Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis

BACKGROUND: Recent reports indicated that nonsense mutations in filaggrin (FLG) found in ichthyosis vulgaris (IV) patients are predisposing factors for atopic dermatitis (AD) with asthma. The exon 3 of FLG contains tandemly repeated, highly homologous, 11-13 sequence units of 972 or 975 bp, each of which corresponds to the coding sequence of the processed filaggrin with slight sequence difference. This unique gene structure has hampered the precise DNA sequence determination. OBJECTIVE: We developed a novel DNA sequencing method "FLG-shotgun" to directly characterize the mutations in Japanese AD patients. METHODS: We examined 24 Japanese AD patients with "FLG-shotgun" method. RESULTS: Multiple units of FLG were amplified by PCR using several sets of common primers for the conserved regions, and DNA sequences of each cloned PCR product were determined. Multiple reads of DNA sequences in both alleles were aligned and re-constructed to cover the entire coding regions. We found three major genotypes (A, B, and C) which represent different numbers (11-13) of homologous sequence units. Furthermore, we found two novel nonsense mutations; one mutation 8666-8667CC>GA on the unit 9 of allele B that causes a nonsense mutation S2899X in two patients and the other mutation 9887C>A on the unit 10 of allele B that causes a nonsense mutation S3296X in two patients. CONCLUSION: We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable tool to further define the nature of the AD phenotype associated with FLG mutations.     

Filaggrin mutations are genetic modifying factors exacerbating X-linked ichthyosis

Mutations inactivating the STS gene cause X-linked ichthyosis (XLI), whereas null mutations in the FLG gene cause ichthyosis vulgaris. Two brothers presented with XLI. One had a typical fine scaling, and the other was much more severely affected. Both patients carried STS missense mutation T165I. Furthermore, the more severely affected patient also carried heterozygous FLG mutation R501X, which was absent from his mildly affected brother. These data suggest that disrupting epidermal differentiation via different pathways can increase phenotypic severity. Owing to the high population frequency of FLG mutations, filaggrin is a possible genetic modifier in other genodermatoses.     

Analysis of Taiwanese ichthyosis vulgaris families further demonstrates differences in FLG mutations between European and Asian populations

Background  Mutations in the gene encoding filaggrin ( FLG ) were identified to underlie ichthyosis vulgaris (IV) and also shown to predispose to atopic eczema. Until now, no FLG mutations have been described in the Taiwanese population.
Objectives  To elucidate filaggrin mutations in the Taiwanese population and further to clarify the population genetics of filaggrin gene mutations in the Asian populations.
Methods  In the present study, 12 individuals from four unrelated Taiwanese IV families were examined for FLG mutations. We carried out comprehensive sequencing of the entire FLG coding region using an overlapping polymerase chain reaction strategy.
Results  We identified three FLG mutations in the Taiwanese IV families. One mutation E1795X was a previously unidentified FLG mutation, which might be specific to the Taiwanese. Interestingly, another FLG mutation 3321delA is prevalent in the Japanese population and the other mutation Q2417X was found in the Singaporean Chinese population. No FLG mutation identified in the white European population was found in the Taiwanese population.
Conclusions  The present findings suggest that the Taiwanese population, as an East Asian group, share FLG mutations with both the Japanese and the Singaporean Chinese population. In addition, these results exemplify differences in the population genetics of filaggrin between Europe and Asia.     

Exacerbation of X-linked ichthyosis phenotype in a female by inheritance of filaggrin and steroid sulfatase mutations

Background

X-linked ichthyosis (XLI) is a relatively common, recessive condition caused by mutations in the steroid sulfatase (STS) gene. Common loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and predispose individuals to atopic eczema.

Objective

To test the hypothesis that co-inheritance of FLG mutations can act as a genetic modifier in XLI.

Methods

An unusually severe XLI phenotype in addition to eczema and mild childhood asthma was investigated in a female Indian patient by fluorescent in situ hybridization (FISH) for the common STS gene deletion. Direct sequencing of the entire FLG gene was also performed.

Results

FISH analysis revealed that the proband was homozygous for the common STS genomic deletion mutation. Further investigation revealed a frame-shift mutation 3672del4 in the gene encoding filaggrin (FLG), leading to premature termination of profilaggrin translation. Interestingly, her father, who had a very typical mild presentation of XLI, did not carry this FLG mutation in addition to his STS deletion. Her mother was a heterozygous carrier of the FLG mutation and consistent with this, had mild symptoms of ichthyosis vulgaris; she was also a heterozygous carrier of the STS deletion.

Conclusion

This is the second reported case of the modifying effects of FLG null alleles on XLI and strengthens the hypothesis that filaggrin defects can synergize with STS deficiency to exacerbate the ichthyosis phenotype.     

On the role of the epidermal differentiation complex in ichthyosis vulgaris, atopic dermatitis and psoriasis

Undisturbed epidermal differentiation is crucial for an intact skin barrier function. The epidermal differentiation complex (EDC) is a cluster of genes on chromosome 1q21 encoding proteins that fulfil important functions in terminal differentiation in the human epidermis, including filaggrin, loricrin, S100 proteins and others. Recently, evidence emerged that variation within EDC genes plays an important role in the pathogenesis of three common skin disorders, ichthyosis vulgaris, atopic dermatitis (AD) and psoriasis. Two loss-of-function mutations in the filaggrin (FLG) gene, R501X and 2282del4, were identified as causative for ichthyosis vulgaris in 15 affected European families, and the mode of inheritance was found to be semidominant. As ichthyosis vulgaris and AD often occur concomitantly in affected individuals, these two mutations were subsequently investigated in AD patients and found to be strongly associated with the disease. Following this first report, seven replication studies have been performed that all confirm an association of these two mutations with AD (or AD subtypes) in several European cohorts. Additionally, two unique loss-of-function mutations in the FLG gene were identified in Japanese ichthyosis vulgaris families and found to be associated with AD in a Japanese cohort. Thus, the FLG mutations are among the most consistently replicated associations for AD. Additionally, linkage analysis has suggested that variation within the EDC might also predispose for psoriasis but the exact susceptibility variation(s) have not yet been elucidated. Taken together, these findings convincingly demonstrate the important role of barrier dysfunction in various common skin disorders.     

Loss-of-function mutations in the filaggrin gene and allergic contact sensitization to nickel

Allergic contact dermatitis is one of the most frequent dermatological problems affecting 7% of the general population. Impaired skin barrier function facilitates the penetration of contact allergens and irritants into the epidermal layer and is regarded as an important cofactor promoting the process of allergic contact sensitization. Filaggrin is crucial for the maintenance of the skin barrier function. Loss-of-function mutations within the filaggrin (FLG) gene are associated with skin barrier diseases such as ichthyosis vulgaris and atopic eczema (AE). To assess the impact of FLG on allergic contact sensitization and plausible intermediate traits, the two prevalent FLG mutations R501X and 2282del4 were typed in 1,502 individuals of the KORA C population-based cohort with extensive dermatologic phenotyping. Associations of FLG mutations with AE could be replicated. Strong associations were seen with dry skin, palmar hyperlinearity, and keratosis pilaris. In addition, an association with contact sensitization to nickel and contact sensitization to nickel combined with intolerance to fashion jewelry, but not with other contact allergens, was observed. From these data, we conclude that a genetically determined FLG deficiency manifests as dry skin and features of ichthyosis vulgaris. In addition, FLG deficiency may also represent a risk factor for contact sensitization to allergens.     

反常性痤疮一家系nicastrin基因突变研究

目的 研究一家系2例汉族反常性痤疮患者γ分泌酶基因的突变.方法 提取家系中5名成员(2例患者、先证者父亲、2例目前未发病者)的外周血DNA,扩增nicastrin蛋白(NCSTN)、早老素(PSEN)1、早老素增强子(PSENEN)、前咽缺陷蛋白(APH1)基因所有外显子和侧翼序列进行测序,并以100例无关系健康人作为对照.同时比较先证者皮损与4个健康对照NCSTN基因mRNA表达差异.结果 检测到2例患者血样DNA存在NCSTN基因中第477位碱基发生C→A的杂合突变,即c.477C＞A,其余3名家系成员及健康对照未发现相应突变;查询美国国家生物技术信息中心网站单核苷酸多态性数据库也未发现此突变.另外,先证者皮损NCSTN mRNA水平较健康对照明显减少.结论 NCSTN基因新的无义突变c.477C＞A是该反常性痤疮家系的致病突变,并可能通过无义介导的mRNA降解途径导致该基因功能失活.     

应用多重连接探针扩增技术检测X-性连锁鱼鳞病一家系STS基因

目的：检测X-性连锁鱼鳞病一家系STS基因突变情况。方法：提取先证者(男，31岁)及其父母外周血DNA，父母均无鱼鳞病临床表现，运用多重连接探针扩增技术检测所有成员的STS基因是否存在外显子缺失，若无外显子缺失，运用聚合酶链式反应特异性扩增STS基因，检测是否存在基因突变。结果：家系中先证者为STS基因半合子缺失，其母为STS基因杂合子缺失，其父亲未发现STS基因突变。家系中仅先证者出现鱼鳞病的临床表现。结论：STS基因缺失是该X-性连锁鱼鳞病患者发病的遗传因素。     

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。     

常染色体隐性遗传性鱼鳞病一家系表型、基因型与皮损超微结构研究

目的 探讨常染色体隐性遗传性鱼鳞病家系临床表型、基因型及超微结构。方法 观察常染色体隐性遗传性鱼鳞病患者临床表现。用PCR扩增TGM1基因15个外显子及其邻近剪切位点,双向直接测序;取先证者背部皮损做透射电镜观察,记录电镜表现特征。结果 先证者临床表现介于板层状鱼鳞病及非大疱性鱼鳞病样红皮病之间,其弟弟为火棉胶婴儿。先证者、其弟及父亲3号外显子第551位碱基胞嘧啶（C）→胸腺嘧啶（T）,其编码的第143位氨基酸由精氨酸变为半胱氨酸（R143C）;先证者、其弟及母亲4号外显子第759位胞嘧啶（C）→胸腺嘧啶（T）,使第212位氨基酸由丝氨酸转变为苯丙氨酸（S212F）。电镜观察发现,先证者皮损不仅有Ⅱ型结构表现,也同时存在Ⅲ型结构特征。结论 该家系患者携带复合杂合突变,R143C属于热点区,S212F为新发现的位点。携带TGM1基因突变的先证者皮损电镜表现为Ⅱ型,但同时发现有Ⅲ型结构存在。     

Siemens大疱性鱼鳞病基因突变研究及文献回顾分析

目的 确定1个Siemens大疱性鱼鳞病(ichthyosis bullosa of Siemens,IBS)家系的致病基因,探讨本病基因型、表型及两者间关系.方法 收集1个IBS家系先证者及其父母的临床资料,采集他们和100例无亲缘关系的健康对照者的外周血标本,提取DNA.应用二代皮肤靶向测序包检测该家系的基因突变,...     

眼皮肤白化病1家系致病基因鉴定

【摘要】 目的 利用全外显子测序及Sanger测序技术对两兄弟眼皮肤白化病患者的（OCA）家系进行致病基因筛选和鉴定。方法 收集1个OCA家系的临床资料，提取家系成员的外周血DNA，通过全外显子组测序技术对先证者的全外显子编码区进行直接测序以寻找可能存在的基因突变，并利用Sanger测序进行一代验证。结果 先证者及其弟弟均表现为全身皮肤、毛发变白，双眼球震颤，畏光，虹膜半透明，结膜充血，双眼屈光不正。先证者父母、祖父母、外祖父母及子女表型均正常，父母非近亲结婚。两兄弟OCA2基因中均出现3个杂合变异，即c.1290T>A无义突变、c.1363A>G错义突变和c.1204T>C错义突变。其中，OCA2 c.1204T>C尚未有报道，为OCA2基因的新突变位点。此外，先证者父亲OCA2基因存在杂合变异c.1204T>C；先证者母亲OCA2基因存在杂合变异c.1290T>A及c.1363A>G；先证者儿子OCA2基因存在杂合变异c.1290T>A；先证者女儿OCA2基因存在杂合变异c.1204T>C，先证者弟弟的女儿OCA2基因存在杂合变异c.1290T>A。结论 本研究中2例OCA2患者均出现3处OCA2基因突变，其中c.1290T>A无义突变可能是导致该家系临床表型的突变位点，这些发现丰富了OCA2的致病基因突变谱。     

Specific filaggrin mutations cause ichthyosis vulgaris and are significantly associated with atopic dermatitis in Japan

Mutations in the gene encoding filaggrin (FLG) have been identified as the cause of ichthyosis vulgaris (IV) and shown to be major predisposing factors for atopic dermatitis (AD). However, these studies have been mainly carried out in European populations. In early 2007, we identified two Oriental-specific FLG mutations in four Japanese families with IV and reported that filaggrin mutations were also significant predisposing factors for AD in Japan. However, the frequency of FLG mutations observed in our Japanese AD cohort (5.6%), was much lower than that seen in Europeans (up to 48%). Here, we studied a further seven Japanese families with IV and identified two additional nonsense mutations in FLG, S2889X, and S3296X. We found that more than 20% of patients in our Japanese AD case series carry FLG mutations, and there is significant statistical association between the four mutations and AD (chi(2) P=8.4 x 10(-6); heterozygote odds ratio 7.57, 95% CI 2.84-23.03). These data emphasize that skin-barrier impairment due to reduced filaggrin expression plays an important role in the pathogenesis of AD and sheds further light on the genetic architecture of atopy in Japan.     

Unique and recurrent mutations in the filaggrin gene in Singaporean Chinese patients with ichthyosis vulgaris

Filaggrin is an abundant protein of the outer epidermis that is essential for terminal differentiation of keratinocytes and formation of an effective barrier against water loss and pathogen/allergen/irritant invasion. Recent investigations in Europe and Japan have revealed null mutations in the filaggrin gene (FLG) as the underlying cause of ichthyosis vulgaris (IV), a common skin disorder characterised by dry skin, palmar hyperlinearity and keratosis pilaris. Following the development of a strategy for the comprehensive analysis of FLG, we have identified five unique mutations and one recurrent mutation in Singaporean Chinese IV patients. Mutation 441delA is located in the profilaggrin S100 domain, whereas two additional frameshift mutations, 1249insG and 7945delA, occur in the first partial filaggrin repeat ("repeat 0") and in filaggrin repeat 7, respectively. Both nonsense mutations Q2147X and E2422X are found in filaggrin repeat 6, whereas R4307X was found on one of the longer size variant alleles of FLG, within duplicated repeat 10.2. Mutation E2422X, previously found in a single Dutch patient, was found in one Singaporean IV patient and at a low frequency in Asian population controls. Our study confirms the presence of population-specific as well as recurrent FLG mutations in Singapore.     

上海某中学青少年特应性皮炎和鱼鳞病FLG基因型的研究

目的 探讨青少年FLG基因型、了解特应性疾病患病率、发展进程及其FLG基因的关系。 方法 上海某中学334例11 ~ 19岁青少年作为研究样本,进行鱼鳞病、特应性皮炎（AD）、哮喘、鼻炎等特应性疾病的临床病史及体征观察、记录。285例进行了外周血采样及FLG基因高频突变筛查。5年后,进行样本人群随访,观察并记录各特应性疾病的临床症状及体征。 结果 5年前,334例青少年中AD19例（5.69%）,寻常性鱼鳞病14例（4.19%）,过敏性鼻炎36例（10.78%）,哮喘4例（1.20%）。在完成FLG基因测序的285例样本中,携带FLG基因高频突变者24例（8.42%）。5年后完成随访265例,失访69例（20.66%）,AD、鱼鳞病、过敏性鼻炎及哮喘的患病人数分别为13例（4.89%）、15例（5.64%）、27例（10.15%）、1例（0.38%）,原19例AD中6例进入完全缓解期,13例SCORAD评分明显下降,原36例过敏性鼻炎中9例症状消失。单纯AD、单纯鱼鳞病、AD合并鱼鳞病的FLG基因高频突变率分别为10.0%、55.6%、40.0%,且患鱼鳞病与FLG基因突变有关（P < 0.001）。结论 青少年中,FLG基因高频突变率为8.42%。FLG基因是寻常性鱼鳞病的半显性遗传因素,中间丝聚合蛋白的表达受多因素影响。     

丝聚蛋白与相关皮肤病发病机制的研究进展

丝聚蛋白通过参与角质形成细胞的终末角化过程,在表皮屏障中发挥重要作用.中间丝聚合蛋白基因突变引起蛋白表达下降,导致多种过敏性疾病.丝聚蛋白基因突变导致特应性皮炎,也是欧洲和亚洲人种寻常型鱼鳞病的主要诱发因素.丝聚蛋白基因与特应性皮炎发病的婴儿期或早期儿童期关系密切,而与晚期儿童期和成人期无关.有研究发现,小鼠体内缺氧诱导因子引起丝聚蛋白表达降低,提示丝聚蛋白可能不是疾病的单一致病因素.     

四种遗传性鱼鳞病基因型与临床表型的相关性分析

目的对四种鱼鳞病(寻常型鱼鳞病;X性-联鱼鳞病;板层状鱼鳞病;大疱性鱼鳞病)的致病基因进行精细定位,并分析其基因型与临床表型的关系。方法对四种鱼鳞病各1例患者进行临床表型分析以及外周血DNA直接测序检测鱼鳞病FLG基因、STS基因、TGM1基因和K1,K10角蛋白基因的突变位点。结果①寻常型鱼鳞病患者在FLG基因的外显子5的第278位有G-T突变,613位有G-A突变。②板层状鱼鳞病患者TGM1基因外显子3的第504位碱基有C-T突变,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位碱基有C-T突变,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。③X-性联鱼鳞病患者STS基因完全缺失。④大疱性鱼鳞病患者外显子5的第242碱基存在A-C突变,外显子6的第599位碱基均存在A-G突变,导致K1蛋白第633位氨基酸由赖氨酸(Lys)变为精氨酸(Arg)。结论鱼鳞病患者临床表型的不同与致病基因的突变位点密切相关。     

X性联锁遗传鱼鳞病一家系的STS基因研究

目的 研究一 X性联锁遗传鱼鳞病(XLI)家系基因突变,探讨基因突变与临床表现的关系,为进一步开展基因诊断和基因治疗奠定基础。方法 应用PCR方法扩增家系中的先证者及其母亲及与该家系无关的50例正常人外周血基因组DNA STS基因的第一外显子和第十外显子。以角蛋白hHb6为引物,作内对照。结果 家系中先证者的STS基因全部缺失,而先证者之母和与该家系无关的50例正常人未发现缺失。先证者及先证者之母的内对照引物PCR扩增后都有产物。结论 该XLI家系存在STS基因缺失,该缺失引发出XLI特有的皮肤病变。     

Atopic eczema and the filaggrin story

The discovery that null mutations in the filaggrin gene (FLG) are associated with atopic eczema represents the single most significant breakthrough in understanding the genetic basis of this complex disorder. The association has been replicated in multiple independent studies during the past 2 years with the use of various methodologies, from populations in Europe, the United States, and Japan. Filaggrin plays a key role in epidermal barrier function, and its association with atopic eczema emphasizes the importance of barrier dysfunction in eczema pathogenesis. This review aims to summarize the current state of knowledge regarding the role of FLG mutations in ichthyosis vulgaris, atopic eczema, and other skin disorders, with an emphasis on potential clinical applications. Further research is needed to clarify the precise role of filaggrin in skin and systemic atopic disease, to pave the way for novel therapeutic interventions.     

A KRT1 gene mutation related to epidermolytic ichthyosis in a Chinese family

We report a Chinese family with members affected by epidermolytic ichthyosis (EI), caused by KRT gene mutations. The proband was a 14‐year‐old boy who had simultaneous appearance of nephroblastoma and epidermolytic ichthyosis (EI). Both the patient and his mother exhibited the specific clinical and pathological manifestations of EI. We analysed all exons and flanking sequences of the KRT1 and KRT10 genes using PCR, and found that the proband and his mother had a G>C transition at nucleotide position 1432 in exon 7 of KRT1, resulting in an amino acid substitution of glutamate (GAA) to glutamine (CAA) at codon 478 (E478Q). The KRT10 gene had no mutations.