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Lack of EVER2 Protein in Two Epidermodysplasia Verruciformis Patients with Skin Cancer Presenting Previously Unreported Homozygous Genetic Deletions in the EVER2 Gene

2011; Elsevier BV; Volume: 132; Issue: 4 Linguagem: Inglês

10.1038/jid.2011.399

1523-1747

Manuela M. Landini, Elisa Zavattaro, Cinzia Borgogna, Barbara Azzimonti, Marco De Andrea, Enrico Colombo, Federica Marenco, A. Amantea, Santo Landolfo, Marisa Gariglio,

Viral-associated cancers and disorders

epidermodysplasia verruciformis human papillomavirus TO THE EDITOR Epidermodysplasia verruciformis (EV) is a rare, lifelong, autosomal recessive skin disease (OMIM number 226400) associated with an unusual susceptibility to infections with ubiquitous beta human papillomaviruses (β-HPVs), but not to infections with other pathogens, including cutaneous and genital HPVs of the alpha, gamma, mu, or nu genera (Jablonska and Majewski, 1994Jablonska S. Majewski S. Epidermodysplasia verruciformis: immunological and clinical aspects.Curr Top Microbiol Immunol. 1994; 186: 157-175Crossref PubMed Scopus (115) Google Scholar; Orth, 2006Orth G. Genetics of epidermodysplasia verruciformis: Insights into host defense against papillomaviruses.Semin Immunol. 2006; 18: 362-374Crossref PubMed Scopus (215) Google Scholar). β-HPVs are evolutionarily distinct from the other HPV genera and are associated with widespread in-apparent or asymptomatic infections in the general population (Bernard et al., 2010Bernard H.U. Burk R.D. Chen Z. et al.Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments.Virology. 2010; 401: 70-79Crossref PubMed Scopus (1221) Google Scholar; Bravo et al., 2010Bravo I.G. de Sanjose S. Gottschling M. The clinical importance of understanding the evolution of papillomaviruses.Trends Microbiol. 2010; 18: 432-438Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In immunosuppressed individuals, however, and in individuals suffering from the rare inherited disease EV, these viruses can replicate unchecked and have been implicated in the development of non-melanoma skin cancer (Akgul et al., 2006Akgul B. Cooke J.C. Storey A. HPV-associated skin disease.J Pathol. 2006; 208: 165-175Crossref PubMed Scopus (189) Google Scholar; Bouwes Bavinck et al., 2011Bouwes Bavinck J.N. Neale R.E. Abeni D. et al.Multicenter study of the association between betapapillomavirus infection and cutaneous squamous cell carcinoma.Cancer Res. 2011; 70: 9777-9786Crossref Scopus (111) Google Scholar; Proby et al., 2011Proby C.M. Harwood C.A. Neale R.E. et al.A Case-Control Study of Betapapillomavirus Infection and Cutaneous Squamous Cell Carcinoma in Organ Transplant Recipients.Am J Transplant. 2011; 11: 1498-1508Crossref PubMed Scopus (103) Google Scholar). The sensitivity of EV patients to β-HPVs has been linked to homozygous mutations in two genes (EVER1 and EVER2; Ramoz et al., 2002Ramoz N. Rueda L.A. Bouadjar B. et al.Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis.Nat Genet. 2002; 32: 579-581Crossref PubMed Scopus (337) Google Scholar). The mutation of either of the EVER genes holds the potential to alleviate EVER-mediated host restriction and favor β-HPV replication and skin carcinogenesis, as is the case in EV patients (Lazarczyk et al., 2009Lazarczyk M. Cassonnet P. Pons C. et al.The EVER proteins as a natural barrier against papillomaviruses: a new insight into the pathogenesis of human papillomavirus infections.Microbiol Mol Biol Rev. 2009; 73: 348-370Crossref PubMed Scopus (113) Google Scholar). In this report, we describe the presence of two previously unreported homozygous mutations in the EVER2 gene in two Italian EV patients, each of whom have already developed more than 10 non-melanoma skin cancer. EV patient 4 (EVpt4) has been previously described by our group (Dell'Oste et al., 2009Dell'Oste V. Azzimonti B. De Andrea M. et al.High beta-HPV DNA loads and strong seroreactivity are present in epidermodysplasia verruciformis.J Invest Dermatol. 2009; 129: 1026-1034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and his clinical picture is presented in Figure 1a. EV patient 5 (EVpt5) is a 58-year-old male who was referred to our hospital with a diagnosis of EV in 2009. Since the age of 42 years, he has had 12 tumors resectioned from his face (forehead; Figure 1b) and back. HPV14 was consistently found in all lesions (CB and MG, manuscript in preparation). HPV DNA analysis of plucked eyebrow hairs revealed the presence of multiple HPV genotypes of the β-genus, with HPV5 and HPV14 viral loads being the highest (Figures 1a and b, below the forehead images). Both patients are HIV-negative. As shown in the representative Bowen's disease tumor sections in Figures 1c and d, the unequivocal histological features of EV (defined by acanthosis containing a majority of cells that show perinuclear halos and blue–gray pallor) are present in both patients' tumors. Genomic DNA extracted from blood samples of both patients was used to perform genetic analysis of the EVER1 and EVER2 genes (see Supplementary Information online). In these patients, no mutations were detected in the EVER1 gene, while they harbor novel invalidating deletions in the EVER2 gene, which create shifts in the reading frame and introduce a premature termination codon as shown in Figure 1e and f. Neither of these frameshift mutations had previously been reported in EV. We confirmed the lack of the EVER2 protein by western blot analysis. As shown in Figure 1h, lysates obtained from keratinocytes isolated from the patients lacked a protein of about 85kDa, the molecular weight expected for the EVER2 protein, which was present in keratinocytes obtained from healthy donors. No extra bands with lower molecular weight were detected in EV patients' extracts, indicating that production of the predicted truncated proteins (Figure 1g) did not occur as a consequence of premature termination codon introduction but rather transcripts are eliminated by nonsense-mediated RNA degradation (Bhuvanagiri et al., 2010Bhuvanagiri M. Schlitter A.M. Hentze M.W. et al.NMD: RNA biology meets human genetic medicine.Biochem J. 2010; 430: 365-377Crossref PubMed Scopus (165) Google Scholar). Download .pdf (.06 MB) Help with pdf files Supplementary Information EVpt4 has consanguineous parents (they were first cousins). He is married to a healthy, unrelated woman, has a son and a daughter, who are both unaffected, and has a brother who is unaffected. To verify the presence of the 13 nucleotide deletion detected in EVpt4, the genomic DNA extracted from blood samples of all of his family members was amplified by exon 4–specific primers and analyzed by agarose gel electrophoresis with high resolution. As shown in Figure 2, both parents carried the 13 nucleotide deletion in the heterozygous state, but not inherited by the other sibling. In contrast, both the proband's son and daughter inherited the mutated allele in the heterozygous state. HPV–DNA genotyping and determination of DNA copy numbers by type-specific Q-PCR protocols were performed with DNA extracted from the plucked eyebrows of all family members (see Supplementary Information online). Some of the genotypes present in the proband were consistently found in all family members, including HPV5 and 20. Of note, the HPV5 viral load in his mother was 15 DNA copies per cell. This finding is consistent with the predominant high viral load for HPV5 reported in all five EV patients enrolled by our group (Azzimonti et al., 2005Azzimonti B. Mondini M. De Andrea M. et al.CD8+ T-cell lymphocytopenia and lack of EVER mutations in a patient with clinically and virologically typical epidermodysplasia verruciformis.Arch Dermatol. 2005; 141: 1323-1325Crossref PubMed Scopus (24) Google Scholar; Zavattaro et al., 2008Zavattaro E. Azzimonti B. Mondini M. et al.Identification of defective Fas function and variation of the perforin gene in an epidermodysplasia verruciformis patient lacking EVER1 and EVER2 mutations.J Invest Dermatol. 2008; 128: 732-735Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar; Dell'Oste et al., 2009Dell'Oste V. Azzimonti B. De Andrea M. et al.High beta-HPV DNA loads and strong seroreactivity are present in epidermodysplasia verruciformis.J Invest Dermatol. 2009; 129: 1026-1034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and suggests that even in the heterozygous genetic background HPV5 replication may be favored by a reduction in EVER2 protein availability. HPV5 and 20 are highly persistent in this family, despite the fact that the family members are of very different ages and live in different houses. The presence of HPV5 with a viral load of four DNA copies per cell in his wife suggests that close and frequent skin contacts with an individual carrying high viral loads for a certain genotype can facilitate virus transmission and sharing of such specific genotype between family members. This report describes a correlation between genetic status and β-HPV carriage in EV family members; to our knowledge this is previously unreported. Collectively, our findings strengthen the hypothesis that the EVER2 gene has a crucial role in the development of EV and in susceptibility to specific β-HPV genotypes. The detection of HPV5 and 20, both of which belong to the β1 species, in all family members, confirms that infection by these subsets of β genotypes is favored by the EV genetic background and is passed between individuals via skin contact (Weissenborn et al., 2009Weissenborn S.J. De Koning M.N. Wieland U. et al.Intrafamilial transmission and family-specific spectra of cutaneous betapapillomaviruses.J Virol. 2009; 83: 811-816Crossref PubMed Scopus (63) Google Scholar). Following the pioneering work by Ramoz et al., 2002Ramoz N. Rueda L.A. Bouadjar B. et al.Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis.Nat Genet. 2002; 32: 579-581Crossref PubMed Scopus (337) Google Scholar, which showed frameshift mutations in the EVER2 gene in an Algerian/Colombian consanguineous EV family, this is the second report to demonstrate an EVER2 autosomal recessive trait in EV. Another three studies have described nonsense mutations in the EVER2 gene: a Chinese EV patient born from consanguineous parents (Sun et al., 2005Sun X.K. Chen J.F. Xu A.E. A homozygous nonsense mutation in the EVER2 gene leads to epidermodysplasia verruciformis.Clin Exp Dermatol. 2005; 30: 573-574Crossref PubMed Scopus (40) Google Scholar), three siblings from a Brazilian family (Rady et al., 2007Rady P.L. De Oliveira W.R. He Q. et al.Novel homozygous nonsense TMC8 mutation detected in patients with epidermodysplasia verruciformis from a Brazilian family.Br J Dermatol. 2007; 157: 831-833Crossref PubMed Scopus (14) Google Scholar), and a Hispanic man (Berthelot et al., 2007Berthelot C. Dickerson M.C. Rady P. et al.Treatment of a patient with epidermodysplasia verruciformis carrying a novel EVER2 mutation with imiquimod.J Am Acad Dermatol. 2007; 56: 882-886Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here, we demonstrate two previously unreported deletions, in exons 4 and 6, respectively, in two Italian EV patients. In one patient, we also show that the EVER2 deletion was carried in the heterozygous state by his parents and inherited in an heterozygous manner by his children. This study provides additional evidence indicating that the lack of the EVER2 protein favors the infection and persistence of specific β1 genotypes, in particular, HPV5, which is often the type presenting the highest viral loads in EV hair bulbs. In addition, the findings of this study strengthen the notion that EVER2 is a key host factor in controlling β-HPV infection and virus-induced skin carcinogenesis in EV patients. We thank the EV patients and their family members for their cooperation, and Mandar Bawadekar for his comments on the manuscript. EZ is funded by Regione Piemonte (LR 4/2006—DR 225/2009). This study was supported by MIUR (PRIN 2008: SL and MG; FIRB—Futuro in Ricerca 2008: MDA), and Fondazione Banca Popolare di Novara BPN (MG). CB is on sabbatical at the NIMR in London on a fellowship from FEMS. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid