【作者】 赵小燕；

【导师】 曾抗；

【作者基本信息】 南方医科大学 ， 皮肤病与性病学（专业学位）， 2014， 博士

【摘要】 毛发上皮瘤(Trichoepithelioma,TE)又名囊性腺样上皮瘤、多发性良性囊性上皮瘤及多发性丘疹性毛发上皮瘤,是一种与毛囊分化有关的皮肤良性附属器肿瘤,1892年首次由Brooke及Fordyce先后报道。该病可分为单发及多发两型,后者多见。单发者少见,不遗传,无家族,通常在成人期发病。相反,多发性家族性毛发上皮瘤(Multiple familial trichoepithelioma,MFT,OMIM#601606),与遗传有关,通常有家族史,呈常染色体显性遗传,常在儿童或青春期发病。MFT多发于20岁以前,女性多见,文献中报道的最小发病年龄为1岁。皮疹表现为小、圆形、光滑发亮、半透明、坚实、正常肤色、境界清楚的丘疹或结节。皮疹平均直径约为2-5mm,通常散在分布,偶可融合成斑块。可发生于面部、头皮、颈部、躯干部等处,其中以面部最常见。此外,还有报道多发性毛发上皮瘤沿不常见的线状分布、皮节或单侧面部分布。MFT特征性临床表现为面中部出现对称性分布的多发性丘疹及结节,多为肤色,呈半透明状,特别好发于鼻唇沟、下颌、上唇,鼻根、额头等处。一般无自觉症状,偶有轻度灼烧感或痒感。无其他相关的系统异常表现。该病虽无生命危险,但因影响患者的美观,可导致自卑等心理问题。本病极少数可恶变成基底细胞癌(basal cell carcinoma, BCC)或毛母细胞癌。也可并发其他皮肤附属器肿瘤如家族性圆柱瘤(Familial Cylindromatosis, FC,OMIM#132700)、Brooke-Spiegler综合征(Brooke-Spiegler syndrome, BSS,OMIM#605041)等。FC患者仅有圆柱瘤的表现。圆柱瘤通常发生在成人早期,主要累及头皮,偶可累及面部。肿瘤若覆盖整个头皮,像头巾样,又称为头巾瘤。BSS患者表现为多种皮肤附属器肿瘤,包括圆柱瘤,毛发上皮瘤和小汗腺螺旋腺瘤。虽然MFT、FC、BSS被描述为独立的疾病,但这些疾病临床表现重叠,且在同一个家系可见这些疾病的不同表现,因此一些学者认为这些疾病可能是同一疾病不同的表型。TE的组织病理上可见真皮上部有多个形态相对单一的基底样细胞构成的细胞团块,周围有丰富的纤维基质包绕伴基质内裂隙。基底样细胞团块主要排列成筛状或网状模式,但有时也可排列成其他模式,包括结节状、串花状。典型的肿瘤团块的病理表现为中间可见一些角质囊肿、退化的毛乳头和胚芽聚集灶,而边缘可见栅栏状排列细胞。TE必须与BCC鉴别,两者很容易混淆。TE组织病理示裂隙发生在基质的胶原纤维间,而BCC裂隙则发生在肿瘤团块与基质之间。此外,免疫组化染色技术亦有利于两者的鉴别。针对TE或BCC表达的CD34与bcl-2抗体免疫组化染色示分布不同。此外,TE的组织病理上,还应与毛母细胞瘤、汗管瘤、微囊肿附属器癌进行鉴别。MFT是一种常染色体显性遗传性疾病。1996年,Harade等首次把MFT定位在染色体9p21,但在该区域未发现易感基因。2004年,张学军等首次将该病的致病基因确定为位于染色体16q12-q13的圆柱瘤(Cylindromatosis,CYLD)基因,该基因也是家族性圆柱瘤与Brooke—Spiegler综合征的致病基因。CYLD基因全长约56kb,包含20个外显子,前三个外显子不编码蛋白质。目前,已报道的各种CYLD基因突变有60余个,其中发生在MFT家系内突变的有20余个。这些突变包括无义突变、错义突变、移码突变及剪切突变,好发于基因羧基端(第9-20外显子)。大多数突变可导致CYLD蛋白的截断。基因型-表型的相关性分析未发现突变类型、位置与患者的表型及严重程度之间有明确的关联。此外,其他学者亦报道过CYLD基因突变检测阴性的病例。研究发现在MFT患者中CYLD基因突变检测阳性率约为44～72%,原因可能是发生在在非编码区(内含子深部、3’UTR或5’UTR)的潜在突变,或为染色体大片段重组或缺失所致；尚有另外一种可能,即MFT具有遗传异质性,存在其他的致病基因,如曾被报道过的染色体9p21区域内。但是,多发性遗传性头颈部丘疹结节的临床及组织病理学鉴别诊断复杂,误诊也有可能。CYLD基因编码CYLD蛋白,又称为泛素特异性加工蛋白酶CYLD和泛素羧基端水解酶CYLD。CYLD蛋白有三个剪切变异,外显子3(位于5’UTR)和外显子7呈选择性剪切。CYLD蛋白全长包含956个氨基酸,而另外两个CYLD同型体因为RNA选择性剪切时仅包含953个氨基酸。CYLD蛋白在人类的脑部、睾丸、骨骼肌、免疫细胞中广泛表达。在人类头皮中CYLD蛋白则表达于毛囊内毛根鞘、小汗腺。CYLD蛋白包含以下结构域：3个细胞骨架相关蛋白-甘氨酸(Cytoskeleton-associated protein-glycine,CAP-Gly)保守结构域(第127-203,232-285与472-540位氨基酸)、2个富含脯氨酸的重复区域(第388-413与446-471位氨基酸)、4个CyS-X-X-CyS区域(第788-856之间氨基酸)和2个泛素羧基端水解酶(Ubiquitin carboxy-terminal hydrolases,UCH)结构域(第593-610与871-889位氨基酸)。第3个CAP-Gly结构域与UCH结构域高度保守。CYLD蛋白为肿瘤抑制蛋白,具有去泛素化酶活性,可负性调节核因子-κB (Nuclear factor-κB,NF-κB)与C-Jun氨基末端激酶(C-Jun NH2-terminal kinase,JNK)信号途径。NF-κB和JNK信号途径对多个生物过程有调整作用,包括免疫与炎症反应、细胞生长、凋亡、肿瘤形成等ψCYLD蛋白的UCH结构域发挥去泛素化酶活性,可对特定蛋白的去泛素化,从而积极参与NF-κB和JNK信号途径。因此CYLD基因失活可增强抑制细胞增殖或促进凋亡的蛋白质降解,导致肿瘤的发生。MFT是皮肤附属器良性肿瘤,一般没必要手术治疗。但是因MFT影响美观,一些患者要求采取某些干预措施。由于皮损数量多,不主张常规手术切除。一般采用其他的剥脱性治疗包括剥脱性铒Yag激光或CO2激光治疗、射频消融术、冷冻或电灼术。但上述治疗有可能导致瘢痕形成或皮损再发,需定期重复上述治疗。近年来研究表明阿司匹林可抑制NF-κB活性。Brummelkamp等发现抑制CYLD的活性可提高细胞抗凋亡的能力,而阿司匹林可拮抗该效应。随后,Fisher和Geronemus报道联合应用阿司匹林325mg,一日2次与阿达木单抗(一种重组人IgGl抗TNF-α单克隆抗体)40mg,每周1～2次,成功治疗一例女性MFT患者。治疗的原理可能为阿司匹林及阿达木单抗可阻断NF-κB活化途径上不同的位点。因为抑制TNF-α的药物物价格昂贵,因此未来研究是否可单独使用其他更有效的非甾体类消炎药取得满意的肿瘤抑制作用是值得关注的。此外,在MFT家系中进行CYLD基因突变分析是开展遗传咨询和产前诊断的重要手段,有助于深入认识MFT基因型-表型之间的相关性,这对MFT患者及其亲属是有益的。而对CYLD基因突变携带者的早期识别可提高临床治疗效果,降低毁容或肿瘤恶性转化等并发症。目的1.报道一个中国MFT家系,应用聚合酶链反应(polymerase chain reaction,PCR)和DNA直接测序的方法对CYLD基因进行突变检测。2.回顾文献报道的发生CYLD基因突变的MFT家系及散发病例,总结MFT临床遗传特征。3.采用抗CYLD抗体免疫组化法来检测毛发上皮瘤肿瘤组织和正常人面部皮肤组织中CYLD蛋白的表达情况。资料与方法患者及家属签署知情同意书,临床及试验研究符合《赫尔辛基宣言》。1.研究对象我们收集了一个来自于中国福建省MFT家系,该家系共3代,包括3例女性患者,2例男性对照,所有患者均经过厦门大学附属第一医院皮肤科专家的确诊。同时,对自2004年以来国内外文献报道的发生CYLD基因突变的MFT的家系或散发病例进行检索。2.组织病理检查对先证者母亲面部皮损进行组织病理切片及HE染色。3.血液和皮肤样本的收集告知患者研究的保密性和自愿性,征得知情同意后,对所有患者和家系中正常人及50例无亲缘关系的正常人进行外周血采集,每例抽取外周血5.0m1于EDTA抗凝管中,—80℃保存备用。收集的毛发上皮瘤组织取自先证者母亲的面部肿瘤处。正常面部皮肤样本则来自一个在我科进行简单的美容手术的年龄匹配的健康对照者。4.CYLD基因突变筛查采用DNA提取试剂盒提取外周血基因组DNA。应用Primer3设计特异性引物,所有的引物由上海英骏生物技术有限公司合成。PCR扩增包括CYLD基因的全部外显子及外显子与内含子交界区的序列。在200μL的微量离心管中进行PCR反应,PCR反应体系50μL,含10x缓冲液(Mg Plus)5μL,引物(25μmol/L)各1μL,Taq酶(购自TaKaRa公司)0.4μL,dNTPs(各2.5mmmol/μL)4μL,基因组]DNA(25ng/μL)5μL；反应条件如下：94℃5min,94℃30s,52-62℃30s,72℃45s,共35个循环,最后72℃延伸5min。反应条件如下：94℃5min,94℃30s,52～62℃30s,72℃45s,共35个循环,最后72℃延伸5min.PCR扩增产物经2%琼脂糖凝胶电泳检测。使用PCR物纯化试剂盒纯化,将纯化产物送上海英骏生物技术有限公司应用ABI Prism377测序仪(美国)进行测序。测序结果采用Chromas软件(Version2.3)对基因序列进行分析,并与家系中的健康人员和50个亲缘关系的正常人序列进行对比。具体步骤如下：4.1外周血DNA提取4.2特异性引物设计合成4.3PCR扩增CYLD基因4.4PCR产物琼脂糖凝胶电泳4.5PCR产物纯化4.6PCR纯化产物测序4.7序列比对分析5.在不同的数据库中对2004年以来国内外文献报道的发生CYLD基因突变的MFT的家系或散发病例进行检索。6.免疫组化法检测CYLD蛋白表达免疫组化法检测患者毛发上皮瘤肿瘤组织、正常人面部皮肤组织CYLD蛋白的表达情况。将石蜡切片脱蜡、水化,3%H2O2室温10min灭活内源性过氧化物酶,蒸馏水洗涤3次；晾干,滴加正常血清封闭液,室温20min,甩去余液；加入适量稀释的兔抗人多克隆CYLD抗体,常温2小时。滴加山羊抗兔IgG,37℃20min,除加入正常血清封闭液外,以上各步间均用PBS(pH7.4)充分洗涤；最后用DAB显色,常规脱水、透明、封片。免疫组化阳性表现为棕褐色,免疫组化阴性对照也是按上述步骤进行操作,除了用PBS替代一抗。结果1.MFT家系临床与遗传特征1.1临床特征该家系共3代,包括3例女性患者,2例男性对照。先证者为4岁女性,足月顺产。2岁时双侧鼻唇沟出现圆形的肤色的约粟粒大小的丘疹。随着年龄的增长,皮疹逐渐增多增大,扩展至面中部,无自觉症状,但影响美观。查体：智力及发育正常,各系统检查无异常,鼻唇沟、鼻部、双眼睑见多个小的圆形的光滑发亮的半透明的坚实的境界清楚的肤色丘疹。皮疹直径约2-3mm。未发现头皮及躯干部肿瘤。家系中患者母亲(图2b)及外祖母有相似的临床表现,但发病年龄较晚,皮损数量较少。1.2遗传特征家系图显示连续三代中有三例女性患者,提示遗传方式可能是常染色体显性遗传或X连锁显性遗传,根据既往的文献报道,考虑常染色体显性遗传的可能。2.组织病理结果先证者母亲皮损组织病理示：真皮内多个基底样细胞组成的肿瘤团块,周围胶原纤维及纤维母细胞包绕,可见角质囊肿。3.PCR扩增结果利用设计的特异性引物,以提取的外周血DNA为模板,对CYLD基因20个外显子进行PCR扩增,均取得到预期的DNA片段。4.测序结果该家系及50例正常人中CYLD基因突变检测未发现外显子及侧翼序列中有突变发生。5.文献总结自2004年以来我们共检索到国内外文献报道的26个MFT家系及散发病例发生了CYLD基因突变,并总结了MFT患者的CYLD基因突变及临床特征。6.免疫组化结果患者毛发上皮瘤肿瘤组织CYLD蛋白表达阳性,分布呈细胞质型,周围的毛囊、小汗腺及表皮全层CYLD蛋白表达亦阳性；正常人面部皮肤组织中毛囊内毛根鞘、小汗腺、表皮基底细胞层CYLD蛋白表达阳性。结论1.我们确诊了一个中国三代MFT家系。该MFT家系中未发现CYLD基因突变位点,免疫组化显示毛发上皮瘤肿瘤组织中CYLD蛋白表达阳性,说明该家系中无CYLD基因突变来限制CYLD蛋白的在肿瘤组织中的表达,提示该MFT家系可能具有遗传异质性,但这需将来采用全基因组扫描技术平台对该家系进行致病基因的重新确定。2.总结的2004年以来MFT患者中CYLD基因突变及临床特征与既往的文献报道一致。因样本量相对较少及缺乏详细的临床资料,未观察到CYLD基因突变-表型之间的相关性。3.在人类正常面部皮肤组织中,免疫组化显示CYLD蛋白表达在毛囊内毛根鞘、小汗腺与表皮的基底细胞层,而毛囊内毛根鞘、小汗腺及表皮基底层均起源于胚胎的体表外胚层,因此推测毛发上皮瘤可能起源于多能性表皮干细胞。更多还原

【Abstract】 Trichoepitheliomas(TE), also named epithelioma adenoides cysticum or multiple benign cystic epithelioma or multiple papular trichoepithelioma, are benign cutaneous adnexal tumors with follicular differentiation.First reported in1892by Brooke, and then subsequently by Fordyce. TE may occur as solitary, or more common, as multiple lesions. Solitary trichoepitheliomas do not appear to be inherited with no family history, and they often develop during the adult years. In contrast, multiple familial trichoepitheliomas(MFT,OMIM#601606) are inherited in an autosomal dominant pattern with positive family history, occuring in early childhood or at puberty.MFT ofen appears under20years old, more common in female than in male patients. In the literature, the youngest reported age of onset is at1years old. The lesions are small, round, smooth, shiny, slightly translucent, firm, skin-colored, circumscribed papules or nodules. The individual lesions average about2-5mm in diameter, usually discrete, occasionally confluent.The face is most frequently affected, whereas other sites such as the scalp, neck or trunk have been described. In addition, there have been several descriptions of unusually arranged linear, dermatomal, or hemifacial multiple trichoepotheliomas. MFT is clinically characterized by development of numerous, symmetrical, slightly translucent, skin-coloured papules or nodules on the central face, especially around the nasolabial folds, the chin, the upper lip, the nasal root, forehead. These patients are asymptomatic, occasionally burning or pruritic. There are no other associated systemic anomalies. Although these lesions are not life-threatening, the effects of MFT can be cosmetically troublesome and psychologically devastating resulting in psychological problems such as low self-esteem. The occasional development of a basal cell carcinoma (BCC) or trichoblastic carcinoma in the setting of MFT has been reported. Patients with MFT are predisposed to combine with other skin appendage tumors such as familial cylindromatosis(FC,OMIM#132700),Brooke-Spiegler syndrome(BSS, OMIM#605041). Patients with FC have only cylindromas. Cylindromas, usually developing from early adulthood, typically occur on the scalp and occasionally on the face. Tumours may come to cover the scalp area, a phenomenon known as’turban tumours’. Patients with BSS develop multiple skin appendage tumors including cylindromas, trichoepitheliomas, and spiradenomas. Although MFT, FC and BSS were originally described as separate entities, these disorders show overlapping phenotypic features, and because different manifestations of each have been described within a single family, many scholars consider these disorders to represent phenotypic variations of the same condition.Histopathologically, the benign tumour consists of islands of relatively monomorphic basaloid cells in the upper dermis surrounded by abundant fibrous stroma with intrastromal clefts. The basaloid aggregations are mainly arranged in a cribriform or retiform pattern but may show other architectural patterns, including nodular, racemiform.They typically show several foci with keratinous cysts, rudimentary follicular papillae, germ and speripheral palisading. Trichoepithelioma must be differentiated from BCC, with which it is frequently confused. In trichoepithelioma, clefts form between collagen fibers in the stroma, while in BCC, clefts form between the tumor islands and stroma. In addition to this, there are histochemical staining techniques that can also differentiate the two. Antibodies against CD34and bcl-2that are expressed by TE or BCC are stained by histochemical method according to their distribution. Moreover the microscopic differential diagnosis of TE should include trichoblastoma, syringoma, and microcystic adnexal carcinoma.MFT is an autosomal-dominant disorder. In1996, Harade et al mapped the first locus for MFT to chromosome9p21, but no susceptibility gene has been identified. In2004, Zhang et al firstly identified mutations in the cylindromatosis gene (CYLD) on chromosome16q12-ql3as the genetic defect of this disease, which is also responsible for FC and BSS. The CYLD gene extends over approximately56kb and is composed of20exons, with the first three exons untranslated. Full-length CYLD is predicted to be a protein of956amino acids, with another CYLD isoform of953amino acids resulting from alternative RNA splicing and skipping of exon7. To date, more than60different germline mutations in the CYLD gene have been described, including more than20germline CYLD mutations identified in cases of MFT. These mutations included nonsense, missense, frameshift and splicing mutations, mainly locating in carboxyl-terminal region (exon9-20). Most of these mutations result in truncated CYLD proteins. Genotype-phenotype analysis has failed to reveal a clear correlation between the types of mutation, their location within the gene and the patients’ phenotype and the disease severity. Moreover, CYLD germline mutation-negative MFT cases were also described by other studies. The putative positive frequency of CYLD mutation in MFT patients is44-72%, probably due to underlying mutations in non-coding regions (introns,3’ UTR and5’UTR) or fragment rearrangements or chromosomal deletions. It still remains unclear whether abnormal function of other genes could also be the cause of MFT, such as reported loci in chromosome9p21.However, the clinical and histologic differential diagnosis of multiple inherited head and neck papules and nodules is complex and misdiagnosis is also a possibility.The CYLD gene encodes the CYLD protein, also known as ubiquitin (Ub)-specific-processing protease CYLD, and Ub carboxyl-terminal hydrolase CYLD. CYLD has three major splice variants. Exon3(in the5’ UTR) and exon7exhibit alternative splicing. Full-length CYLD is predicted to be a protein of956amino acids, with another CYLD isoform of953amino acids resulting from alternative RNA splicing. CYLD is widely expressed in brain, testis, skeletal muscle and immune cells.In human skin, prominent CYLD protein immunoreactivity was seen in the inner root sheath of hair follicles, and the eccrine glands from normal human scalp. The CYLD protein contains the following motifs:three cytoskeleton-associated protein-glycine conserved (CAP-Gly) domains (aa127-203,232-285and472-540); proline-rich repeat (aa388-413and446-471); four Cys-X-X-Cys pairs (between aa788and856); a split domain with similarity to ubiquitin carboxy-terminal hydrolases (UCH) domains (aa593-610and871-889). The third CAP-Gly domain and the UCH catalytic domains are highly conserved.The CYLD protein functions as a tumor braker, a deubiquitinating enzyme that negatively regulates the nuclear factor-κB (NF-κB), c-Jun NH2-terminal kinase (JNK) signaling pathways. The NF-κB and JNK signaling pathways regulate diverse biological processes, including the immune and inflammatory response, cell growth, apoptosis, and tumour formation. CYLD has a UCH domain characteristic of deubiquitinating enzymes, and this is essential for CYLD to remove ubiquitin from certain proteins that positively mediate signaling through the NF-kappaB and JNK pathways. Therefore, inactivation of the CYLD gene may contribute to oncogenesis by enhancing the degradation of proteins that suppress cell proliferation or promote apoptosis.MFT is a benign cutaneous adnexal neoplasm, and there is no imperative for surgical treatment. However, MFT can be cosmetically troublesome and many affected patients desire some type of intervention. Because of multiple lesions, conventional excision is not usually indicated. Other ablative approaches, including ablative erbium:Yag Or CO2laser surgery, radiofrequency ablation, cryosurgery or electrosurgery, have been employed. However, most of the aforementioned therapies can lead to scar formation, or lesions may recur, which need repeated treatment at regular intervals. Recent studies show that Aspirin is capable of inhibiting NF-KB activation. Brummelkamp et al. showed that inhibition of CYLD increases resistance to apoptosis, and the effect can be counteracted by aspirin derivatives. Fisher and Geronemus later reported successful treatment of a woman with MFT by aspirin325mg twice a day and adalimumab (a human recombinant IgGl anti-TNF monoclonal antibody)40mg1-2times weekly. The treatment was based on that, aspirin and adalimumab were given in an attempt to block two different points in the NF-κB activation pathway. As TNF-a inhibitors are very costly, it would be worthwhile to find out whether other more potent NSAIDs used alone could achieve satisfactory tumour-suppression effects in the furure.In addition, Mutation analysis of the CYLD gene in MFT pedigrees is the important tool for genetic counseling and prenatal diagnosis and makes a contribution to the worldwide knowledge of genotype-phenotype correlation in MFT, which is beneficial to patients with MFT as well as to their relatives.Moreover, early identification of CYLD gene mutation carriers may improve the therapeutic management to decrease complications such as disfigurement or malignant transformation.Objective1.To report a Chinese family of MFT and to explore the genetic mutation of CYLD gene by polymerase chain reaction (PCR) and direct sequencing.2.To summarize and analyse the clinical and genetic features of MFT cases by a literature review of CYLD gene mutations in MFT families and sporadic cases.3.To evaluate the expression of CYLD protein in trichoepithelioma tumour tissue by immunohistochemistry with anti-CYLD antibody.Materials and methodsThe patients and their family members signed our informed consent and all of the clinical and experimental research meet the Helsinki declaration. All family members were received careful physical examination by experienced dermatologists.1.Subject investigatedWe collected a Chinese MFT family from Fujian province of China. The family of three generations with MFT that includes three affected females and two unaffected males was identified by experienced dermatologists at the Dermatology department of the First Affiliated Hospital of Xiamen University. At the same time, we reviewed the literatures about CYLD gene mutations in MFT families and sporadic cases since2004.Histopathological examinationFor histopathological examination, the section obtained from facial lesion of the proband’s mother were stained with H&E staining.3. Blood and skin tissue samples collectionAfter obtaining informed consent and notifying patients of their right to confidentiality and the voluntary nature of the study, five millilitres of peripheral blood from available family members and50unrelated and unaffected people were collected in EDTA anticoagulant tubes, and kept frozen at-80℃until DNA extraction. Trichoepithelioma tissue was taken from a skin tumour on the face of the proband’s mother. A normal skin specimen of face was obtained from a age-matched healthy control who underwent a simple plastic surgery procedure at our department.4.CYLD gene mutation analysisGenomic DNA was extracted from each blood sample using a whole blood genomic DNA extraction kit, according to the manufacturer’s instructions. The specific primers were designed according to Primer3Input. All the primers were synthesized by Shang hai Invitrogen Biotechnology Co.LTD. All of the exons of CYLD and its boundary regions were amplified by PCR as according to standard literature methods. PCR was carried out in a200μL Eppendorf tube containing5μL of IOxPCR buffer(Mg Plus),1μL of each primer(25μmol/L),0.4μL of Taq DNA polymerase (TaKaRa, TaKaRa Biotech, Dalian,China),4μL dNTP Mixtures (each2.5mmol/μL),5μL of genomic DNA(25ng/μL) within a total volume of50μL.The PCR conditions were as follows:Taq activation at94℃for5min,followed by35cycles each having denaturation at94℃,30s, anneal52-62℃for30s, and extension at72℃for45s and a final5min extension at72℃. PCR amplification products were detected by2%agarose gel electrophoresis and were purified using PCR purification kit.The purified product were sended to Shang hai Invitrogen Biotechnology Co.LTD. for the sequencing. Direct sequencing was performed using a DNA sequencing system (ABI Prism377sequencer, U.S.A). The sequencing results were analysed using Chromas software (Version2.3) and compared with those of unaffected members and50unrelated and unaffected samples. Specific steps are as follows: 4.1DNA extraction from peripheral blood4.2Design and synthesis specific primers4.3PCR amplification of CYLD gene4.4PCR product agarose gel electrophoresis4.5PCR product purification4.6PCR purification product sequencing4.7Sequence alignment and analysis5. A review of the literature about MFT families and sporadic cases with CYLD gene mutations in different databases.6. Immunohistochemical method to detect CYLD protein expressionWe conducted immunohistochemistry to examine the expression of CYLD in the trichoepithelioma tumour tissue of the patient and in normal skin of human face. Paraffin sections were dewaxed, hydrated and incubated in3%H2O2for10min to inactivate endogenous peroxidase at room temperature. The sections was washed with PBS three times. Dry, add normal serum blocking solution at room temperature for20min, to absorb excess liquid. Add proper diluted primary antibody(rabbit antihuman CYLD polyclonal antibody) at normal temperature for2hours. Add goat anti-rabbit1gG secondary antibody, at37℃for20min. In addition to adding normal serum blocking solution, each step with PBS (pH7.4) thoroughly washing. Diaminobenzidine(DAB) was used as the chromogen. The Stained sections were conventionally dehydrated, transparent and sealed. Immunoreactivity appeared as brown staining. Immunohistochemical controls were routinely performed following the same procedures, except that PBS was substituted for the primary antibody.Results1. Clinical and genetic features of the MFT family1.1Clinical features The family of three generations with MFT included three affected females and two unaffected males. The proband was a4-year-old girl, born following a normal pregnancy and delivery. At age2, she began to develope round, skin-colored, miliary sized papules on the nasolabial folds. During the past two years, the papules enlarged in sizes and increased in number gradually on the central area of the face, which were asymptomatic but cosmetic troublesome. On physical examination, there were normal intelligence, physical development and no systematic anomalies. The numerous small, round, smooth, shiny, slightly translucent, firm, circumscribed, discrete, skin-colored papules were present on the face, favoring the nasolabial folds, nose and eyelids. The individual lesions average2-3mm in diameter. There were no scalp and neck tumours. Her mother and grandmother also had similar papules on the face, with later age of onset and fewer lesions.1.2Genetic featuresThe pedigree revealed three affected females belonging to three consecutive generations, suggesting that inheritance pattern may be autosomal dominant or X-linked dominant inheritance. We considered the possibility of autosomal dominant inheritance according to previous reports.2.Histopathological resultBiopsy of a skin lesion of the proband’s mother revealed dermal tumors with multiple nests of basaloid cells, collagen fibers and fibroblasts surrounding the tumor islands in a concentric array. Keratinous cysts may be seen.3.PCR amplification resultsThe specific primers were designed and genomic DNA was used as a template for the PCR amplification of all the20exons of the CYLD gene. We obtained the expected DNA fragments.4.Sequencing results We carried out mutation scanning and detected no germline mutation in all exons and exon-intron boundaries of the CYLD gene in available family members and50unrelated and unaffected people5.Literature summaryWe reviewed26MFT families and sporidic cases with CYLD gene mutations reported in the literature since2004, and summarized the mutations and clinical findings in patients with MFT.6.Immunostaining resultsTumour tissue sections displayed prominent cytoplasmic CYLD protein immunoreactivity in trichoepithelioma tumour tissue, the root sheath of hair follicles and eccrine glands surrounding it, and all layers of the epidermis. In normal human face skin, prominent CYLD protein immunoreactivity was seen in the inner root sheath of hair follicles, the eccrine glands, and the basal layer of the epidermis.Conclusions1.A Chinese family of three generations with MFT was identified. No germline mutation in the CYLD gene was detected in this family. Immunostaining results confirmed CYLD expression in trichoepithelioma tumour tissue, which evidenced no CYLD mutations in this family limiting its expression in tumor tissue. It possibly suggests genetic heterogeneity of the MFT family. The genome-wide scanning technique will be used to identify the causative gene for this Chinese MFT pedigree in the future.2.The mutation characteristics of CYLD gene and clinical findings in patients with MFT since2004were comparable with the previously reported literature.Thus far, there is no evident genotype-phenotype relationship in CYLD mutations and phenotype, due to the relative small sample and the lack of the full clinical informations.3.In normal human face skin tissue, immunohistochemical staining revealed expression of CYLD protein in the inner root sheath of hair follicles, the eccrine glands, and the basal layer of the epidermis, which originated from the embryonic surface ectoderm.Therefore, it is speculated that trichoepithelioma may originate from pluripotent epidermal stem cells.更多还原