| 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. |
