毛囊角化病患者ATP2A2基因新剪切位点突变的检测研究

目的:检测1例严重表型毛囊角化病患者ATP2A2基因的突变.方法:采用聚合酶链反应及直接测序法对该患者进行ATP2A2基因的突变位点检测,运用反转录技术检测突变导致的RNA水平的变化,同时对120名无血缘关系健康者作为对照进行测序验证.结果:通过筛查在患者ATP2A2基因中发现一个新的剪切位点突变IVS18+5G＞C,反转录分析证实该突变的发生导致ATP2A2基因转录后在其突变等位基因的18号和19号外显子之间插入了27个核苷酸.结论:该例毛囊角化病患者的检测结果进一步扩充了ATP2A2基因的突变库,并为阐明ATP2A2基因的突变导致该病发生的分子机制提供了帮助.     

毛囊角化病一家系及三例散发患者ATP2A2基因突变研究

目的：对毛囊角化病（Darier's disease, DD）一家系及三例散发患者进行ATP2A2基因的突变分析。方法：收集先证者及其家系成员、散发病例的临床资料和外周血,采用PCR技术扩增ATP2A2基因所有编码区及侧翼序列,用Sanger法测序检测潜在的突变,选取与患者无亲缘关系的100例健康人作为对照,同时对已报道的ATP2A2基因突变进行文献回顾。结果：家系中三例患者均检测出ATP2A2基因第5号外显子c.380 G>A(p.G127D)新发错义突变;散发患者S1检测出第13号外显子C.1676G>A(p.R559Q)错义突变,散发患者S2检测出第14号外显子c. 2001C>T(p.D667D)同义突变,散发患者S3未检测出突变。结论：本研究中共发现三个突变,其中c.380G>A(p.G127D)在中国人群中首次报道,拓展了ATP2A2的基因突变谱。     

毛囊角化病一家系中ATP2A2基因新的剪接突变

目的: 检测毛囊角化病一家系中ATP2A2基因新的剪接突变。方法: 提取家系中2例患者和2名正常人外周血DNA,采取聚合酶链反应技术对ATP2A2基因进行扩增,并对其产物进行测序,以100名正常人作对照。结果:该家系中患者ATP2A2基因的第13号内含子第1761+2位碱基由胸腺嘧啶(T)转化为胞嘧啶(C)。结论: 该家系发病可能是由ATP2A2基因发生剪接突变所致。     

毛囊角化病ATP2A2基因突变的检测

目的:检测2例散发及2个家系毛囊角化病患者ATP2A2基因的突变。方法:采用聚合酶链反应扩增患者和健康对照个体ATP2A2基因的全部外显子,并进行DNA测序,检测该基因突变。结果:共发现1个新的点突变(TVS11+32 C→T)和2个已发现的错义突变(R131Q,N767S)。结论:毛囊角化病具有遗传异质性。     

一毛囊角化病家系ATP2A2基因突变检测

目的:检测一毛囊角化病家系的ATP2A2基因突变。方法:提取2例患者外周血DNA,采用聚合酶链式反应及DNA直接测序方法,检测患者ATP2A2基因突变。结果:该家系患者及其两女儿存在ATP2A2基因的碱基缺失突变,即ATP2A2基因第10个外显子1220位开始缺失了AA 2个碱基。而该家系中其他正常者未发现此突变。结论:ATP2A2基因第10个外显子1220delAA突变可能与该家系患者临床表型有关。     

Hailey-Hailey病四个家系的ATP2C1基因突变分析

目的检测Hailey-Hailey病(HHD)4个家系的致病基因ATP2C1,鉴定其突变位点和突变类型。方法采集4个HHD家系成员共9例患者和6名正常人,与100名无关健康对照者外周静脉血各2 ml,提取全基因组DNA。运用聚合酶链反应(PCR)扩增ATP2C1基因的全部28个外显子及其侧翼内含子序列,扩增产物纯化后进行DNA直接测序,BLAST比对分析其突变位点和突变方式。结果在9例HHD患者中共检出了3个ATP2C1基因致病突变:c.888_889ins T(p.296Tfs X2)、c.1330del C(p.443Qfs X33)和c.2416CT(p.Arg806X)。在4个HHD家系的6名正常者和100名健康对照者中均未发现上述突变。结论在9个HHD家系患者中存在2个移码突变(c.888_889ins T和c.1330del C)及1个无义突变(c.2416CT),其中2个移码突变为首次报道。这些突变的发现有助于HHD的诊断,并丰富了HHD相关ATP2C1突变数据库。     

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.     

哈萨克族毛囊角化病一家系ATP2A2基因突变研究

目的 探讨哈萨克族毛囊角化病一家系患者ATP2A2基因突变.方法 收集哈萨克族毛囊角化病49人家系的临床资料,采集44名家系成员和100例无亲缘关系健康人外周血,提取基因组DNA.采用PCR和DNA测序对该家系进行ATP2A2基因突变检测.结果 该家系毛囊角化病遗传方式属于常染色体显性遗传.家系中11例患者在ATP2A2基因12号外显子的剪切位点发生杂合突变(1288-1G→A),即第1288-1位碱基由G突变为A,而家系中33例正常成员及100例健康对照均未发现该突变.结论 该家系毛囊角化病发病可能是由ATP2A2基因12号外显子的剪切位点发生杂合突变(1288-1G→A)所致.     

毛囊角化病ATP2A2基因突变分析

目的检测毛囊角化病患者ATP2A2基因的突变。方法提取全部患者及健康对照个体的外周血DNA,采用聚合酶链反应扩增ATP2A2基因的全部外显子,并进行DNA测序。结果在收集到的2个家系和3例散发患者中共发现3个突变,包括1个缺失突变(1622delAACA),1个插入突变(180insCTTAA)和1个错义突变(698GT),均为未见报道的突变。在100例正常对照中均未发现上述突变。结论收集到的毛囊角化病患者存在ATP2A2基因的突变,这些突变可能会影响角质形成细胞中钙离子的转运,使表皮细胞的连接和分化出现异常。     

毛囊角化病ATP2A2基因突变的检测

目的 :检测1个家系及1例散发毛囊角化病患者ATP2A2基因突变情况。方法 :提取家系中2例患者、5例正常人及1例散发患者的外周血DNA,采用聚合酶链反应(PCR)对其ATP2A2基因进行扩增,并对其扩增产物进行测序,检测该基因突变点。对照组为100例无血缘关系的健康人。结果:发现1个新的错义突变(R603I)和1个已报道的错义突变(R131Q)。结论:该研究发现毛囊角化病患者存在ATP2A2基因的突变,这些突变可能影响肌浆/内质网钙ATP酶(SERCA)2的结构和功能,使表皮细胞的连接和分化出现异常,最终导致疾病的发生。     

Mutation analysis of the ATP2A2 gene in Taiwanese patients with Darier's disease

BACKGROUND: Darier's disease (DD) is an autosomal dominant skin disorder characterized by abnormal keratinization and acantholysis. Pathogenic mutations in the ATP2A2 gene encoding SERCA2, a calcium pump of the sarco/endoplasmic reticulum, have recently been identified. OBJECTIVES: To identify mutations of the ATP2A2 gene in Taiwanese patients with DD. METHODS: Mutation analysis of genomic DNA was performed on five families with DD and two sporadic cases. All 21 exons and the flanking intron boundaries were amplified and followed by direct sequencing. Restriction fragment analysis or direct sequencing in each family and in normal controls further verified the mutations. RESULTS: Mutations in the functional domains of the ATP2A2 gene were identified and verified in all seven pedigrees. They consisted of four mis-sense mutations (R131Q, P680L, G703S, G807R), one altered splice-site mutation (2980 + 5insA) and one frameshift deletion mutation (1457-1458delAG). Of these, R131Q, which was reported twice previously, was detected in two unrelated families. The remaining five were novel mutations. CONCLUSIONS: Six pathogenic mutations in the ATP2A2 gene were identified in seven Taiwanese DD pedigrees. The results confirmed that most mutations in the ATP2A2 gene are private and of the mis-sense type.     

Mutational analysis of the ATP2A2 gene in two Darier disease families with intrafamilial variability

BACKGROUND: Darier disease (DD), an autosomal dominant genodermatosis characterized by warty papules and plaques over seborrhoeic areas, is caused by mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca(2+) ATPase type 2 isoform (SERCA2). While markedly different clinical severity within DD-affected family members is known, the pathomechanism has not been elucidated. OBJECTIVES: Based on the hypothesis that multiple ATP2A2 mutations might contribute to the pathomechanism, we have analysed two DD families in which the clinical severity differs markedly within a single pedigree, and, as controls, eight DD families without differing clinical severity. METHODS: All the exons and intron-exon borders of ATP2A2 were directly sequenced from the genomic DNA extracted from all the subjects. RESULTS: We identified the heterozygous mutations, G233R in pedigree 1 and C318R in pedigree 2, respectively, whereas no other ATP2A2 mutations in any of severely affected individuals were found. In eight DD pedigrees as control, we have found M1V, N39D, L180R, A838P and 2170 insertion G in each of five pedigrees, but no mutation was found in three DD pedigrees. CONCLUSIONS: Our results together with previous data indicate that the distribution of mutations is scattered over the entire ATP2A2 without any, as yet, discernible 'hotspots'. The mutations in pedigrees 1 and 2 with intrafamiliar clinical differences occurred around the Ca(2+)-binding sites on SERCA2, which might be associated with differences in clinical severity. These variations in ATP2A2 mutations alone cannot account for the clinical heterogeneity within DD pedigrees.     

Acrokeratosis verruciformis of Hopf showing P602L mutation in ATP2A2 and overlapping histopathological features with Darier disease

The relationship between acrokeratosis verruciformis (AKV) of Hopf and Darier disease (DD) has been debated for several decades. Both diseases are now thought to result from mutations in the same gene, that is, the ATP2A2 gene encoding the sarco (endo) plasmic reticulum Ca ATPase2 pump (SERCA2), although their histopathological features are different. We sought to detect possible overlapping histopathological features between AKV and DD. Fourteen members of a family affected by AKV were analyzed for the underlying molecular genetic derangement, and 3 cases were studied histopathologically using multiple step sections. A heterozygous P602L mutation in ATP2A2 was identified as the underlying cause in this family. This mutation and a heterozygous A698V were previously described in AKV. Both mutations were not among the 162 mutations in ATP2A2, which were reported to date in DD. The histopathological study demonstrated in several consecutive step sections of 2 of the 3 studied cases, foci of small suprabasal clefts with acantholytic keratinocytes, some of which were mildly dyskeratotic. These focal features were reminiscent of the basic histopathological characteristics of DD. These shared histopathological features of AKV with DD suggest that AKV and DD are allelic disorders with variable expression of the same disease, although identical mutations in ATP2A2 in AKV and DD were not reported to date.     

家族性良性天疱疮患者ATP2C1基因突变研究

目的 探讨3个家族性良性天疱疮家系和1例散发患者的ATP2C1基因突变。方法 采取家系中患病成员外周血,应用外周血细胞DNA抽提、PCR扩增和DNA直接测序等方法检测ATP2C1基因突变情况,用反向测序验证突变,用100例无血缘关系个体作正常人对照。结果 在2个家族性良性天疱疮家系和1例散发患者中发现3个未曾报道的错义突变。家系1第20外显子2048位碱基G→A,导致错义突变R619K;家系2第8外显子853位碱基A→C,导致错义突变T221P;散发患者第23外显子2323位碱基T→C,导致错义突变Y711H。家系中非患病成员和100例无血缘关系正常人均未发现这些改变。在1个家族性良性天疱疮家系未检测到基因突变。结论 发现家族性良性天疱疮3种新的ATP2C1基因突变位点。     

Darier disease--novel mutations in ATP2A2 and genotype-phenotype correlation

Darier disease (DD) is with a frequency of up to 1 in 36,000 a relatively common genodermatosis with autosomal dominant inheritance and late age of onset. The progressive skin manifestations are variable, but often debilitating and disfiguring, and may be associated with a wide range of neuropsychiatric problems, such as epilepsy and depression. On histology, acantholysis and dyskeratosis are prominent findings, implicating impaired functionality of desmosomes. Recently, mutations in the ATP2A2 gene encoding SERCA2, a calcium pump of the endo/sacrcoplasmic reticulum, have been identified as the molecular basis of DD. This slow-twitched calcium ATPase has two splice variants, one of which is highly expressed in epidermis, and maintains low intracellular calcium levels by facilitating transport of cytosolic calcium into the endoplasmic reticulum. Thus, it may confer a direct effect on the established calcium-dependent assembly of desmosomes. We screened ATP2A2 in a cohort of 24 DD families using conformation sensitive gel electrophoresis and direct sequencing, and detected 14 distinct mutations, 9 of which were novel. The mutational spectrum included 9 missense mutations, 1 nonsense mutation, 3 small in-frame deletions, and a 19-basepair insertion. Mutations were scattered over the entire gene with a slight preponderance in the first 8 exons, and affected exclusively residues conserved among all SERCAs. In addition, we found 2 silent polymorphisms, 1 of which occurred in 4 unrelated families. Comparison of molecular data and phenotypic features, such as severity and type of disease, occurrence of mucosal involvement, or association with neuropsychiatric disorders, did not reveal an obvious genotype-phenotype correlation in our cohort.     

家族性良性天疱疮ATP2C1基因突变研究

目的 探讨家族性良性天疱疮5个散发病例的ATP2C1基因突变.方法 5例来自门诊的散发病例,采集外周血,提取基因组DNA,采用PCR和DNA直接测序的方法,检测5个散发家族性良性天疱疮患者的ATP2C1基因突变,在100例正常人对照中予以验证.结果在5例具有典型临床表现,经皮肤病理和免疫病理确诊的散发家族性良性天疱疮患者,检测到5个未曾报道的ATP2C1基因突变位点,包括1个缺失突变(2025delG),3个错义突变(L269R,C348R,A651D),和1个无义突变(Q259x).100例正常人对照中均未检测到上述突变.结论 发现家族性良性天疱疮新的ATP2C1基因突变位点.     

家族性良性天疱疮ATP2C1基因突变研究

目的 探讨家族性良性天疱疮5个散发病例的ATP2C1基因突变。方法 5例来自门诊的散发病例,采集外周血,提取基因组DNA,采用PCR和DNA直接测序的方法,检测5个散发家族性良性天疱疮患者的ATP2C1基因突变,在100例正常人对照中予以验证。结果 在5例具有典型临床表现,经皮肤病理和免疫病理确诊的散发家族性良性天疱疮患者,检测到5个未曾报道的ATP2C1基因突变位点,包括1个缺失突变（2025delG）,3个错义突变（L269R,C348R,A651D）,和1个无义突变（Q259X）。100例正常人对照中均未检测到上述突变。结论 发现家族性良性天疱疮新的ATP2C1基因突变位点。     

Rothmund-Thomson综合征一例基因检测分析

目的:对一例自幼身材矮小、发育迟缓、周身皮肤异色症的患儿进行基因检测,以明确诊断确定病因.方法:收集患儿临床资料,提取患儿及其父母外周血DNA,采用全外显子组测序检测潜在的基因突变,并通过Sanger测序进行验证.结果:该患儿在RECQL4基因上携带两个复合杂合突变:(c.1579dupA)(p.T527fs)和(c....