Report of Three Novel Germline CYLD Mutations in Unrelated Patients

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Year: 2016

Report of three novel germline CYLD mutations in unrelated patients with Brooke-Spiegler syndrome, including classic phenotype, multiple familial trichoepitheliomas and malignant transformation

Tantcheva-Poór, Iliana ; Vanecek, Tomas ; Lurati, Massimo C R ; Rychly, Boris ; Kempf, Werner ; Michal, Michal ; Kazakov, Dmitry V

Abstract: Brooke-Spiegler syndrome is a rare autosomal-dominant genetic disorder characterized by multiple adnexal tumors, including cylindromas, spiradenomas, spiradenocylindromas and trichoepitheliomas. It is caused by germline CYLD mutations commonly leading to a premature stop codon. We here report on 3 novel CYLD mutations in 3 unrelated BSS patients, including the classic phenotype, multiple familial trichoepitheliomas phenotype and malignant transformation. These included c.18211826 + 1delinsCT/L607Ffs∗9, c.2666A > T/p.D889V andc.2712delT/p.905Kfs∗8.ByextendingthespectrumofCY LDmutations,

DOI: https://doi.org/10.1159/000437303

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-115928 Journal Article Published Version

Originally published at: Tantcheva-Poór, Iliana; Vanecek, Tomas; Lurati, Massimo C R; Rychly, Boris; Kempf, Werner; Michal, Michal; Kazakov, Dmitry V (2016). Report of three novel germline CYLD mutations in unrelated patients with Brooke-Spiegler syndrome, including classic phenotype, multiple familial trichoepitheliomas and malignant transformation. Dermatology, 232(1):30-37. DOI: https://doi.org/10.1159/000437303 Original Paper

Dermatology 2016;232:30–37 Received: March 13, 2015 DOI: 10.1159/000437303 Accepted after revision: June 13, 2015 Published online: September 2, 2015

Report of Three Novel Germline CYLD Mutations in Unrelated Patients with Brooke-Spiegler Syndrome, Including Classic Phenotype, Multiple Familial Trichoepitheliomas and Malignant Transformation

a b, c d f Iliana Tantcheva-Poór Tomas Vanecek Massimo C.R. Lurati Boris Rychly e b b, c Werner Kempf Michal Michal Dmitry V. Kazakov a b Department of Dermatology, University Hospital, Cologne, Germany; Sikl’s Department of Pathology, c Charles University in Prague, Medical Faculty in Pilsen, and Bioptical Laboratory, Pilsen , Czech Republic; d e Private Dermatology Practice, Lugano, and Kempf und Pfaltz, Histologische Diagnostik, Zurich, Switzerland; f Cytopathos Laboratory, Bratislava, Slovak Republic

Key Words Introduction Adnexal tumors · Spiradenoma · Spiradenocylindroma · Cylindroma · Trichoblastoma · Trichoepithelioma · Brooke-Spiegler syndrome (BSS) is a rare autosomal- CYLD gene · Carcinoma dominant genetic disorder characterized by multiple adnexal tumors, including cylindromas, spiradenomas, spiradenocylindromas and trichoepitheliomas (cribriform Abstract trichoblastomas). The tumors are mostly localized in the Brooke-Spiegler syndrome is a rare autosomal-dominant ge- head and neck area and may undergo a malignant trans- netic disorder characterized by multiple adnexal tumors, in- formation in 5–10% of cases [1–4] . In addition, BSS pa- cluding cylindromas, spiradenomas, spiradenocylindromas tients may very rarely develop tumors of the major (pa- and trichoepitheliomas. It is caused by germline CYLD mu- rotid) and minor salivary glands, and exceptionally in the tations commonly leading to a premature stop codon. We breast [5–7] . here report on 3 novel CYLD mutations in 3 unrelated BSS Familial cylindromatosis and multiple familial tricho- patients, including the classic phenotype, multiple familial epitheliomas (MFT) are nowadays considered phenotyp- trichoepitheliomas phenotype and malignant transforma- ic variants of BSS: 1 patient may develop various types of tion. These included c.1821_1826+1delinsCT/L607Ffs 9, tumors and/or different phenotypes may occur in the c.2666A>T/p.D889V and c.2712delT/p.905Kfs 8. By extend- same family [8–16] . All three conditions are caused by ing the spectrum of CYLD mutations, better understanding* germline mutations in the same CYLD gene [17–20] . of the molecular mechanisms of BSS can be gained,* which At the time of writing, a total of 95 distinct germline might later assist in finding new treatment options. CYLD mutations have been reported [17, 21–28] . CYLD © 2015 S. Karger AG, Basel is a tumor suppressor gene located on chromosome 16q

© 2015 S. Karger AG, Basel Dmitry V. Kazakov, MD 1018–8665/15/2321–0030$39.50/0 Sikl’s Department of Pathology, Charles University Medical Faculty Hospital Alej Svobody 80 E-Mail [email protected] CZ–304 60 Pilsen (Czech Republic) www.karger.com/drm E-Mail kazakov @ medima.cz Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM Color version available online Color version available online

Fig. 1. Patient 1. Multiple skin-colored papules on the face favoring Fig. 2. a, b Patient 1. Cribriform trichoblastoma (trichoepithelio- the nasolabial folds and eyelids (covered). ma). Cribriform and solid basaloid cell aggregates focally resembling follicular germ structures surrounded by specific follicular stroma resembling follicular papillae.

abc

Fig. 3. Family pedigree of patient 1 (a ), patient 2 (b ) and patient 3 (c ).

Brooke-Spiegler Syndrome Dermatology 2016;232:30–37 31 DOI: 10.1159/000437303 Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM Table 1. Main clinicopathological data

Case Sex/age, Nationality/ Clinical features Available histology CYLD mutation years ethnicity

1 F/29 Swiss multiple paranasal papules 2 trichoepitheliomas c.2666A>T 2 M/60 Czech multiple confluent scalp tumors, 1 BCAC-HG evolving from c.2712delT the biggest one measuring 5 × 3 × 2.5 cm, spiradenocylindroma; with recent rapid growth 1 spiradenoma; 1 cylindroma 3 F/76 Slovak multiple tumors on the face and 2 spiradenocylindromas; c.1821_1826+1delinsCT scalp 1 trichoepithelioma

which has 20 exons and encodes a protein that negatively regulates the NF-κB and JNK pathways [29] . Most germline mutations reported to date are frameshift and nonsense mutations commonly leading to a premature stop codon. We here report on 3 BSS patients (including 1 with a malignant phenotype) from 3 unrelated families with novel germline CYLD mutations, thus extending the cat- Color version available online alogue of known germline CYLD mutations.

Patients and Methods

Patients The main clinical data, histopathological and molecular findings are summarized in table 1 . Patient 1. A 29-year-old woman presented with a few papules on her face, favoring the nasolabial folds and eyelids. She started developing them at the age of 13 years ( fig. 1 ). A shave biopsy of two papules revealed cribriform trichoblastoma (trichoepithelioma) in each of the specimens ( fig. 2 ). The family pedigree showed an autosomal dominant mode of inheritance (fig. 3 a). Patient 2. A 60-year-old man had multiple partly confluent tumors on his scalp (fig. 4 ) which first appeared at the age of 40 years. Some of the tumors had grown faster in the last 2 years, with the largest one (right temporal area) reaching the size of 5 × 3 × 2.5 cm. Two out of four specimens showed stereotypical features of a cylindroma and a spiradenoma. The other two specimens revealed malignant transformation of a preexisting spiradenocylindroma featuring nests and sheets of medium-sized pleomorphic Fig. 4. Patient 2. Multiple variably sized red to bluish confluent basaloid cells with an infiltrative growth pattern and plentiful mi- nodules on the scalp. toses. The stroma was partly fibrous, partly myxoid with a retrac- tion phenomenon. Lymphocytes were missing and the malignant moiety of the tumor was diffusely positive for p53 ( fig. 5, 6). The mother of the patient suffered from similar tumors on her scalp (fig. 6 b). Molecular Analysis Patient 3. A 76-year-old woman presented with multiple skin After obtaining the patients’ informed consent, analysis of tumors on her face and scalp, which had appeared at the age of germline CYLD mutations was performed as described previous- 17 years. Two biopsies were taken, which revealed a spiradenocyl- ly [30] . Briefly, DNA was extracted from peripheral blood using indroma and cribriform trichoblastoma (trichoepithelioma) next a QIAsymphony DNA Mini Kit (Qiagen, Hilden, Germany) on to an incipient spiradenocylindroma. Family members were af- an automated extraction system (QIAsymphonySP, Qiagen) ac- fected in an autosomal-dominant fashion (fig. 3 c). cording to the manufacturer’s protocol. Coding sequences and

32 Dermatology 2016;232:30–37 Tantcheva-Poór et al. DOI: 10.1159/000437303 Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM Color version available online

Fig. 5. Patient 2. a Spiradenocylindroma composed of small nodules arranged in jigsaw pattern (left) corresponding to cylindromatous moiety in close proximity to large nodular aggregates representing spiradenomatous portion (right). b Close-up of the cylindromatous part: note conspicuous droplets of hyaline basement membrane material. c Transitional area with cylindromatous jigsaw pattern but numerous intratumoral lymphocytes, an essential component of a spiradenoma. d Area with sharp demarcation of spiradenomatous (upper right) and cylindromatous (lower left) d components.

exon-intron junctions were amplified by HotStar Taq DNA Results Polymerase (QIAntigen, Hilden, Germany). PCR products were purified and sequenced bidirectionally, using the Big Dye Ter- Three novel CYLD gene mutations were found and minator Sequencing Kit (Applied Biosystems, Carlsbad, Calif., USA) . In silico analysis of the impact of the mutation on protein the proteins were in silico predicted. These included structure and function was performed by PolyPhen-2 and c.1821_1826+1delinsCT/L607Ffs 9, c.2666A>T/p.D889V PROVEAN. and c.2712delT/p.905Kfs 8 ( fig. 7 ). The prediction of * * Brooke-Spiegler Syndrome Dermatology 2016;232:30–37 33 DOI: 10.1159/000437303 Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM Color version available online

a b

Fig. 6. Patient 2. a, b Malignant tumor evolving from a preexisting spiradenocylindroma. Remnants of spiradenomatous component can be recognized (a, upper portion) in close proximity to irregu- lar infiltrative nodules (a, lower portion) composed of pleomorphic medium-sized to large basaloid cells with plentiful mitoses and necrotic cells (b). c Strong p53 expression in the malignant c part of the tumor.

protein functionality by PolyPhen-2 and by PROVEAN radenocylindroma, both sporadic and associated with revealed a malignant status of missense mutation (‘prob- BSS, this was the second most common type of a malig- ably damaging’, score 0.999, and ‘deleterious’, respective- nant pattern after low-grade salivary gland-type BCAC ly). [31] . In the same study, a correlation between the histo- logic pattern and clinical course was found in the sense that in patients with BCAC-HG, a more aggressive course Discussion is to be expected. The malignant portion was strikingly positive for p53, as is sometimes seen in these lesions In this study, we have reported three novel CYLD mu- [32] . tations in 3 unrelated BSS patients. Our patients have a CYLD has no mutation hotspot, but the three novel variable phenotypic expression ranging from tiny pap- germline mutations we identified are located in the usu- ules on the face representing trichoepitheliomas in pa- ally mutated parts of the CYLD gene, namely between ex- tient 1 (MFT phenotype) to multiple tumors on the scalp ons 9 and 20 (fig. 8 ) [27] . Two mutations represent a in patients 2 and 3. Additionally, in patient 2, malignant splice-site (patient 1) and a frameshift (patient 3) muta- transformation of spiradenocylindroma has occurred, tion, resulting in a premature stop codon and, thus, sub- and the tumor was classified as a salivary gland-type bas- stantially disturbing the protein structure. In patient 1, a al cell adenocarcinoma-like pattern, high-grade (BCAC- missense mutation was found, meaning that a change in HG). In a series of 24 malignant tumors evolving from a single amino acid had occurred. Mutations that change preexisting benign spiradenoma, cylindroma and spi- an amino acid can differ in their impact on protein func-

34 Dermatology 2016;232:30–37 Tantcheva-Poór et al. DOI: 10.1159/000437303 Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM c.1821_1826+1delinsCT Color version available online

c.2666A>T

c.2712delT

Fig. 7. Sequences of the three novel mutations identified in our study (c.1821_1826+1delinsCT/L607Ffs*9 (case 3), c.2666A>T/p.D889V (case 1) and c.2712delT/p.905Kfs*8 (case 2).

tion. In our case, the in silico analysis supports the claim clinical and histomorphologic data may eventually help of causality of this mutation. in developing prognostic criteria. By extending the spec- Using the current analysis (sequencing of coding se- trum of CYLD mutations a better understanding of the quences and exon-intron junctions), germline CYLD mu- molecular mechanisms of BSS can be gained. This might tations are presently detected in about 76% of cases (the later assist in identifying new treatment options. mutation frequency is about 85% for classic BSS and 44% for MFT) [17, 33]. Mutations in introns leading to intronic exonization and large deletions have been reported Disclosure Statement in some of those ‘negative’ cases [30, 34]. So far, no geno- The authors have nothing to disclose to the editor regarding type-phenotype correlation has been found [11, 17, 35] . any direct or indirect financial implication that publication of this Bigger cohorts with accurate correlation of molecular, paper may have for them, their relatives or their institution.

Brooke-Spiegler Syndrome Dermatology 2016;232:30–37 35 DOI: 10.1159/000437303 Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM –34111_*297858del378779 914-6398_1769del13642ins20 AUG

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Color version available online

561dupT

1027dupA 1096del2 1569T>G 1112C>A 1628del2 2042A>G 1135G>T 1830_1831insA 2065_2066delCT 1681_1682delTT 2253delG 2546G>A 1843delT 2068_2069delTT 1682T>A 2255delT 2552_2553insA 1859_1860delTG insC 1684+1G>A 2259dupT 2569C>T 1863insA 2070delT 2272C>T 2602G>T 1893_1906del14 2081delT 1139-148A>G 2288_2289delTT 2613C>G 1935_1936insT 2104_2105insA 1178_1179delCA 2290del5 2641delG 2104delA 1207C>T 2299A>T 2655G>A 2108G>C 1364_1365delAA 2305delA 2662_2664delTTT 2109-2A>C 2666A>T 1392_1393dupT 1950-2A>T 2305_2306insC 1455T>A 2317G>A 1950_1953delGATA 2686+60_*3340del5362 1455T>G 2116_2117 2330_2331delTA 1758insGATA 1961T>A 2687G>C 1462delA insATTAG 2335G>A err 1776delA 2012_2021del10 2711C>T 1518+2T>C 2119C>T 2339T>G 1787G>A 2041+1G>T 2355_2358delCAGA 2712delT 2128C>T 2350+5G>A 1821_1826delinsCT 2409C>G 2713C>T 2138delA IVS12+1G>A 2449delT 2729dupC 2154insT 1826+1G>T 2460delC 2806C>T 2155dupA 1826+2T>G 2467C>T 2814_2817delGCTT 2164C>A 2469+1G>A 2822A>T 2170_2171insTC 2172delA 2214delT 2240A>G 2240_2241delAG

Fig. 8. Presently identified germline mutations and their distribution in the CYLD gene. Novel mutations are identified in red (modified from Grossmann et al. [17] ) .

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Brooke-Spiegler Syndrome Dermatology 2016;232:30–37 37 DOI: 10.1159/000437303 Downloaded by: Universität Zürich, Zentralbibliothek Zürich 130.60.47.22 - 5/12/2016 12:02:13 PM