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Nov 30, 2006 - and gene map information of EVER1 and EVER2 genes. We thank David K. Watson for the expert technical help. REFERENCES. Azzimonti B ...


Novel Homozygous Frameshift Mutation of EVER1 Gene in an Epidermodysplasia Verruciformis Patient Michael D. Gober1,3, Peter L. Rady2,3, Qin He2, Stephen B. Tucker2, Stephen K. Tyring2, Anthony A. Gaspari1 Epidermodysplasia verruciformis (EV) is a rare genetic skin disease with an autosomal recessive trait, and the patients have susceptibility to a specific group of human papillomavirus genotypes. Recently germline mutations in EVER1/2 genes have been detected in EV patients with different ethnic origins. In this study, we have applied PCR, single-stranded conformational polymorphism analysis, and sequencing as well as restriction fragment length polymorphism analysis for identifying potential mutation(s) of EVER genes in an EV patient and in the parents of Pakistani origin. A novel homozygous frameshift mutation (T base deletion at nucleotide position 968 of DNA) has been detected in the EVER1 gene of the patient. The parents carried this mutated allele in a heterozygous form. This is the third report on the presence of EVER1 mutations in an EV patient, and this result supports better understanding, diagnosis, and genetic counseling of EV patients. Journal of Investigative Dermatology (2007) 127, 817–820. doi:10.1038/sj.jid.5700641; published online 30 November 2006

INTRODUCTION Epidermodysplasia verruciformis (EV) is a rare genodermatosis with an autosomal recessive trait. The EV patients have abnormal susceptibility to a specific group of human papillomavirus (HPV) genotypes (Orth et al., 2001). The infection with these EV-associated HPV genotypes contributes to the development of flat wart like and pityriasis versicolor like lesions, Bowen type squamous cell carcinomas in situ, and ultimately invasive squamous cell carcinomas (Orth, 1987). More recently, EV genes, EVER1 and EVER2, were identified; mutations of these genes are responsible for development of EV disease. EVER1 and EVER2 genes are located on chromosome 17q25. Complementary DNAs of full-length EVER1 and EVER2 transcripts were characterized from lymphoblastoid cell lines and from normal skin (Ramoz et al., 2002). Two further case reports have confirmed the presence of EVER1 or EVER2 mutations in EV patients (Tate et al., 2004, Sun et al., 2005) and one article reported the lack of mutation of these genes in an EV patient (Azzimonti et al., 2005). In this case report we describe the presence of a novel frameshift mutation of the EVER1 gene in an EV patient and in the parents. 1

Department of Dermatology, University of Maryland, Medical Center, Baltimore, Maryland, USA and 2Department of Dermatology, University of Texas Health Science Center, Houston, Texas, USA


These authors contributed equally to this study

Correspondence: Dr Anthony A. Gaspari, Department of Dermatology, Medical Center, University of Maryland, 405 W Redwood St Sixth Floor Baltimore, Maryland 21201, USA. E-mail: [email protected] Abbreviations: EV, epidermodysplasia verruciformis; HPV, human papillomavirus Received 5 July 2006; revised 14 September 2006; accepted 5 October 2006; published online 30 November 2006

& 2006 The Society for Investigative Dermatology

CLINICAL HISTORY This is a 34-year-old Pakistani man who presented to the outpatient department with a history of multiple verrucous lesions on his face, torso, and extremities that first appeared at age 14. The patient’s parents are brother and sister and the patient is the only family member affected. He is married to a healthy, unrelated woman, and has one son who is unaffected. The lesions on his face, chest, abdomen, and back were brown and pink verrucous plaques resembling flat warts. On his forearms and knees, he presented with multiple verrucous papules and plaques resembling common warts. He also presented with multiple pink and dark brown palmar hyperkeratotic plaques. The patient had no other disorder and was diagnosed with EV based on the clinical and histopathological findings of moderate hyperkeratosis and acanthosis and with an irregular granular zone, occasional perinuclear halos, and characteristic enlargement of the keratinocytes (Figure 1). Biopsy specimens from this patient did not exhibit features of squamous cell carcinoma or Bowen’s disease. Before diagnosis of EV, the patient was presumed to have an atypical presentation of psoriasis and was treated with acitretin 10 mg daily for which he saw the best clinical results. The patient stopped therapy owing to insomnia, which he attributed to the medication. The patient was subsequently treated with trials of topical tretinoin 0.1% or oral isotretinoin 40 mg daily with only mild improvement. Recently disease has worsened and the patient’s treatment with acitretin therapy (10 mg daily by mouth) was again administered. Multiple HPV infections were detected in the wart biopsies of the patient. One of the flat warts contained HPV-17, and known but not fully characterized DL473 (NCBI ID: 5830225) and SK3 (ID: 8650474) HPV DNA. The other flat wart carried DL473 and SK3 HPV isolates. The common wart contained HPV-17 DNA. Additionally the wart samples


MD Gober et al. Novel Homozygous Frameshift Mutation






MspI S1









Figure 1. Flat wart biopsy from the patient’s back demonstrates histologic features of EV. Note the moderate hyperkeratosis and acanthosis as well as irregular granular zone, perinuclear halos, and intranuclear vacuoles consistent with the diagnosis of EV. Bar ¼ 100 mm.

carried a novel HPV DNA isolate designated to GRT04 (NCBI-GenBank ID: DQ641480) which had 83% sequence identity to HPV38 (ID: 1020234) in the L1 region. RESULTS The single-strand conformation polymorphism analysis indicated the presence of a mutation in exon 9 of the EVER1 gene (data not shown). The sequencing of the PCR fragment revealed a homozygous T base deletion at the nucleotide position 968 of EVER1 complementary DNA (NCBI ID: AY099356) (Figure 2a). The computer assisted open reading frame analysis predicted that this frameshift mutation results in a premature termination codon at nucleotide c.1045. According to the rules for nomenclature the mutation can be designated as c.968delT (L323fsX26) (den Dunnen and Antonarakis, 2001). The computer assisted restriction map analysis of the exon 9 specific PCR fragment revealed that the mutation created a specific MspI restriction enzyme site. The restriction fragment length polymorphism analysis using the MspI enzyme confirmed the presence of c.968delT in the patient (Figure 2b). Similar analysis indicated that the patient’s parents are heterozygous for c.968delT (Figure 2b). DISCUSSION In our study, we found a homozygous frameshift mutation of the EVER1 gene in a patient of Pakistani descent who was born from consanguineous parents (Figure 2; Table 1). The computer analysis of the mutation predicts a premature termination codon at nucleotide c.1045. Although this premature termination codon may trigger nonsense-mediated messenger RNA decay, a well-established mechanism involved in preventing translation of truncated proteins due to premature termination codons, approximately 5–25% of messenger RNA containing premature termination codon escape nonsense-mediated messenger RNA decay suggesting a low level of truncated EVER1 protein (approximately 350 amino acids long) with altered function may be expressed in this patient (Kuzmiak and Maquat, 2006). Although this is early evidence of the c.968delT (p.L323fsX26) mutation, other EVER1 and EVER2 mutations 818

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Figure 2. Identification of a homozygous frameshift mutation in exon 9 of the EVER1 gene. (a) The automated sequencing of the PCR fragment derived from exon 9 of the EVER1 gene of the patient (P) detected a homozygous thymine deletion at position 968 (c.968delT). The wild-type (WT) sequence is also shown. (b) RFLP analysis of PCR fragment amplified from exon 9 of the EVER1 gene utilizing MspI restriction enzyme. c.968delT created an MspI cleavage site and the enzyme digestion resulted in 155- and 115-bp-sized fragments of the 270-bp-sized PCR product derived from the patient (lane P) and from the positive control (lane, PC). The positive control DNA was a cloned PCR fragment from the patient containing the mutated allele with the T deletion (pCR4-TOPO plasmid, Invitrogen Co). The deletion was verified by sequencing in this positive control. The 270-bp-sized PCR fragment from negative control DNA (lane, WT) was not digested by MspI enzyme. The undigested 270 bp PCR fragment from a negative control (lane UD) is also shown. A negative PCR reagent control (no DNA) (lane R), fx174 RF DNA marker (Invitrogen Co. Carlsbad, CA) (lane S1) and 25 bp DNA ladder marker (Invitrogen Co.) (lane S2) can be seen on the gel. The RFLP analysis also served as a confirmation of the presence of the c.968delT mutation in the family members. The digested PCR samples from the patient’s mother (M) and father (F) showed three fragments (270, 155, and 115 bp sizes) indicating that they are both c.968delT mutation heterozygotes.

Table 1. Summary of EVER1 mutations reported in EV Mutation

Position (cDNA)





280 (C–T)


Ramoz et al. (2002)


744 (C–A)


Tate et al. (2004)

Splice site (acceptor)

IVS8-2 (A–T)


Tate et al. (2004)


This study


Ramoz et al. (2002)

Frameshift Nonsense

968 (T deletion) 1726 (G–T)

cDNA, complemenatry DNA; EV, epidermodysplasia verruciformis; IVS, intervening sequence.

have been identified. Ramoz et al. (2002) discovered the EVER1 and EVER2 genes, and identified nonsense mutations in two Algerian and two Colombian consanguineous EV families. This pioneering work was followed by the finding of two new heterozygous mutations (combination of a splice

MD Gober et al. Novel Homozygous Frameshift Mutation

site and a nonsense mutation) in the EVER1 gene of a Japanese EV patient (Table 1). Sun et al. (2005) reported a novel homozygous nonsense mutation in the EVER2 gene of a Chinese EV patient born from a consanguineous marriage. Interestingly, a recent report could not find a mutation of the EVER genes in an EV patient (Azzimonti et al., 2005), suggesting a second genetic locus may be involved EV disease pathogenesis (Ramoz et al., 2000). Putative EVER1 and EVER2 proteins share 28.4% amino acids and belong to the transmembrane channel gene family, and are also designated as TMC6 and TMC8 (Keresztes et al., 2003; Kurima et al., 2003). Both are integral membrane proteins and are localized in the endoplasmic reticulum. The role of EVER1 and EVER2 gene products in EV pathogenesis is still unclear. They may be involved in controlling the HPV pathogenesis in epidermal keratinocytes or play a role in the innate or adaptive immune responses which may control the clearance of EV-HPV-infected keratinocytes (Ramoz et al., 2002). In conclusion, we report a novel frameshift mutation of the EVER1 gene in an EV patient of Pakistani descent. This mutation study provides additional information to the understanding of the role of the EVER1 gene in EV pathogenesis. The restriction fragment length polymorphism analysis can serve as a quick screening procedure and may help with the genetic counseling of the relatives within this EV family.

CP69 were used. Isolation and sequence analysis of HPV-PCR products were performed as described earlier (de Oliveira et al., 2004).


The sequencing indicated the presence of a homozygous T base deletion in exon 9 of the EVER1 complementary DNA (nucleotide position: 968) from the patient (Figure 2). The restriction enzyme map of this gene region was analyzed by GeneRunner for Windows, version 3.05, software (Hastings Software Inc., Hastings, NY). The evaluation indicated that the mutation will create a specific MspI cleavage site. The PCR fragments derived from the patient and parents were digested with MspI. The digested PCR products were run on 2.5% SeaKem LE agarose (Cambrex BioScience Rockland Inc, Rockland, ME) gel electrophoresis and visualized on a UV transilluminator (Figure 2).

Patients A family with a sibling affected with EV disease was studied. The parents had a consanguineous marriage. Blood samples were obtained from the parents and from the sibling with EV disease. Biopsies from two flat warts and a common wart of the EV patient were taken for HPV testing. The patient and his parents were recruited into the study after giving informed consent and this study was conducted following Institutional Review Board (IRB) approval and followed the Declaration of Helsinki Principles.

Detection of mutation within EVER1 and EVER2 genes PCR fragments for the exons of EVER1 and EVER2 genes of the EV patient were amplified individually using sequence information as described earlier (Ramoz et al., 2002). Additional sequence information of primers and gene maps was kindly provided by Gerard Orth and Michel Favre (Department of Virology, Institute Pasteur, Paris, France). For screening of mutations, single-strand conformation polymorphism analysis of the exon specific PCR fragments was utilized via the Genephor DNA Separation System and Precast GeneGel Excel 12.5/24 gels (GE Healthcare, Piscataway, NJ). The gels were silver-stained applying the Plus One DNA silver-staining kit and Processor-Plus instrument (GE Healthcare). After the single-strand conformation polymorphism screening a mutation was verified with automated sequencing of the PCR fragment derived from the exon 9 of EVER1 gene (ABI Prism 3100 Genetic Analyzer, Applied BioSystems, Foster City, CA). The sequencing reactions were performed by the Sequencing Core Facility of the Department of Microbiology & Molecular Genetics, UT-Medical School, Houston.

Restriction fragment length polymorphism analysis and confirmation of the mutation identified by sequencing within the exon 9 of the EVER1 gene from the patient and the parents

Histology Tissue from flat wart biopsies were fixed in formalin, embedded in paraffin, sectioned onto glass slides, and stained with hematoxylin and eosin.

CONFLICT OF INTEREST The authors state no conflict of interest.


DNA extraction DNA was extracted from the whole-blood samples of the parents, peripheral blood mononuclear cells, and the biopsies of the patient using a DNA extraction kit (Puregene, Gentra Systems Inc, Minneapolis, MN). The quality of the DNA extracted for PCR procedures was assessed by amplification of the reference control gene b-globin (Resnick et al., 1990).

Determination of HPV sequences by PCR and sequencing The nested PCR method was utilized as previously described by Berkhout et al. (1995). For the first step, PCR amplification was performed using two degenerate consensus primers termed CP65 and CP70, with annealing sites located in the L1 open-reading frame. The CP65/70 primer set amplifies the complete set of EV-HPV types. For the second PCR step, two nested primers termed CP66 and

We gratefully thank Dr Gerard Orth and Dr Michel Favre for the sequence and gene map information of EVER1 and EVER2 genes. We thank David K. Watson for the expert technical help.

REFERENCES Azzimonti B, Mondini M, De Andrea M, Gioia D, Dianzani U, Mesturini R et al. (2005) CD8+ T-cell lymphocytopenia and lack of EVER mutations in a patient with clinically and virologically typical epidermodysplasia verruciformis. Arch Dermatol 141:1323–5 Berkhout RJ, Tieben LM, Smits HL, Bavinck JN, Vermeer BJ, ter Schegget J. (1995) Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients. J Clin Microbiol 33:690–5 de Oliveira WR, He Q, Rady PL, Hughes TK, Neto CF, Rivitti EA et al. (2004) HPV typing in Brazilian patients with epidermodysplasia verruciformis: high prevalence of EV-HPV 25. J Cutan Med Surg 8:110–5


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den Dunnen JT, Antonarakis SE (2001) Nomenclature for the description of human sequence variations. Hum Genet 109:121–4 Keresztes G, Mutai H, Heller S (2003) TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins. BMC Genomics 4:24 Kurima K, Yang Y, Sorber K, Griffith AJ (2003) Characterization of the transmembrane channel-like (TMC) gene family: functional clues from hearing loss and epidermodysplasia verruciformis. Genomics 82:300–8


Ramoz N, Rueda LA, Bouadjar B, Montoya LS, Orth G, Favre M (2002) Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis. Nat Genet 32:579–81 Ramoz N, Taieb A, Rueda LA, Montoya LS, Bouadjar B, Favre M et al. (2000) Evidence for a nonallelic heterogeneity of epidermodysplasia verruciformis with two susceptibility loci mapped to chromosome regions 2p21–p24 and 17q25. J Invest Dermatol 114:1148–53

Kuzmiak HA, Maquat LE (2006) Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges. Trends Mol Med 12:306–16

Resnick RM, Cornelissen MT, Wright DK, Eichinger GH, Fox HS, Ter Schegget J et al. (1990) Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J Natl Cancer Inst 82:1477–84

Orth G (1987) Epidermodysplasia verruciformis. In: The Papillomaviruses. The Papovaviridae. (Salzman NP, Howley PM, eds), Vol. 2 New York: Plenum Press, 199–243

Sun XK, Chen JF, Xu AE (2005) A homozygous nonsense mutation in the EVER2 gene leads to epidermodysplasia verruciformis. Clin Exp Dermatol 30:573–4

Orth G, Favre M, Majewski S, Jablonska S (2001) Epidermodysplasia verruciformis defines a subset of cutaneous human papillomaviruses. J Virol 75:4952–3

Tate G, Suzuki T, Kishimoto K, Mitsuya T (2004) Novel mutations of EVER1/ TMC6 gene in a Japanese patient with epidermodysplasia verruciformis. J Hum Genet 49:223–5

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