类脂质蛋白沉积症家系ECM1基因突变检测

[摘要] 目的 检测一类脂蛋白沉积症家系细胞外基质蛋白1（ECM1）基因的突变,并探讨中国类脂蛋白沉积症家系ECM1基因的突变特点。方法 PCR扩增家系成员ECM1基因的全部外显子,并行DNA测序。以100例无关正常人作对照。结果 先证者及其胞兄的ECM1基因第六外显子均检测出第658位碱基发生T G纯合性突变,使220位的半胱氨酸转变为甘氨酸（p.C220G）。其父母均为该突变的杂合性携带者,该突变在100例无关正常对照中未被检测出。结论 p.C220G为引起本家系临床症状的特异性突变。     

类脂质蛋白沉积症两家系调查及ECM1基因突变检测

目的:检测类脂质蛋白沉积症二家系中细胞外基质蛋白(ECM1)基因突变位点。方法:提取1号家系先证者及其母亲,2号家系先证者、父母、配偶及儿子外周血DNA。PCR技术扩增ECM1基因编码序列,采用一代Sanger法对PCR扩增产物进行测序。结果:1号家系先证者在7号外显子发现已知突变(纯合突变c.960GA),其母亲为杂合携带者;2号家系先证者为遗传复合体,是上述突变位点的杂合携带者,此外在3号外显子上存在1个插入突变c.142insC。结论:类脂质蛋白沉积症存在遗传异质性。     

一类脂蛋白沉积症家系ECM1基因突变检测

目的：报道1例类脂蛋白沉积症家系,并对其家系成员的细胞外基质蛋白1（ECM1）基因突变进行分析。方法：PCR,DNA直接测序以及RFLP对患者的ECM1编码区进行了基因突变分析。结果：先证者及其胞姐在ECM1基因6号染色体上均发现纯合性单核苷酸颠换c.658T〉G,产生纯合错义突变p.C220G。该家系中父母二人均为此突变的杂合子,该突变在100个非相关对照中未被检测出。结论：p.C220G突变是引起该家系临床病变的特异突变,不是多态性变化。     

类脂蛋白沉积症一家系调查及基因突变检测

【摘要】 目的 报道1例来自山东的类脂蛋白沉积症家系,研究其家系成员的细胞外基质蛋白（ECM1）基因突变。 方法 类脂蛋白沉积症家系3代8名成员,有2例患者,分别为先证者（Ⅲ1）及其母亲（Ⅱ2）,均表现为睑缘丘疹、舌系带增厚变短及声音嘶哑。间接喉镜检查显示声带浸润增厚。睑缘及喉部黏膜组织病理检查均提示真皮透明蛋白样物质沉积,PAS染色强阳性,淀粉酶染色阴性,病理诊断符合类脂蛋白沉积症。采用DNA直接测序检测家系8例成员ECM1基因,巢式PCR琼脂糖凝胶电泳对患者的ECM1编码区进行基因突变分析。取100例正常汉族人基因组DNA样品做对照。 结果 2例患者均为遗传复合体,先证者母亲（Ⅱ2）ECM1的两个等位基因分别存在3个错义突变（p.P169T , p.A44T,p.R392W）。先证者除了从其母亲遗传来的一个错义突变外（p.P169T）,还有从其父亲（Ⅱ1）遗传来的一个同义突变（c.879G > A）,这个突变产生一个潜在的剪切受体位点“AG”,巢式PCR琼脂糖凝胶电泳结果和PCR产物测序结果证实,该同义突变导致ECM1基因的剪切发生改变。 结论 本类脂蛋白沉积症家系存在新的ECM1基因同义突变导致的剪切突变。     

板层状鱼鳞病两家系TGM1基因的突变

目的:检测2个板层状鱼鳞病家系TGM1基因的突变情况.方法:采用PCR-DNA直接测序方法明确两个家系TGM1基因突变位点,并以免疫组化的方法检测其中一家系患者皮肤中转谷氨酰胺酶的活性.结果:在1个家系的患者中发现TGM1基因第13号外显子第2060位碱基发生G→A纯合突变,使密码子CGT→CAT,导致R687H突变:在另一个家系的患者中发现TGM1基因第4号内含子第一个核苷酸发生G→T及第5号外显子第760位碱基发生G→A杂合突变,分别导致IVS4 1G＞T及D254N的复合杂合突变,其父母均为相应突变的携带者.在50名无关正常人中未发现相同突变.R687H纯合突变患者皮肤转谷氨酰胺酶的活性降低.结论:R687H纯合突变及D254N和IVS4 1G＞T复合杂合突变为新发现的TGM1基因突变位点,可能是引起两家系板层状鱼鳞病患者的病因.     

寻常型鱼鳞病一家系Filaggrin基因突变检测

目的探讨一家系寻常型鱼鳞病(ichthyosis vulgaris,IV)丝聚合蛋白(filaggrin,FLG)基因的突变。方法提取IV患者及其家庭成员和100例健康对照者基因组DNA,采用PCR及直接测序法,对FLG基因已报道的13个突变位点(3321delA,441delA,1249insG,E1795X,S3296X,R501X,2282del4,R2447X,S2889X,7945delA,3702delG,Q2417X,R4307X)进行测序。结果三代7位成员中4例IV患者同时检测到FLG(441delA)基因突变。结论患者FLG(441delA)基因突变可能导致其发病。     

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。     

4例可变性红斑角化症的基因突变检测

目的:检测4例可变性红斑角化症患者的基因突变位点.方法:应用DNA直接测序法检测基因的突变.结果:发现1个新的突变位点,1个已报道突变位点.结论:错义突变c.403C＞G和c.292C＞T是导致该3例患者发生可变性红斑角化症的特异突变.     

家族性良性慢性天疱疮ATP2C1基因突变检测

目的:检测家族性良性慢性天疱疮ATP2C1基因突变.方法:提取20例患者和100例健康对照者的外周血DNA,采用聚合酶链反应(PCR)扩增ATP2C1基因的全部外显子,用直接测序法进行DNA测序,检测ATP2C1基因突变.结果:20例患者中检测到9种突变,包括4种缺失突变(167或168de1C,2374delTTTG,2454delT,2558delTGGGACAATT),3种错义突变(I711R,Q737R,1580V),2种无义突变(R55X,R799X),健康对照者中未检测到上述突变.其中167或168de1C,2454delT,2558de1T-GGGACAATT,I711R,Q737R为新发现的突变位点.结论:家族性良性慢性天疱疮存在遗传异质性.     

双下肢浅表播散型汗孔角化症一例基因突变检测

目的：检测一例局限于双下肢的散发浅表播散型汗孔角化症患者致病基因突变。方法：提取患者外周血DNA,在Illumina HiSeq测序平台进行全基因组外显子测序,将测序结果与既往报道的汗孔角化症致病基因比对,采用Sanger测序对发现的突变位点在100例患者中进行验证。结果：最终将DSP的致病基因锁定为位于12号染色体的MVK基因,检测到10号内含子c.1040-2A>C突变位点,在全部正常对照中未检测到该位点。经检索,该突变为浅表播散型和播散性浅表性光线型汗孔角化症的共同突变位点。结论：本家系中MVK基因突变位点(c.1040-2A>C)与浅表播散型汗孔角化症发病相关。     

Detection and comparison of two types of ATP2C1 gene mutations in Chinese patients with Hailey–Hailey disease

The gene ATP2C1 is identified as the defective gene in Hailey–Hailey disease (HHD). The nonsense and missense are two common types of mutations and have ,respectively, been detected in many HHD patients. The aims of our study were to identify the pathogenic ATP2C1 abnormality in Chinese HHD patients, and to compare nonsense and missense mutations in vivo to provide further understanding of the molecular and the physiological basis of HHD. The nucleotide sequencing of the ATP2C1 gene was performed in HHD patients, unaffected family members and 100 unrelated individuals. Meanwhile, we detected and analyzed the clinical manifestations, the expression of ATP2C1 mRNA and hSPCA1 protein in the two types of mutations. Three heterozygous mutations were identified, including a previously reported nonsense mutation (R799X), two novel missense mutations (D644G) and (R417K). The results of comparisons between two types of mutations showed that the common clinical features, the similarly low-level expressions of ATP2C1 mRNA and hSPCA1 protein, but the ATP2C1 mRNA expression of nonsense mutation was lower than missense mutation and even less than half the level of normal people. Our findings expand the known spectrum of ATP2C1 mutations in HHD. We supported the haploinsufficiency theory as prevalent mechanism in both types of mutations, and believed that the differences of ATP2C1 mRNA expressions in peripheral blood may relate with the type of mutation and reflect the state of illness of patients.     

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.     

Treatment of lipoid proteinosis with acitretin in two patients from two unrelated Chinese families with novel nonsense mutations of the ECM1 gene

Lipoid proteinosis is a rare recessive genetic disorder caused by loss‐of‐function mutations to chromosome 1 at 1q21, the extracellular matrix protein 1 (ECM1) gene. Two children with lipoid proteinosis were reported from two unrelated Chinese families, both manifesting with a typical hoarse voice, white acne‐like atrophic lesions and scarring on the skin, and beaded papules around the eyelids. The diagnosis had been confirmed by laboratory tests, skin biopsy and laryngoscope examination. Genomic DNA sequencing was performed for both children and their family members. The two children were treated with acitretin for 6 months and followed up for 1 year. Genomic DNA sequencing of the ECM1 gene showed a novel homozygous nonsense mutation of C1522>T (p.R508X) at exon 10 in one patient, and a novel compound heterozygote for a nonsense/frame‐shift combination of mutations of R281X/1596delG at exons 7 and 10 in the other patient. The symptom of hoarse voice was improved by 6‐month treatment with acitretin, while there was no improvement in the skin lesions. These results demonstrated that acitretin treatment may have efficacy for some of patients with lipoid proteinosis, with superior effect on laryngeal symptoms than skin lesions. However, the conclusive therapeutic effect and underlying mechanisms remain to be further investigated.     

Lipoid proteinosis presenting with an unusual nonsense Q32X mutation in exon 2 of the extracellular matrix protein 1 gene

Lipoid proteinosis (LP) is a rare disorder characterized by extensive hyaline‐like deposits on the skin, mucous membranes and various internal organs with varying clinical manifestations. The disorder has been recently shown to result from loss‐of‐function mutations in the extracellular matrix protein 1 gene (ECM1) on 1Q21. The two cases reported here had typical clinical and histological features consistent with LP. Direct sequencing of amplified DNA from the second patient showed a single nucleotide substitution (C > T) at nucleotide 94 within exon 2 of the ECM1 gene, nonsense mutation Q32X. This is the second case reported of LP with involvement of exon 2 of ECM1.     

Homozygous missense mutation in the ECM1 gene in Chinese siblings with lipoid proteinosis

Lipoid proteinosis is caused by loss-of-function mutations in the glycoprotein extracellular matrix protein 1 (ECM1). We report here mutation analysis of the ECM1 gene in a Chinese family with lipoid proteinosis. A 10-year-old boy presented with a hoarse voice, acneiform scars and yellow skin nodules, as well as beaded eyelid papules and a thickened sublingual frenulum. Skin biopsy showed widespread deposition of hyaline material in the dermis and thickened basement membrane. His elder sister had the same clinical manifestations. The coding region of ECM1 was amplified and sequenced and both affected siblings were shown to have a novel homozygous single nucleotide substitution, c.658T>G, in exon 6, which converts cysteine to glycine, designated p.C220G. Both parents were heterozygous for this mutation which was not detected in 100 control chromosomes. Missense mutations in the ECM1 gene are an unusual finding in lipoid proteinosis, but this case adds to the spectrum of disease-associated mutations in this rare genodermatosis.     

Novel CDH3 mutations in hypotrichosis with juvenile macular dystrophy

BACKGROUND: Hypotrichosis with juvenile macular dystrophy (HJMD) is a rare autosomal recessive disorder characterized by sparse and short hair, heralding progressive degeneration of the retinal pigment epithelium, which leads to blindness by the second decade of life. The disorder is caused by mutations in CDH3, a gene encoding P-cadherin, a major component of adherens junctions. Most HJMD cases reported to date have been shown to be caused by homozygous CDH3 mutations segregating in consanguineous families. AIM AND METHODS: To elucidate the genetic basis of HJMD in two nonconsanguineous families, we established the coding sequence of CDH3 in four patients and their healthy siblings. RESULTS. The four patients demonstrated markedly variable degrees of visual acuity impairment. Novel biallelic recessive mutations were identified in all affected individuals. One patient in the first family was found to carry two heterozygous mutations, IVS2 + 1G-->A and p.E504K; the other three patients in the second family were compound heterozygous for a missense mutation, p.H575R, and a nonsense mutation, p.R221X. CONCLUSION: This paper expands the spectrum of known mutations in CDH3 and points to the existence of clinical heterogeneity in this syndrome.     

Mutation analysis of the ATP2C1 gene in Taiwanese patients with Hailey-Hailey disease

BackgroundHailey-Hailey disease (HHD) is an autosomal dominant disorder with recurrent pruritic vesicles and erosions, and scaly erythematous plaques, particularly involving intertriginous areas such as the neck, axillae, groins and perineum. Histopathology shows intraepidermal vesiculation with acantholysis in the suprabasal layer. It is caused by heterozygous mutations in the ATP2C1 gene, which encodes for the human secretory pathway Ca²⁺/Mn²⁺ ATPase 1. In this study, we analyze the mutations of the ATP2C1 gene in 26 Taiwanese patients with HHD.MethodsIn total, 21 familial cases from seven families and 5 sporadic cases (including 7 previously reported) were retrieved from the medical records. The diagnosis of HHD was made based on the characteristic clinical features and histopathological evidence. All 27 exons and flanking intron boundaries were amplified by polymerase chain reaction and the products were analyzed by direct sequencing.ResultsWe identified three nonsense mutations (R39X, R468X, R783X), two splice-site mutations (483 + 2t→a, 832G→A), four deletion mutations (nt884-904del, 1459delCTCA, 1874delA, 1975delA) and one missense mutation (A730T). Two unrelated families with nonsense mutation R783X had the comorbidity of chronic schizophrenia since the third decade.ConclusionsWe report two novel mutations (832G→A and 1874delA) of ATP2C1 involved in HHD. The nonsense mutation R783X might represent a mutational “hotspot” in the ATP2C1 gene. The present study demonstrates that a spectrum of ATP2C1 gene mutations is present in Taiwanese HHD patients.