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Identification of Defective Fas Function and Variation of the Perforin Gene in an Epidermodysplasia Verruciformis Patient Lacking EVER1 and EVER2 Mutations

2007; Elsevier BV; Volume: 128; Issue: 3 Linguagem: Inglês

10.1038/sj.jid.5701124

1523-1747

Elisa Zavattaro, Barbara Azzimonti, Michele Mondini, Marco De Andrea, Cinzia Borgogna, Valentina Dell’Oste, Massimo Ferretti, Stefania Nicola, Giuseppe Cappellano, Adriana Carando, G Leigheb, Santo Landolfo, Umberto Dianzani, Marisa Gariglio,

Parvovirus B19 Infection Studies

autoimmune lymphoproliferative syndrome epidermodysplasia verruciformis human papillomavirus interleukin perforin gene TO THE EDITOR Epidermodysplasia verruciformis (EV), an infrequently reported lifelong clinical entity, is characterized by abnormal susceptibility to human papillomaviruses (HPVs). The natural course of the disease may at times be punctuated by the transformation of EV into squamous cell carcinoma; the lesions are preferentially located on sun-exposed sites. Nonsense mutations in two adjacent novel genes, named EVER1 and EVER2, have recently been associated with the disease in some consanguineous families and sporadic cases (Ramoz et al., 2002Ramoz N. Rueda L.A. Bouadjar B. Montoya L.S. Orth G. Favre M. Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis.Nat Genet. 2002; 32: 579-581Crossref PubMed Scopus (311) Google Scholar; Orth, 2006Orth G. Genetics of epidermodysplasia verruciformis: insights into host defense against papillomaviruses.Semin Immunol. 2006; 18: 362-374Crossref PubMed Scopus (198) Google Scholar). Despite these findings, we have recently described an EV case with a lack of EVER gene mutations and a remarkable CD8+ T-cell lymphocytopenia (Azzimonti et al., 2005Azzimonti B. Mondini M. De Andrea M. Gioia D. Dianzani U. Mesturini R. 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). Although EV has recently been classified as a primary deficiency in innate immunity to specific HPV genotypes (Notarangelo et al., 2004Notarangelo L. Casanova J.L. Fischer A. Puck J. Rosen F. Seger R. et al.Primary immunodeficiency diseases: an update.J Allergy Clin Immunol. 2004; 114: 677-687Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), with the central role assigned to keratinocytes, the molecular mechanisms underlying abnormal susceptibility to a single type of weakly pathogenic infectious agent are still unclear. A 59-year-old woman was admitted to our hospital with a diagnosis of EV. Physical examination revealed a limited number of multiple, flat, whitish and reddish papular lesions on the hands and forearms (Figure 1a and b) and a few pityriasis versicolor-like lesions on the trunk (Figure 1c). Surprisingly, the patient had never developed either cutaneous premalignant or malignant lesions, even in sun-exposed areas; thus, her forehead did not present any erythematous lesions. As reported in Figure 1d and e, biopsy specimens from lesions of the forearm and hand showed hyperkeratosis, acanthosis, and numerous large cells with pale staining of the cytoplasm and perinuclear vacuolization in the spinous and granular layers, resembling the typical histological features of EV (de Oliveira et al., 2003de Oliveira W.R. Festa Neto C. Rady P.L. Tyring S.K. Clinical aspects of epidermodysplasia verruciformis.J Eur Acad Dermatol Venereol. 2003; 17: 394-398Crossref PubMed Scopus (80) Google Scholar). This study was approved by the Research Ethics Committee "Maggiore Hospital" Novara and conducted according to the Declaration of Helsinki Principles. Written informed consent was obtained from the patient. HPV DNA analysis was performed on samples collected with prewetted cotton-tipped swabs from different sites of the skin and on a formalin-fixed, paraffin-embedded papular lesion from the forearm. The results obtained by PCR analysis are shown in Table 1.Table 1HPV DNA presence in skin samples from different sites1Skin samples collected with a saline-soaked cotton-tipped swab and from paraffin-embedded surgical specimen.SampleHPV typeSkin swabs Hand (papular lesion)5, 14 Neck (pityriasis versicolor-like lesion)14 Forehead (no lesion)5, 14 Cheek bone (no lesion)5, 14, 38Surgical specimen Forearm (papular lesion)14, 15, 241 Skin samples collected with a saline-soaked cotton-tipped swab and from paraffin-embedded surgical specimen. Open table in a new tab Genetic analysis of EVER1 (20 exons) or EVER2 (16 exons) genes did not detect any mutation that would result in a loss of function. We only found a nonsynonymous heterozygous single nucleotide polymorphism in the EVER2 gene (rs7208422) in position 1289 of the cDNA (numbering based on GenBank accession no. NM_152468), which caused the N306I amino-acid substitution at the protein level. Analysis of serum Ig levels by ELISA showed that the patient displayed mildly decreased levels of IgA and normal amounts of IgM, IgG, and IgE (Table 2). Immunophenotype analysis of peripheral blood mononuclear cells by two-color immunofluorescence and flow cytometry (Table 2) showed a low reduction of helper T cells (CD3+CD4+) and a striking expansion (9%) of an atypical T-cell population expressing the TCRαβ, but double-negative for both CD4 and CD8 (double-negative T cells). The patient did not develop a contact allergy to dinitrochlorobenzene sensitization, and the purified protein derivative intradermal test was also negative. Analysis of cytokine secretion of peripheral blood T cells stimulated with anti-CD3 mAb showed a higher production of IL-10 in comparison to healthy controls (Figure 2a). Secretion of IL-4, IL-5, and tumor necrosis factor-α was also significantly increased. The proliferative response and natural killer activity were in the normal range (data not shown).Table 2In vivo immunological analysis of the EV patientParameterStudy patientNormal rangeSerum IgM931Values are expressed as international units (IU) per ml.40–230 IgG1,050700–1,600 IgA482Absolute number lower than the normal range.70–400 IgE25<150PBMC immunophenotype T cells CD3+4653Values are expressed as median number of cells per μl (percentage of peripheral blood mononuclear cells out of the total mononuclear cell population); similar results were obtained from blood sample analyses performed over a 1-year period. (404Median percentage of lymphocytes.)425–503 CD3+CD4+2302Absolute number lower than the normal range. (20)280–332 CD3+CD8+2265Absolute number higher than the normal range. (19)163–213 TCRα/β+ CD4/CD8 DN1055Absolute number higher than the normal range. (9)14–28 CD3+/CD25+1615Absolute number higher than the normal range. (14)114–149 CD3+/CD28+254 (22)180–312 CD3+/CD45+RA509 (44)616–676 CD3+/CD45+RO540 (46)344–539 B cells CD19+/CD20+217 (19)110–243 NK cells CD16+/CD56+29 (2)15–47 Monocytes CD14+78 (7)54–104 Skin DTH response DNCB—+++ PPD—+++DTH, delayed-type hypersensitivity; DNCB, dinitrochlorobenzene; PBMC, peripheral blood mononuclear cells; PPD, purified protein derivative.Numbers in bold indicate statistically significant difference compared to the normal range.1 Values are expressed as international units (IU) per ml.2 Absolute number lower than the normal range.3 Values are expressed as median number of cells per μl (percentage of peripheral blood mononuclear cells out of the total mononuclear cell population); similar results were obtained from blood sample analyses performed over a 1-year period.4 Median percentage of lymphocytes.5 Absolute number higher than the normal range. Open table in a new tab DTH, delayed-type hypersensitivity; DNCB, dinitrochlorobenzene; PBMC, peripheral blood mononuclear cells; PPD, purified protein derivative. Numbers in bold indicate statistically significant difference compared to the normal range. Expansion of double-negative T cells is a hallmark of the autoimmune lymphoproliferative syndrome (ALPS), an inherited disease mainly due to impaired apoptosis triggered by the Fas (CD95) death receptor (Straus et al., 1999Straus S.E. Sneller M. Lenardo M.J. Puck J.M. Strober W. An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome.Ann Intern Med. 1999; 130: 591-601Crossref PubMed Scopus (245) Google Scholar; Dianzani et al., 2003Dianzani U. Chiocchetti A. Ramenghi U. Role of inherited defects decreasing Fas function in autoimmunity.Life Sci. 2003; 72: 2803-2824Crossref PubMed Scopus (47) Google Scholar; Worth et al., 2006Worth A. Thrasher A.J. Gaspar H.B. Autoimmune lymphoproliferative syndrome: molecular basis of disease and clinical phenotype.Br J Haematol. 2006; 133: 124-140Crossref PubMed Scopus (132) Google Scholar). Functional analysis of the Fas gene, performed by using in vitro-activated peripheral blood T cells stimulated with an anti-Fas mAb, revealed defective Fas function. By contrast, etoposide-induced cell loss, a topoisomerase II inhibitor chemotherapeutic agent that triggers a death pathway independent of Fas, was in the normal range (Figure 2b). Evaluation of Fas expression in these cells by immunofluorescence and flow cytometry detected normal levels (data not shown). Despite the double-negative T-cell expansion and defective Fas function, the patient did not show any clinical feature of ALPS. ALPS is often caused by mutations of the Fas gene (TNFRSF6), but mutations of the FasL (TNFSF6), caspase-10 (CASP10), or caspase-8 (CASP8) genes have also been occasionally reported (Dianzani et al., 1997Dianzani U. Bragardo M. DiFranco D. Alliaudi C. Scagni P. Buonfiglio D. et al.Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lymphoproliferation.Blood. 1997; 89: 2871-2879PubMed Google Scholar). Moreover, we have recently shown that mutations of the perforin gene (PRF1) and polymorphisms of the osteopontin gene (SPP1) may cooperate with defective Fas function in development of ALPS-like diseases (Clementi et al., 2004Clementi R. Dagna L. Dianzani U. Dupre L. Dianzani I. Ponzoni M. et al.Inherited perforin and Fas mutations in a patient with autoimmune lymphoproliferative syndrome and lymphoma.N Engl J Med. 2004; 351: 1419-1424Crossref PubMed Scopus (55) Google Scholar, Clementi et al., 2006Clementi R. Chiocchetti A. Cappellano G. Cerutti E. Ferretti M. Orilieri E. et al.Variations of the perforin gene in patients with autoimmunity/lymphoproliferation and defective Fas function.Blood. 2006; 108: 3079-3084Crossref PubMed Scopus (50) Google Scholar). No mutation was detected in TNFRSF6, CASP10, and CASP8, whereas a missense variation in PRF1, that is, a C/T substitution at position 272 of the cDNA (numerations are referred to cDNA clone M28393, ATG=+1), which caused an A91V amino-acid substitution, was found. She did not carry the ALPS-associated allele of SPP1, as she was homozygous for +1239A. In summary, this study describes an EV patient lacking mutations of the EVER genes, but carrying several immunological alterations typically found in patients with ALPS, such as peripheral blood expansion of double-negative T cells, decreased Fas function, and a strikingly increased secretion of IL-10. In the study patient, no mutations were detected in the Fas (ALPS type I) and caspase-10 or caspase-8 gene (ALPS type II). Therefore, her defect might be similar to that of ALPS type III, that is, ALPS lacking known mutations and presumed to carry unknown mutations of genes involved in the Fas signaling pathway. Moreover, the patient displayed the A91V variation of the perforin gene that has been associated with an incomplete variant of ALPS. A number of reports on viral disease models, in which mice with deficiencies in one or more components of the two cytolytic pathways were used, demonstrated that perforin and Fas concur in virus clearance (Kagi et al., 1994Kagi D. Ledermann B. Burki K. Seiler P. Odermatt B. Olsen K.J. et al.Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice.Nature. 1994; 369: 31-37Crossref PubMed Scopus (1493) Google Scholar; Balkow et al., 2001Balkow S. Kersten A. Tran T.T. Stehle T. Grosse P. Museteanu C. et al.Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.J Virol. 2001; 75: 8781-8791Crossref PubMed Scopus (77) Google Scholar). In this context, it can be postulated that both Fas function and perforin defects may contribute to the abnormal susceptibility of the study patient to cutaneous HPV infection, possibly by decreasing viral clearance in vivo. Consistent with a role of defective cell-mediated cytotoxicity in some EV patients, we previously reported an EV patient displaying a dramatic CD8+ T-cell lymphocytopenia (Azzimonti et al., 2005Azzimonti B. Mondini M. De Andrea M. Gioia D. Dianzani U. Mesturini R. 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). Thus, a common pathogenetic mechanism can be found in the reduced number or decreased cytotoxic function of CD8+ T cells that may impair the immune defenses against HPV and lead to lifelong infection. It remains to be explained why a cell-mediated cytotoxicity defect would selectively favor cutaneous HPV infection. One possibility is that the putative mild cytotoxicity defect, carried by the EV patient, may reveal itself only in the skin environment, which is a peculiar lymphoid tissue. A second, non-mutually exclusive possibility is that additional genetic or environmental factors may be present in such patients. The authors state no conflict of interest. We thank M. Pippione, University of Turin, for providing the biopsy specimens from 1982. This work was supported by Regione Piemonte "Ricerca Sanitaria Finalizzata e Applicata (CIPE)," Special Oncology Project "Compagnia di San Paolo," MIUR PRIN projects, and Telethon Grant E1170 (Rome). M.M. is a recipient of a research fellowship from "Fondazione Internazionale di Ricerca in Medicina Sperimentale Turin," and V.D.O. is a recipient of a PhD fellowship from "Fondazione Cassa di Risparmio di Vercelli." Supplementary text. 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