合子。结论 板层状鱼鳞病患者转谷氨酰胺酶1的活性完全缺失,是其转谷氨酰胺酶1基因的无义突变,引起编码的蛋白缺陷。     

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。     

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.     

三个表皮松解性掌跖角化症家系KRT9基因突变的研究

目的 探讨表皮松解性掌跖角化症家系的KRT9基因突变与临床表现的关系。方法 PCR扩增KRT9基因编码氨基酸的7个外显子,对扩增产物进行变性高效液相色谱分析、DNA测序。结果 在所研究的3个EPPK家系中,发现KRT9基因第1外显子第497位核苷酸A缺失并插入GGCT,导致角蛋白9分子第166位酪氨酸缺失并插入色氨酸和亮氨酸,即Y166delinsWL。片段特异性PCR证实了该突变不是一个常见的多态性,而是国际中间纤维突变库(http://www.interfil.org)中未报道过的一种新突变。结论 KRT9基因497delAinsGGCT突变可能是部分中国人EPPK患者发病的遗传基础。     

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。     

Hartnup病一家系基因突变研究

目的: 研究一Hartnup病家系的氨基酸转运蛋白基因(SLC6A19)的突变.方法: 提取Hartnup病患者及家族成员的基因组DNA,采用聚合酶链反应(PCR)扩增SLC6A19基因所有的外显子,并对PCR产物进行测序序列分析.结果: Hartnup病患者SLC6A19基因存在异常:第6外显子第850位碱基由鸟嘌呤变为腺嘌呤,使第284位氨基酸由甘氨酸(G)转变为精氨酸(R),即G284R错义突变.其弟与患者突变相同.其父母均为G284R突变杂合子.结论: 该Hartnup病家系由氨基酸转运蛋白基因(SLC6A19)的G284R错义突变所致.     

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.     

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。     

板层状鱼鳞病TGM1基因突变研究

目的 探讨一个板层状鱼鳞病家系转谷氨酰胺酶1基因(TGM1)的突变.方法 提取板层状鱼鳞病患者及家族成员的基因组DNA,采用PCR扩增TGM1基因所有的外显子及其邻近的剪切点并进行双向直接测序,并对TGM1基因的同源性进行分析.结果 板层状鱼鳞病患者TGM1基因存在异常:外显子3的第504位碱基由胞嘧啶突变为胸腺嘧啶,使第142位氨基酸由精氨酸(R)转变为半胱氨酸(C),即R142C错义突变;外显子7的第1122位碱基由胞嘧啶突变为胸腺嘧啶,使348位氨基酸由精氨酸(R)突变为终止密码(R348X),导致其编码的蛋白缺失了C端的470个氨基酸.其父亲为R142C杂合子,母亲为R348X突变杂合子;R142C错义突变位于TGM1基因保守区域.结论 该板层状鱼鳞病患者存在转谷氨酰胺酶1基因的R142C错义突变和R348X无义突变.     

DNA-based prenatal diagnosis in a Chinese family with xeroderma pigmentosum group A

BACKGROUND: Xeroderma pigmentosum (XP) is a group of autosomal recessive diseases characterized by hypersensitivity to ultraviolet rays. Among its eight complementation groups, XP group A (XPA) is the most severe type. The XPAC gene has been identified as the defective gene in XPA patients. OBJECTIVES: To examine genomic DNA from a Chinese family with XPA, to determine the XPAC mutation and, after genetic counselling, to undertake DNA-based prenatal diagnosis in a subsequent pregnancy. METHODS: Fetal DNA was extracted from amniotic fluid and used to amplify exon 5 of XPAC containing the potential mutation. Direct sequencing and restriction endonuclease digestion were used for prenatal diagnosis. RESULTS: We identified a homozygous nonsense XPAC mutation of 631C-->T, which results in an R211X mutation in XPA protein, in the proband. Both her parents are heterozygous. Prenatal diagnosis demonstrated a heterozygous sequence predicting an unaffected child, and a healthy girl was born. CONCLUSIONS: These data provide the first example of a DNA-based prenatal test for genodermatosis in China.     

Severe neurological abnormalities associated with a mutation in the zinc-finger domain in a group A xeroderma pigmentosum patient

All the reported Japanese patients with group A xeroderma pigmentosum (XP) have two or three mutations at codon 116 in exon 3, codon 228 in exon 6, and the splicing acceptor site of intron 3 of XP group A complementing (XPAC) gene. A homozygote (XP390S) with a nonsense mutation at codon 228 has less severe neurological abnormalities than patients with the splicing mutation at the acceptor site of intron 3. As homozygotes for the nonsense mutation at codon 116, which truncates a carboxyl-terminal site of XPAC protein at an early part of its zinc-finger domain, have not been reported previously, the possible severity of associated neurological abnormalities was not known. We report a group A XP patient, XP180S, who had neurological abnormalities which were more severe than those in patients homozygous for the splicing mutation. The polymerase chain reaction product from exon 3 of the patient's XPAC gene was digested completely into three fragments by MseI restriction endonuclease. Thus, the patient was homozygous for the mutation at codon 116.     

Compound heterozygosity for the Xeroderma pigmentosum complementation group A gene associated with a mild phenotype

We describe a case of a 7-year-old boy diagnosed as xeroderma pigmentosum complementation group A (XPA). Severe photosensitivity developed at 5 months after birth, and at a visit to our hospital at the age of five years, multiple brownish freckles were present on the face. XPA complementing (XPAC) gene compensated the ability of DNA repair after UV-irradiation of the fibroblasts. PCR-RFLP and DNA sequencing analyses revealed compound heterozygosity for a splicing mutation (IV3 -1G => C) at the splicing acceptor site of intron 3 and a nonsense mutation (Arg228 => stop) in exon 6. The former mutation can be detected by a restriction enzyme Alw NI and the latter detected by Hph I. Neither obvious neurological symptoms nor malignant skin tumors were noted. This genotype is associated with milder clinical symptoms than homozygosity for the IV3 -1G => C mutation.     

Correlation of the clinical manifestations and gene mutations of Japanese xeroderma pigmentosum group A patients

Summary The gene responsible for xeroderma pigmentosum (XP) group A has recently been cloned and designated XPA gene. Previous studies have shown that most Japanese XPA patients have homozygous mutations for the splicing site of intron 3 of the XPA gene, which was recognized by restriction endonuclease (RE) AlwNI (AlwNI mutation). Other mutations found to date have been the nonsense mutation at codon 228 in exon 6, recognized by RE Hphi (HphI mutation), and at codon 116 in exon 3, recognized by RE Msel (Msel mutation). Using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis, we examined the point mutations of the XPA gene in 16 XPA patients, their parents, and their four asymptomatic siblings. We found that eight patients were homozygous for the AlwNI mutation, two were compound heterozygotes for the AIwNI mutation and the Hphl mutation, one was a compound heterozygote for the AIwNI mutation and the MseI mutation, three were compound heterozygotes for the AlwNl mutation and an unidentified mutation, and two were compound hcterozygotes for the Hphl mutation and an unidentified mutation, investigation of their clinical features suggested that the four patients who were heterozygous for the Hphl mutation and the AlwNI or an unidentified mutation had milder clinical manifestations such as later development of skin cancers and milder neurological deterioration, than those patients who were either homozygous for the AlwNI mutation or heterozygous for the AlwNI mutation and MseI mutation. PCR-RFLP analysis of the XPA gene in the four asymptomatic siblings of the XPA patients revealed that two were carriers of the mutated XPA allele, one was not a carrier, and one was not diagnosed because of the presence of an unidentified mutation. These data indicate that determination of the point mutation of the XPAC gene is important in predicting the clinical course in XPA patients. In addition, this method is useful for the detection of asymptomatic carriers in affected families, who have not been identified with conventional techniques.     

A novel XPA gene mutation and its functional analysis in a Japanese patient with xeroderma pigmentosum group A

Most Japanese patients with xeroderma pigmentosum group A (XPA) have the homozygous intron 3 splicing mutations (AlwNI mutation). Here, we report a Japanese XPA patient, XP79KO, a compound heterozygote with a newly identified T to G transversion at splice donor site in intron 1 in one allele, and with the AlwNI mutation in another allele in the XPA gene. The mutation in intron 1 creates two new abnormal splice sites that resulted in two types of aberrant mRNA. These abnormal splicings cause frameshifts that make stop codons downstream. No XPA protein was detected in XP79KO fibroblasts.     

Weber-Cockayne亚型单纯型大疱性表皮松解症一家系报告

目的:研究Weber-Cockayne亚型单纯型大疱性表皮松解症(EBS-WC)一家系的基因突变,并进行产前诊断。方法:应用PCR及DNA直接测序方法明确突变位点,针对所发现的突变以限制性内切酶片段长度多态性(RFLP)分析加以验证,在此基础上于妊娠24周时对从胎儿羊水所提取的DNA进行测序及酶切验证。结果:该家系患者存在角蛋白(keratin,KRT)5基因突变:第7外显子第1388位碱基由胸腺嘌呤突变为胞嘧啶,导致第463位氨基酸由亮氨酸变为脯氨酸(L463P)。50名健康对照者不存在此突变。羊水细胞DNA不存在此突变的胎儿,出生后未患大疱性表皮松解症。结论:KRT5第7外显子的突变是引起该家系临床症状的特异性突变。     

Reinvestigation of the classification of five cell strains of xeroderma pigmentosum group E with reclassification of three of them

Xeroderma pigmentosum is a photosensitive syndrome caused by a defect in nucleotide excision repair or postreplication repair. Individuals of xeroderma pigmentosum group E (xeroderma pigmentosum E) have a mild clinical form of the disease and their cells exhibit a high level of nucleotide excision repair as measured by unscheduled DNA synthesis, as well as biochemical heterogeneity. Cell strains from one group of xeroderma pigmentosum E patients have normal damage-specific DNA binding activity (Ddb+), whereas others do not (Ddb-). Using a refinement of a previously reported cell fusion complementation assay, the previously assigned Ddb+ xeroderma pigmentosum E strains, XP89TO, XP43TO, and XP24KO, with various phenotypes in DNA repair markers, were reassigned to xeroderma pigmentosum group F, xeroderma pigmentosum variant, and ultraviolet-sensitive syndrome, respectively. The Ddb- xeroderma pigmentosum E strains, XP82TO, and GM02415B, which showed almost normal cellular phenotypes in DNA repair markers, however, remained assigned to xeroderma pigmentosum group E. With the exception of the Ddb+ strain XP89TO, which demonstrated defective nucleotide excision repair, both Ddb- and Ddb+ xeroderma pigmentosum E cells exhibited the same levels of variation in unscheduled DNA synthesis that were seen in normal control cells. By genome DNA sequencing, the two Ddb- xeroderma pigmentosum E strains were shown to have mutations in the DDB2 gene, confirming previous reports for XP82TO and GM02415B, and validating the classification of both cells. As only the Ddb- strains investigated remain classified in the xeroderma pigmentosum E complementation group, it is feasible that only Ddb- cells are xeroderma pigmentosum E and that mutations in the DDB2 gene are solely responsible for the xeroderma pigmentosum E group.