RESEARCH ARTICLEOFFICIAL JOURNAL

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Correlation of Phenotype/Genotype in a Cohort of 23Xeroderma Pigmentosum-Variant Patients Reveals 12 NewDisease-Causing POLH Mutations

Kristina Opletalova,1,2 † Agnes Bourillon,3 † Wei Yang,4 Caroline Pouvelle,1,5 Jacques Armier,1 Emmanuelle Despras,1,5

Ludovic Martin,6 Christine Mateus,2 Caroline Robert,2 Patricia Kannouche,1,5 Nadem Soufir,3,7 and Alain Sarasin1,8 ∗

1Laboratory of Genetic Stability and Oncogenesis, UMR8200 CNRS, University Paris-Sud and Institut Gustave Roussy, Villejuif, France;2Department of Dermatology, Institut Gustave Roussy, Villejuif, France; 3Laboratoire de Genetique, Hopital Bichat, APHP, Paris, France;4Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland; 5Equipe labellisee Ligue Contre le Cancer “TLSpolymerases and cancer”, University Paris-Sud and Institut de Cancerologie Gustave Roussy, Villejuif, France; 6Service de Dermatologie, CHUAngers, Angers, France; 7Inserm U976, Centre de Recherche Sur la Peau, Hopital Saint Louis, APHP and University Paris, Paris, France;8Department of Genetics, Institut Gustave Roussy, Villejuif, France

Communicated by Rolf H. SijmonsReceived 11 July 2013; accepted revised manuscript 2 October 2013.Published online 15 October 2013 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22462

ABSTRACT: Xeroderma pigmentosum variant (XP-V) is a raregenetic disease, characterized by some sunlight sensitivity and pre-disposition to cutaneous malignancies. We described clinical andgenetic features of the largest collection ever published of 23 XP-V patients (ages between 21 and 86) from 20 unrelated families.Primary fibroblasts from patients showed normal nucleotide ex-cision repair but UV-hypersensitivity in the presence of caffeine,a signature of the XP-V syndrome. 87% of patients developedskin tumors with a median age of 21 for the first occurrence.The median numbers of basal-cell carcinoma was 13 per patient,six for squamous-cell carcinoma, and five for melanoma. XP-V isdue to defects in the translesion-synthesis DNA polymerase Polηcoded by the POLH gene. DNA sequencing of POLH revealed29 mutations, where 12 have not been previously identified, lead-ing to truncated polymerases in 69% of patients. Four missensemutations are correlated with the protein stability by structuralmodeling of the Polη polymerase domain. There is a clear re-lationship between the types of missense mutations and clinicalseverity. For truncating mutations, which lead to an absence of orto inactive proteins, the life-cumulated UV exposure is probablythe best predictor of cancer incidence, reinforcing the necessityto protect XP-Vs from sun exposure.Hum Mutat 35:117–128, 2014. C© 2013 Wiley Periodicals, Inc.

KEY WORDS: DNA repair; UV; trans-lesion polymerases;skin cancers; melanomas; POLH

Additional Supporting Information may be found in the online version of this article.†These authors are considered as the joint first coauthors.∗Correspondence to: Alain Sarasin, UMR8200 CNRS, Institut Gustave Roussy, PR2,

114, rue Edouard Vaillant, 94805 Villejuif, France. E-mail: alain.sarasin@gustaveroussy.fr

Contract grant sponsors: Centre National de la Recherche Scientifique (Paris,

France); the Agence Nationale pour la Recherche (Paris); the Association des Enfants

de la Lune (Tercis, France); La Ligue Nationale contre le Cancer (“Equipe labellisee”),

Paris, France; the Institut National du Cancer (INCa, PLBio-2010), Paris; the Agence

Nationale de la Recherche (N◦ ANR-09-PIRI-001), Paris.

IntroductionA variety of exogenous (radiations, chemicals, drugs) and en-

dogenous (free radicals) agents are constantly attacking genomes ofliving cells. Numerous DNA repair pathways can eliminate specificlesions. Their biological importance is demonstrated by the exis-tence of dramatic diseases, caused by a deficiency in one of thesepathways [Hoeijmakers, 2001]. For example, classical xerodermapigmentosum (XP) is an autosomal, recessive, genetic disorder withan extremely high sensitivity to UV that leads to a more than 2,000-fold increase in skin cancers [Cleaver, 1968; Stary and Sarasin, 2002].The majority of these patients are deficient in the nucleotide excisionrepair (NER) pathway, which removes the majority of damage dis-turbing the DNA structure. If complete repair of DNA lesions doesnot occur before the S-phase, some noncoding lesions can block theprogression of replication fork [Lehmann et al., 1975; Sarasin andHanawalt 1980]. Such blockage induces a DNA damage response,involving signals and defects threatening cellular viability. One wayto eliminate this replication blockage is to tolerate the lesions, forexample, by translesion DNA synthesis (TLS) mode [Kannoucheet al., 2004; Yoon et al., 2009]. TLS has been demonstrated by theisolation of several specialized low-fidelity DNA polymerases able toreplicate damaged strand, forming the Y-family of polymerases. Themost understood one is the polymerase eta (Polη; MIM #603968),which is a 713 AA protein with a MW of 78 kDa coded by thePOLH gene (human homolog of the yeast RAD30 gene) [Johnsonet al., 1999; Masutani et al., 1999a]. Its major function is to repli-cate through cyclobutane pyrimidine dimers (CPD), produced byUV rays, in an error-free manner. In the absence of Polη, replica-tion forks are blocked for a longer period of time at UV-induceddamage, expecting the arrival of other TLS polymerases. Theseare less efficient and more mutagenic when replicating thyminedimers, which are the most frequent UV-induced DNA modifica-tions [Gueranger et al., 2008; Kannouche and Stary 2003; Stary et al.,2003]. Polη is the only TLS polymerase, which defect is associatedwith cancer. Its absence gives rise to the cancer-prone syndromexeroderma pigmentosum variant (XP-V; MIM #278750). This is arare, genetic disease transmitted as an autosomal, recessive mode.XP-V patients represent approximately 20% of XP patients aroundthe world [Lehmann et al., 1975]. Usually, XP-V patients are notvery sensitive to sunlight in their infancy, but their photosensitivity

C© 2013 WILEY PERIODICALS, INC.

increased after the age of 15–20 years, without really sunburning andare then developing multiple skin cancers, although the symptomsare less severe and appear later in life than for classical XP patients.XP-V cells exhibit a normal NER, but a defective postreplicationrepair following UV-irradiation because they are unable to rapidlybypass thymine dimers. This defect can be corrected in vitro with thewild-type Polη. Oncogenic mutations located at pyrimidine dimerssites have been reported in the skin tumors of XP-V patients [Giglia-Mari and Sarasin, 2003]. Polη is, however, a relatively “error-prone”polymerase, which can insert an incorrect nucleotide when replicat-ing other DNA lesions than CPD or on undamaged DNA [Masutaniet al., 1999b]. This intrinsic characteristic may be associated with thefact that Polη is also involved in somatic hypermutagenesis and isnecessary for the Class Switch pathway [Faili et al., 2004 and 2009].About 50 germline mutations of the POLH gene have been reportedin the literature. The majority of them are deletions or give rise tostop codons and therefore to the truncation or absence of the Polηprotein [Biertumpfel et al., 2010; Broughton et al., 2002; Inui et al.,2008; Johnson et al., 1999; Masaki et al., 2008; Masutani et al., 1999a;Tanioka et al., 2007].

We report here detailed clinical characterization of 23 XP-V pa-tients (from 20 families) diagnosed in our laboratory, in the last 20years, as well as biological and genetic characteristics of patients’cells, aiming to find out a relationship between the type of ger-minal mutations and clinical course. This is, to our knowledge, thelargest series and the most clinically characterized of published XP-Vpatients.

Materials and Methods

Patients

The clinical features of 23 patients belonging to 20 unrelated fam-ilies have been retrospectively reviewed. These patients have hadclinical diagnosis of putative XP-V in various hospitals in Franceand in North Africa. The molecular diagnosis has been made inour laboratory between 1985 and 2012. Clinical analyses have beendetailed in the Results section and summarized in Supp. Table S1.The Informed signed consent for skin biopsy and for genetic in-vestigation was obtained from all patients or their parents by thedermatologists in charge of the skin biopsy. The French Agency ofBiomedicine (Paris, France) has approved this study to AS.

Cellular Analysis

Primary fibroblasts isolated from unexposed skin biopsies fromalmost all XP-V patients were cultivated in MEM plus 10% FCS andantibiotics and analyzed at low passages. Normal skin fibroblasts(198VI, 405VI, 873VI, and 950VI), isolated and cultured in ourlaboratory in the same conditions, were used as controls. EBV-transformed lymphoblastoid cell lines were also produced and usedwhen skin biopsies were unavailable.

NER was characterized by post-UV unscheduled DNA synthe-sis [Sarasin et al., 1992] and post-UV cell survival with or with-out caffeine by measuring DNA synthesis as we already published[Broughton et al., 2002]. Basically, growing fibroblasts were UVC-irradiated at doses up to 5 J/m2 with mainly 254 nm at a fluency of0.3 J/m2/sec. Caffeine (1.5 mM; C0750-50G; Sigma, saint-Quentin,France) was added just after the irradiation and DNA synthesis wasquantified 48 hr after irradiation using 10 μCi/ml MeH3-Thymidine(NET 027Z001MC; PerkinElmer, Courtaboeuf, France) given for

3 hr. Cells were scraped off the plates into 2% SDS and the ra-dioactivity was measured in a liquid scintillator. Results were givenas % of DNA labeling as compared with unirradiated cells.

siRNA Transfection

siRNA was used to transiently downregulate the expression ofPOLH (5′ GAAGUUAUGUCCAGAUCUU 3′; Eurogentec, Angers,France) gene. Cells were transfected with 30 nM of siRNA us-ing Interferin reagent (Polyplus; Ozyme, Montigny-Le-Bretonneux,France) according to the manufacturer’s instructions, and weretrypsinized 48 hr after transfection. A unique siRNA was used inthese experiments because it has been already characterized in thelaboratory as able to knock down specifically Polη [Despras et al.,2010] and it was only used, as a control, to show the disappearanceof the Pol η band in Western analysis.

Western Blot

Cells were directly lysed in lysis buffer (50 mM Tris pH 7.5,20 mM NaCl, 10 mM MgCl2, 0.1% SDS), antiproteases (Roche,Meylan, France) and DNAse (Benzonase; Novagen, Merk-Millipore,Molsheim, France). Aliquots were kept for Bradford quantifica-tion (Biorad, Marnes-La-Coquette, France). Laemmli 5x was addedand proteins were denatured 10 min at 90◦C. The equivalent of40 μg of proteins per sample was separated on SDS-PAGE. Mem-branes were blotted with antibodies directed against the follow-ing proteins: PCNA (PC-10; Santa Cruz Biotechnology, Tebu, LePerray-en-Yvelines, France), Polη (two different antibodies wereused: ab17725 that recognized residues 650 to C-ter; Abcam, Coger,Paris, France and PA5–29063 that recognized within amino acids 12–292; Thermo Scientific Rockford, IL). After washing, the membraneswere incubated with horseradish peroxidase-conjugated antirabbit-IgG (1:5,000) or antimouse-IgG (1:5,000) (Dako, Trappes, France),and signals were visualized using enhanced chemiluminescence(Amersham, Velizy, France). To quantify the half-life of Polη, lym-phoblastoid cell lines were treated or not by 10 mM of MG132(Calbiochem) for 3 hr and then by 25 mg/l of cycloheximide for 1,3, or 5 hr before protein extraction.

DNA and RNA Sequencing

Purification of DNA and RNA from growing primary fibroblastswas done using the Nucleospin Tissue and Nucleospin RNAII kitsrespectively, according to the manufacturer conditions (Macherey-Nagel, Duren, Germany).

On the POLH gene (6p21.1; NM 006502.2) coding, exons andexon–intron junctions were amplified with 12 couples of primersselected by using Exon Primers USC Genome Browser primer(http://genome.ucsc.edu/cgi-bin/hgGateway/). A 60◦C hybridiza-tion temperature was used for all PCRs. PCR mix comprised 25 nggenomic template DNA, 1.5 ml 10× PCR buffer, 1.8 μL 5 mMMgCl2, 0.6 ml dNTP 5 mM, 6 pmol each of PCR primer and 0.5 UAmpli Taqgold R©. Samples were denatured for 7 min at 96◦C andwere passed through 35 cycles of amplification: 30 sec of denat-uration at 96◦C, 30 sec of primer annealing at 60◦C, 30 sec ofelongation at 72◦C, and a final elongation for 10 min at 72◦C.The amplifications were carried out in 0.2 ml Eppendorf R© tubesin an Eppendorf R© thermocycler. PCR products were controlled ona 2% agar gel and were purified by EXOSAP-IT R©. A sequencing

118 HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014

Figure 1. Characteristics of some xeroderma pigmentosum variant patients described in this study. A: Squamous cell carcinoma (SCC) in the16-year-old XP865VI patient (picture from Dr. Raoul Triller, Levallois-Perret, France). B: SCC of the external auditory canal and basal cell carcinoma(BCC) under the right ear in the XP853VI patient, whose skin presented multiple scars and atrophy (Picture from the plastic surgery departmentof Prof. Maurice Mimoun, Saint-Louis Hospital, Paris, France). C and C′: Deep actinic damage, telangiectasias, and pigmented lesions, whichoccurred in childhood in the XP888VI patient and his sister XP968VI (picture from Dr. Christine Chiaverini, University hospital of Nice, France). D andD′: Melanoma on the arm (red arrow) of the 37 years old XP606VI patient, difficult to distinguish among multiple disseminated pigmented lesionsand scar after excision of melanoma in the middle of the back of the same XP606VI patient, respectively. Re-excision of the melanoma outlinedalong the scar (picture from Dr. Francoise Boitier, dermatology department, Cochin hospital, Paris). E: Lentigines and depigmented macules onphoto-exposed parts of the body of the XP961VI patient of African origin with black skin (picture from Dr. Caroline Robert, dermatology department,Gustave Roussy Institute, Villejuif, France).

reaction was performed, in one sense, on 8900 Fast thermal cyclerApplied Biosystem R©, using 10 ng PCR-purified products and a Big-dye terminator cycle kit Applied Biosystem R©. Sequence analysis wasperformed on an ABI-Prism 3130 Applied Biosystem R© and was readwith Seqscape R©. The DNA mutation numbering system is based onthe cDNA sequence. The new variants reported in this article havebeen submitted to the locus-specific database: http://www.lovd.nl.Three in silico prediction tools were used to predict the functionalimpact of nonsynonymous variants:

SIFT prediction (Sorting Intolerant From Tolerant, http://sift.jcvi.org/www/SIFT_enst_submit.html): SIFT takes a query se-quence and uses multiple alignment information to predict toler-ated and deleterious substitutions for every position of the querysequence. Positions with normalized probabilities less than 0.05 arepredicted to be deleterious, those greater than or equal to 0.05 arepredicted to be tolerated. We used the “By Default” parameters.

PolyPhen-2 (Polymorphism Phenotyping v2, http://genetics.bwh.harvard.edu/pph2/) is a tool that predicts possible impactof an amino-acid substitution on the structure and functionof a human protein using straightforward physical and com-parative considerations. PolyPhen-2 server has been updated toutilize version 2.2.2 of the software, protein sequences fromUniProtKB/UniRef100 Release 2011 12, structures from PDB/DSSPSnapshot 03-Jan-2012 (78,304 entries) and UCSC MultiZ multiplealignments of 45 vertebrate genomes with hg19/GRCh37 humangenome.

SNPs3D (http://www.snps3d.org/) is a Website that assignsmolecular functional effects of nonsynonymous SNPs based onstructure and sequence analysis. We used the stability model basedon the hypothesis that many disease SNPs affect protein functionprimarily by decreasing protein stability (See Supp. Table S2).

Modeling the Mutant Polη Structure

The structural model was generated using the coordinates de-posited in Protein Data Bank (PDB accession code 3MR2 and Py-MOL program (www.pymol.org) [Biertumpfel et al., 2010].

Results

Clinical Description of the XP-V Patients

We have retrospectively reviewed the clinical features of 23 pa-tients (Fig. 1) from 20 unrelated families, living in France but origi-nated from France (six patients), North Africa (11 from Algeria andthree from Tunisia), Turkey (one patient), Kosovo (one patient),and Congo (one patient). They were managed in nine Departmentsof Dermatology and/or Plastic surgery in French hospitals (Insti-tut Gustave Roussy, Villejuif; University hospitals Necker, Bichator Saint-Louis, Paris; University hospitals of Creteil, Nice, Tours,Angers and Reims). Two were managed in Hospitals of Tunis andAlgiers. One patient was managed by a dermatologist out of hospitalin Clermont-Ferrand, France.

The major clinical data are reported in Supp. Table S1. The medianage of the patients was 44 years (21–86 years) at the time of this study(2012, excluding the two deceased patients). There were 12 men and11 women. Consanguinity is present in 11 families and unknown inthree cases, with parents being first-degree cousins in eight cases.

The median age of the first symptoms is 7 years, (extreme 2–20),but exact age is known only for 16 patients. In five other patients,first symptoms appeared in childhood without precision of age andthe data are unknown for two patients. The median age of clinicalXP-V diagnosis was 22 years (5–50). The data are unknown for

HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014 119

two patients. The median and extreme ages of occurrence of thefirst cancer were 21 years (7–45) and the first cancer was SCC insix cases, BCC in six, SCC and BCC simultaneously in three cases,BCC and melanoma simultaneously in one case, melanoma in onecase and the histological type was unknown in two cases. Threepatients did not develop skin cancer at 32, 44, and 46 years old,respectively. The median and extreme ages of occurrence of the firstBCC, SCC, and melanomas were respectively: 26 years (12–45), 22years (7–54), and 32 years (17–64). The median numbers of BCC,SCC, and melanoma per patients were 13, 6, and 5, respectively.Regional and/or distant metastases were noted in three patients withSCC or melanoma. Four patients presented with extracutaneoustumors: three of them were malignant (lung carcinoma, sarcomaof the cheek, and retroperitoneal liposarcoma), one was benign(kidney angiomyolipoma). Xerosis was found in six patients (26%),data not available for five patients. Photophobia was present infive (22%) patients, but was not major (data NA for one patient).Ocular tumors (including eyelids skin tumors) were reported innine (39%) patients, and benign naevi were found in two patients.Ocular tumors excluding eyelids occurred in two patients. None ofthe patients had neurological symptoms or presented abnormalitiesfollowing clinical neurological examination.

Twenty-one patients are still living but four of them are lostfor follow-up at the age of 7, 22, 33, and 56 years. Two patientscommitted suicide at 18 and 54 (Supp. Table S1).

The patients, listed in Table 1 and Supp. Table S1, have beenordered by severity of symptoms. In order to better correlate phe-notype and genotype, we classified the XP-V patients into threegroups of aggressive, medium, and mild symptoms. This classifica-tion was done with specialized cancer clinicians and dermatologistsby taking into account the skin abnormalities, the age of the diag-nosis, and at the time of this study, the age at first symptom andat first tumor, the number, and type of tumors and the total sunexposure as indicated by the patient himself. We have taken intomuch consideration the number of tumors in each patient takinginto account their age at the time of this study. The median numberof epithelioma per patient was 41, 15, and 1 for aggressive, medium,and mild symptoms, respectively. For malignant melanoma, thesenumbers were 5, 2.5, and 0, respectively.

Patients with “Aggressive” Symptoms

XP28VI was diagnosed at the age of 11 years (freckles of the face,arms and thorax; actinic keratoses; atypic naevi). Her mother hada lentigo maligna melanoma, her brother presented skin tumorssimilar to neurofibromas or myxomas. She developed skin cancersmainly on the face (BCC, SCC, and melanomas). Three neurofi-bromas appeared at the age of 17 years (right and left forearms,right supra-clavicular region). Three years later, she underwentright axillary adenectomy for lymph node metastases of melanoma,but also metastases in the scapular subcutaneous tissue. She hasnever relapsed since. She had also a sarcoma of the right cheek (at16 years), which relapsed 1 year after surgery and finally was success-fully treated by complete excision. She protected herself by clothes,cream, and she did not go outside with high sun exposure, but onlyafter the age of 18 years.

XP75VI deceased at the age of 18 years. He presented nine SCCof the face (nose, upper and lower lip, right inferior eyelid, bothcheeks, chin); cancer recurrence appeared in most of these SCCsin spite of surgical treatment. At the age of 15 years, he underwentlymph node dissection: histological examination showed one lymphnode metastasis of SCC in 25 lymph nodes obtained. The recurrence

of the lower lip SCC was treated by brachytherapy by 192Ir, with noreported abnormal reaction. The patient developed a conjuctival,scleral, and episcleral SCC of the right eye, treated by surgery and ra-diotherapy. He suffered from psychical disorders, which conductedto a suicide.

XP127VI committed suicide at the age of 54 years. He presentedmultiple BCC and SCC on the exposed parts of the body. Thesecancers were treated by surgery and/or radiotherapy (conventionalor brachytherapy) with no reported abnormal reaction. He had alsoetretinate and topical treatments (5-fluorouracil and imiquimod),without effect on development of new carcinomas. He applied pho-toprotection after the age of 25 and stopped working outside. Hehad 16 melanomas, one of them was conjunctival, and he presentedlocoregional and lymph node metastases of a melanoma of thetemple.

First cancer in XP603VI was a melanoma on the left leg at the ageof 32 years. Since she presented 24 melanomas mostly of SSM type,all of them localized on legs, except one on the arm. Most of them hadBreslow´s thickness inferior to 1 mm and were classified Clark I orII, except one, which was 1.5 mm thick and was Clark IV. Completeexcisions of all these melanomas with recommended margins wereperformed and she never developed metastases. She presented alsoseven SCC on the face and limbs and two BCC on the face. Morethan 20 excisions were performed since the age of 32 years. Most ofthe excised lesions were atypic nevi. However, genetic examinationshowed the absence of germinal mutation of the CDKN2A-CDK4-P14 genes. She had multiple freckles and actinic keratoses on thesun-exposed parts of the body, in spite of everyday use of SPF creamsince the age of 25 years.

XP819VI had a family history of XP. He presented multiple carci-nomas, mainly on the face and dorsal parts of the hands and arms.He worked as a driver and most of the tumors were localized on theleft part of his face. When he changed his work and started workingat night, he did not develop any skin cancer during 11 years. Butlater he developed so many tumors that, at the age of 56 years, hehad a skin graft of all the affected parts of the face (both cheeks,forehead, and upper lip). The face was resurfaced by split-thicknessskin grafts taken from the buttocks (skin nonexposed to UV radi-ation). Thirteen months later the facial resurfacing was a success[Tayeb et al., 2011].

In XP853VI, XP-related lesions appeared before the age of14 years and lead to nasal amputation at the age of 14 years. He devel-oped multiple carcinomas of the face, some of them treated regularlyby electrocoagulation. At the age of 28 years, he presented a volu-minous exophytic lesion of the left external canthus and left cheek.The histology showed desmoplastic melanoma confluent with SCCand the SCC relapsed after each excision and conducted to left or-bital exenteration. A BCC at the age of 33 years, conducted to leftear amputation. A SCC of right inferior eyelid and a SCC of rightacoustic duct progressed locally with tissular destruction (ear, or-bital cavity, and sinus ethmoidal). He received also chemotherapy(bleomycine), in order to stop the progression of his carcinomas,which was ineffective. Locally, the carcinomas were destructive, buthe never presented metastases of SCC or melanoma. The lesions pre-dominated on sun-exposed parts of the body and he never protectedhimself of sun exposure. Carcinomas of the face are progressing andsurgical treatment is no more possible (the patient had multipleexcisions with reconstructions by flaps) and he is on palliative care.

XP965VI was diagnosed at the age of 22 years. She was born inKosovo from parents with no known consanguinity. Her parentsand her three brothers aged 20, 16, and 12 years were still in Kosovoand considered as unaffected (not examined). The diagnosis of XPhad been suspected 1 year before because of the development of

120 HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014

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a huge, rapidly progressive, mediofacial tumor that invaded andruined the nose. On skin examination the tumor was single butall sun-exposed areas (face, posterior, and lateral aspects of theneck, posterior aspects of both forearms and hands) were coveredby hundreds of solar lentigos despite minor photosensitivity. Eyeexamination was within normal limits with the exception of dryness.The nose was amputated and histological examination providedwith the diagnosis of squamous cell carcinoma. This patient startedto apply sun protection.

Patients with “Medium” Symptoms

XP51VI exhibited his first cancer at the age of 18 years, whichwas an infiltrating SCC of the upper lip, treated by surgery withflap reconstruction and adenectomy (complete excision, absence ofmetastases). He was treated intermittently by etretinate. Unfortu-nately, the follow-up is not available since the age of 22 years. Hissister died at the age of 26 years of XP and there were two othermembers of the paternal family suffering from XP.

In XP86VI, first cancer was a tumoral nodular lesion of the nose atthe age of 22 years. Clinical examination showed hyperpigmentationof the face, eyelids, hands, neck, and lips and multiple SCC and BCC(dorsal side of the hand, eyelid, cheeks, nose). All these lesions weretreated by surgery.

Diagnosis in XP422VI was delayed and clinically suspected onlyat the age of 44 years. The sister of her grandmother died of cu-taneous lesions. Her medical history was marked by an immunedefect, asymptomatic hypogammaglobulinemia (IgG and IgA). Sheunderwent the excisions of more than 50 cutaneous lesions duringthe follow-up (mostly actinic keratoses, atypic nevi, seborrheic ker-atoses, and 25 SCC and 13 BCC). She developed eight melanomas(Clark II and III) in the last 13 years.

XP606VI and XP737VI are sisters whose parents were cousins.XP606VI considered her sun exposure being important and hada lot of sunburns in her life. First cancer was SSM of the thigh(Breslow 0.1 mm, Clark II). She had since four other melanomas(neck, breast, back, arm); all of them were SSM, one in situ andthree with Breslow’s depth inferior to 1 mm, Clark II or III. Allfive were treated surgically, without progression. She presented alsoa SCC and a lot of atypical nevi. Search for germinal mutation ofCDKN2A-CDK4-P14 genes was negative.

XP878VI presented between the age of 17 and 22 years, morethan 20 BCC of different histological types (superficial, nodular,sclerodermiform). Since the age of 22 years, more than 30 excisionswere performed on his face. Histological examination showed for allof them BCC, relapse of already treated BCC or new BCC. Surgicalmargins were not always optimal because of the important numberof lesions on the face. He presented also dysplastic actinic keratoseswithout transformation to SCC and had no melanoma. His med-ication included acitretinoine per os (15 mg/day) taken after thediagnosis of XP-V without obvious effects.

XP966VI and XP967VI are sisters from consanguineous parentsof French ancestry used to live in countryside as farmers. XP966VI,born in 1926, was not suspected to have XPV before the age of 50years and the molecular demonstration was only provided at theage of 85 years. Yet, she had developed all her life long seven skinmelanomas and dozens of actinic keratoses, squamous cell carci-nomas, and basal cell carcinomas. All skin neoplasms were strictlyrestricted to sun-exposed areas. On recent clinical examination,solar lentigos were quite uncommon, actually difficult to identifybecause of the numerous scars and skin grafts. No visceral or osseousdissemination of any skin neoplasms were found.

Patients with “Mild” Symptoms

XP62VI developed a SCC of the right cheek with extension tothe nose at the age of 7 years. It was treated by radiotherapy firstin Algeria, with no abnormal response, and then surgery (com-plete excision and reconstruction by skin graft) was carried out inFrance. His sun exposure was considered important and he neverprotected himself from North-African sunshine. His medical his-tory was marked by lymph node and lung tuberculosis treated inchildhood. Unfortunately the follow-up since the age of 7 years isnot available.

XP546VI had two brothers and two sisters all of them sufferingfrom XP. He developed five SCC (nose and lips) and three ker-atoacanthomas (forehead, lip, cheek). Many lesions were treatedby cryotherapy or electrocoagulation without histological examina-tion. He presented actinic keratoses of the face as well as trichoblas-tomas. Lung carcinoma, probably due to smoking, at the age of 43years, was successfully treated by regular polychemotherapy. He hadalso viral hepatitis at the age of 45 years.

XP606VI and XP737VI are sisters whose parents were cousins.XP737VI had a phenotype of multiple nevi and lentigos, dissemi-nated on the whole body except the posterior sides of her legs. XPVbiological assay was done because of her sister’s disease and similarphenotypes. She never had malignant skin lesion but, at the age of 39years, she presented a retroperitoneal liposarcoma (grade I) treatedby surgery and radiotherapy without abnormal response. She is incomplete remission. She declared that she protected herself muchbetter than her sister and never had any sunburning.

XP843VI was frequently exposed to sun in childhood. The diag-nosis of XPV was suspected at the age of 16 years because of keratosiclesions of the face. He had an extreme photosensitivity with photo-phobia and conjunctival erythema. He also had two operations onan eye, at the age of 16 and 23 years, for unknown reasons. No skinbiopsy was available for this patient.

XP865VI has been living in France since his birth and spent fivesummers in Algeria, but globally his sun exposure was not impor-tant. He presented a SCC of the nose at the age of 16 years. Geneticexamination showed absence of germinal mutation of the CDKN2A-CK4-P14 genes. Dermatological follow-up was performed twice ayear since the diagnosis of SCC and XP-V and a few actinic ker-atoses of the face were treated. The compliance to the treatmentand prevention is not so good and the patient does not apply anyphoto-protection.

XP872VI suffered, since the age of 10 years, from photosensitivitywith sunburns following even a small sun exposure, and then poik-ilodermia and actinic keratoses appeared before 15. The diagnosis ofXP-V was suspected at the age of 21 years. She consulted for an ery-thematous keratosis and squamous lesion of the right inferior eyelidand the biopsy showed Bowen’s disease (intraepidermal squamouscell carcinoma). Surgery and clinical follow-up were performed andshe protected herself from sunlight by clothes, creams, and did notleave home during noontime.

XP888VI and XP968VI are siblings born from consanguineousparents from Algeria. They both received high sun exposure duringtheir childhood in Algeria. XP888VI was even more exposed duringhis life because of his outside job. His first symptoms (deep actinicdamage, telangiectasia, pigmented lesions) appeared precociouslyin the childhood. The diagnosis of XP-V was suspected at the ageof 30 years when he consulted for a BCC of the forehead. AnotherBCC of the temple was treated surgically at the age of 31 years. Thepatient presented also a SSM (melanoma in situ) of the shoulder atthe age of 30, treated by complete excision with no progression. Hissister (XP968VI) developed numerous widespread lentigos strictly

122 HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014

limited to sun-exposed skin areas. Once in France, she got intensiveeducation for sun protection. Up to now, careful screening of theskin failed to reveal any skin malignancy.

XP961VI was African and had lentigines and depigmented mac-ules on sun-exposed parts of the body since the age of 20 years.However, the diagnosis was made lately, at the age of 42 years. Shedid not develop skin cancer at all, despite important sun exposurewithout photoprotection (lived in Congo at the equator level). Thisis probably linked to her black skin, which is very protector againsttumors, even in an XP background [Cartault et al., 2011]. Her 10-year-younger sister, however, suffered from multiple ocular tumors,which is also typical of black-skinned XP patients.

XP966VI and XP967VI are sisters from consanguineous parentsof French ancestry used to live in countryside as farmers. XP967VIwas born in 1940. She was suspected to have XPV when she was 37.She developed widespread lentigos and solar keratoses. Sun protec-tion and long-term oral medications with various retinoids did notprevent first basal and squamous cell carcinomas to occur (1986).An intraepidermal melanoma was diagnosed in 2004.

Characterization of Cells Isolated from the XP-V Patients

DNA repair analysis

XP-V cells exhibit a normal NER level after UV irradiation, but asignificant decrease of cell survival after UV irradiation in the pres-ence of caffeine. Indeed, caffeine aggravates the replication defect inthe absence of Polη, possibly by inhibiting the Chk1–dependentpathway and leading to a destabilization of replication forks[Despras et al., 2010]. For each patient, we cultivated primary der-mal fibroblasts from unexposed skin biopsy. Level of NER followingincreasing doses of UVC (UDS: unscheduled DNA synthesis) andcell survival in the presence of caffeine were performed. All patient’scells exhibited normal level of NER quantified by UDS (data notshown) and a specific reduction of post-UV survival in the presenceof caffeine. Representative experiment for increased UV-sensitivityby caffeine is shown in Figure 2A, as an example, and quantificationof caffeine sensitivity for nine XP-V cell lines compared with twowild-type cell lines is shown in Figure 2B. In some cases, analyses ofTranscription-Coupled Repair by quantifying the Recovery of RNASynthesis were carried out, showing normal level of TC-NER, asexpected, in these XP-V cell lines (data not shown). By definition,all these analyses signed the XP-V cell phenotype.

This diagnosis was confirmed by the presence of POLH mutationsfor all patients leading to an absence, in the majority of patient cells,of the Polη protein or to its reduced level.

mRNA and protein studies

We first used, by Western analysis of fibroblastic protein extracts,an antibody against the C-ter part of Polη because of its high effi-ciency. Figure 2C shows seven XP-V cells with no detectable Polηprotein and four XP-V cells with a faint band (observed only forlong exposure time) at the expected 80 kDa size, whereas in wild-type cells a unique and intense band was found (Fig. 2C). Theidentity of this band with Polη was demonstrated in wild-type cellstransfected with a siRNA produced against the POLH gene. In thisexperiment, we used a unique siRNA because it has been alreadytested in our laboratory [Despras et al., 2010] and it is used only toshow the disappearance of the protein band. As shown in Figure 2C,the 80 kDa band almost completely disappears in the siRNA-treatedcells indicating we are looking to the real Polη protein.

Among the 4 faint bands, two were due to a very poor tran-scription caused by homozygous point mutation in the promoter ofPOLH gene (XP606VI and XP737VI) and two were due to homozy-gous (XP853VI) or heterozygous (XP603VI) missense mutations ofPOLH, leading to unstable proteins.

Results are identical using EBV-transformed lymphoblastoid cellsfrom XP-V patients (Fig. 2D and Supp. Fig. S1) with no detectableprotein due to a stop codon (XP968VI) or with a weaker thanwild-type band due to one AA in-frame deletion (XP966VI). Asan example, we analyzed by RT-PCR the level of POLH mRNA inthese two cell lines to ensure that the low level of protein foundin the XP966VI cells was not due to mRNA degradation. Indeed,similar level of mRNA was found in XP968VI and XP966VI byRT-PCR as compared with control lymphoblastoid cells (data notshown). This result suggests that the low level of polymerase shouldbe due to protein instability and not to mRNA degradation, asalready suggested by Inui et al. (2008). We, then, measured thehalf-life of the Polη protein in the presence of cycloheximide forthe mutated XP966VI. Figure 2E shows that the half-life is abouttwo times shorter for this mutant as compared with WT cells. Thisresult indicates that, while the mRNA is normal, the mutated proteinexhibits an unstable phenotype.

We also used a new antibody able to recognize the N-ter of thePolη. In the majority of XP-V cells with truncated protein, no bandswere detected by Western, indicating that the mutated protein wasunstable (Supp. Fig. S1). However, Western blots revealed truncatedproteins at the expected sizes for XP62VI, XP127VI, XP603VI, andXP968VI (Supp. Fig. S1). Because these truncated proteins do notharbor the NLS signal, or the PCNA-binding domain, we believethese truncated Polη do not intervene in the TLS process. Anyway,correlation between presence or absence of detectable truncatedproteins with severity of symptoms is not obvious.

Mutation analysis on the POLH gene

We have sequenced the POLH gene as described in Materials andMethods. The causative mutations were identified in all 23 XP-Vpatients (Table 1). Overall 29 mutations were found, including 25mutations for unrelated families: three large exonic deletions, eightsmaller deletions, three insertions, three nonsense mutations, fivemissense mutations, including one missense on the physiologicalstop codon leading to a longer protein with another stop codoneight amino-acids downstream (XP872VI), two splicing mutations,one AA in-frame deletion. The location of the mutations along thePOLH gene and the expected sizes of the corresponding mutatedproteins are given in Figure 3.

Mutations were previously identified in eight patients (seeTable 1 for references) but the exact genomic deletion boundarieswere not known and have been characterized in this analysis. In15 new patients (but 12 unrelated families), sequencing and CGHanalysis revealed 12 new mutations, not already described in theliterature: including four missense mutations in three patients, twosplicing mutations, one insertion, four deletions including threelarge homozygous deletions, and one –1 AA in frame deletion. Finemapping by CGH array of exonic 6 and 10 deletions revealed thatthese deletion sizes were respectively 745 and 3,763 bp (Table 1). Allmissense mutations were predicted to be deleterious by SIFT andPolyphen bioinformatic programs. New nonpreviously describedmutations are indicated in Table 1 (2nd column: (“yes”)). Thesenew variants have been deposited to the locus-specific database:http://www.lovd.nl/POLH.

HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014 123

Figure 2. Characterization of XP-V cells. A: DNA synthesis following increased doses of UVC with or without caffeine in an XP-V cell line(XP546VI) and a wild-type cell line (405VI). The XP-V fibroblast is sensitive to UV especially in the presence of caffeine. Experiments have beendone twice with three samples for each point. The SD is around 15%. B: Cell survival, measured as described in Materials and Methods, following3 J/m2 UVC in the presence or absence of caffeine. Results are given in% of unirradiated wild-type cells in the absence of caffeine. WT∗ indicatesthe average of results using two WT different cells (405VI and 198VI) and four different experiments. All XP-V cell lines are hypersensitive to UV inthe presence of caffeine. C: Detection of Polη protein levels from XP-V and wild-type primary fibroblasts. SiNT and siPOLH indicate the transfectionin the wild-type 198VI cells of siRNA control or specific toward the POLH gene, respectively. The complete loss of the protein band followingtransfection with siPOLH demonstrates that we are looking to the Polη protein (arrow). Two different exposure times have been shown to detectsmall amount of the Polη protein in some XP-V cells. The small band bigger than Polη corresponds to the ubiquitinated form of Polη (Ub-polη)[Bienko et al. 2010]. 198VI, 405VI, 873VI, and 950VI are control cells for comparison with XP-V cells isolated from the studied patients. γ -tubulin hasbeen used as a loading control. The asterisk on the XP603VI lane indicates the mutated Polη protein. D: Detection of Polη protein levels from XP-Vand wild-type lymphoblastoid cells. Same legend as in Figure 2C except that β-actin was used as loading control. 974VI, 975VI, and 976VI are controlcells, whereas 973VI are XP-C cells showing that in this class of NER-deficient cells the level of Polη is normal. XP966VI harbors a limited amountof Polη protein in agreement with its Val 37 deletion that is located in the active site and renders the protein unstable. XP968VI exhibits a truncatedprotein not visible with this antibody (See Supp. Fig. S1). E: Quantification of the half-life time of the mutated XP966VI Polη protein. LymphoblastoidXP966VI and control 974VI cell lines were treated by cycloheximide (CHX) for various times as described in Materials and Methods. Western blotswere quantified using the Image J software. Numbers under the Polη bands correspond to the quantification of the amount of protein as comparedwith the β-actin loading controls. Regression analysis were done on two independent experiments showing R2 = 0.966 for the XP966VI cells andR2 = 0.958 for the 974VI control cells. t1/2 for wild-type cells was around 4.7 hr, whereas it was about 2.6 hr for the mutated XP966VI protein.

Discussion

Similarity and Differences of Clinical Symptoms betweenPatients: A Common Phenotype for Diagnosis?

Our patients seem to have in common the first symptoms oftheir disease: lentigines and pigmented lesions predominant on UV-

exposed parts of the body and appearing in childhood, as early as7 for the median age. A few years later appear actinic keratoses andpoikilodermia usually before the age of 20 years. However, thesepatients usually lived a normal outside life before the age of 20 yearsand the clinical diagnosis of XP-V is delayed because the patientsdo not consult a dermatologist before having skin tumors. Also,symptoms of classical XP as photophobia and ocular symptoms are

124 HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014

Figure 3. POLH mutations in the described XP-V patients and predicted sizes of Polη. The top part of the figure corresponds to deletion ofone or two exons and point mutation of the splicing site in intron1 in some XP-V patients. Then below, the three possible transcripts and thelocation of intron/exon are framed. The different domains of the Polη are shown in the red bar. The N-ter 400 AA are conserved in the Y-family ofpolymerases and the active polymerase domain corresponds to the first 432 AA. The C-terminal part contains the regulation domains including thesequences necessary for nuclear localization (NLS), an ubiquitin-binding Zn finger (UBZ), and a PCNA Interacting Peptide motif (PIP-box) [Bienkoet al. 2005; Kannouche et al. 2001]. The mutations for each allele (1) or (2) of the XP-V patients are indicated (arrows) and the theoretical size ofthe corresponding protein is given on the right as a number of amino acids. In the absence of allele numbering, the corresponding mutation ishomozygous, according to Table 1. For the five mutant lines at the bottom of the figure, green color indicates a wild-type size of the mutant proteins,orange indicates a shorter size, and blue a longer size than the wild-type one.

Figure 4.

Figure 4. A structural model of Polη polymerase domain with thepoint mutations in the described XP-V patients. Representation of themissense mutations relative to the DNA substrate (orange templateand yellow primer strand), the active site (pink carbon and red oxygenatoms with two green Mg2+ ions) and important residues surroundingthe active site (light blue carbon with red oxygen and blue nitrogenatoms). The protein backbone is shown in semi-transparent warm-likeC-alpha trace with four structural domains (palm, thumb, finger, and littlefinger labeled). The four missense mutations found in the described XP-Vpatients are highlighted in magenta stick-and-ball and semi-transparentspheres. R93P is closest to DNA substrate and has the most severephenotype. G295R is more disruptive to the protein structure than V266Dand V37 deletion. Although a charged side chain (Asp in V266D) is placedin the hydrophobic core, Val and Asp have similar volume and the proteinapparently tolerates the mutation better than G295R by keeping Aspprotonated thus uncharged [Chimenti et al. 2012]. The already publishedmissense mutations R111H, R361S, G263V, F290S, and A264P (see forreview, Biertumpfel et al. 2010] have been indicated in cyan sticks andlabels. Two hotspots of POLH missense mutations could be describedaround R93P, R111H, and R361S and around V266D, G263V, F290S, G295R,and A264P.

HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014 125

usually absent. Clinical courses of XP-V were very different in ourseries of patients, and the severity of the disease and number of skintumors seem to be correlated to the level of sun exposure duringlifetime. For this reason, the patient’ age of the clinical diagno-sis varies largely from one individual to another (between 5 and50 years old) with a median at 22. The median age of occur-rence of the first nonmelanoma skin cancer (NMSC) was 24 years(7–54 years) in our XP-V cohort, which lies in between thecorresponding age in classical XP (9 years) and in the American gen-eral population (67 years) [Bradford et al., 2011]. For melanoma,the median age of first occurrence in XP-V is 32 years (17–64 years),whereas it is 22 years for classical XP and 55 years for the generalpopulation [Bradford et al., 2011]. In classical XP and in XP-V pa-tients, NMSC occur at a younger age than melanoma, which is thereverse in the general population. It is very important for cliniciansto think at the diagnosis of XP-V in pediatric patients showingunusual responses to short sun exposure, even in the populationswhere this disease is rare and despite of mild symptoms, in order toprevent skin tumor appearance by protecting patients.

Analysis of Cellular and Biological Data to Confirm theDiagnosis

Once the clinical suggestion of XP-V has been proposed by clini-cians, biological confirmation is done by the combination of normalpost-UV unscheduled DNA synthesis, partial cellular UV-sensitivitybut hypersensitivity to UV in the presence of caffeine. These bio-logical assays sign the XP-V phenotype, which is then confirmed byWestern blots of the protein and sequencing of the POLH gene. Allthese analyses allowed us to confirm the XP-V syndrome for the 23presented patients and to find new mutations.

As also found in previous reports [Broughton et al., 2002; Inuiet al., 2008; Johnson et al., 1999; Masaki et al., 2008; Masutani et al.,1999a; Tanioka et al., 2007], the vast majority of POLH mutationsgive rise to truncated proteins, which are not detectable on the West-ern blots, even when using an antibody that recognized the N-terpart of the polymerase. More than 50% of them are deletions offull exons (exons 6 and 10). In two cases (XP819VI and XP865VI),we have determined the size of the genomic deletions that extendfrom 745 and 3,763 bp (Table 1). Only five new missense muta-tions have been found at the homozygous (XP843VI, XP853VI,XP872VI) or heterozygous (XP603VI) state. The Polη protein con-taining missense mutations are observed on Western blots but atvery low level compared with wild-type cells indicating unstableproteins (Fig. 2C).

Correlation Phenotype/Genotype

A review of the literature, allowed us to collect POLH mutationsfor 85 patients including ours, comprising about 56 different muta-tions [Broughton et al., 2002; Inui et al., 2008; Johnson et al., 1999;Masaki et al., 2008; Masutani et al., 1999a; Tanioka et al., 2007].We report here 12 new mutations comprising missense, splicing,deletion, and insertion mutations in roughly the same percentage asthose already published. The fact that we found 12 new mutationscompared with the 56 already described in the last 12 years indicatesthat probably other mutations do exist among not-yet diagnosedpatients. Except deletion of exon 10 that may represent a foundereffect in North Africa, there is no clear hotspot of mutations, whichare basically scattered along the POLH gene. This is different fromthe prevalent XPC or XPA mutations found on the same populationfrom North Africa [Soufir et al., 2010].

To better correlate phenotype and genotype, we divided the XP-Vpatients in terms of mild, medium or aggressive symptoms (Table 1and Supp. Table S1) as described in the Results section. Of the 29mutations reported in Table 1, seven mutations (point mutationsor one AA in-frame deletions) allow the production of a smallamount of Polη. Modeling the effects of four point mutations onthe polymerase active site allowed us to predict the stability andeventually the activity of the polymerase and to correlate them withthe aggressiveness of the disease (Fig. 4).

XP603VI patient harbors one truncated protein after the catalyticcore (P576). This nonfunctional copy lacks all the C-terminal mo-tifs required for nuclear localization and binding to PCNA [Bienkoet al., 2005; Kannouche et al., 2001]. The second allele leads to a full-length protein containing the G295R mutation, which is located inthe middle of an alpha helix in the thumb domain and requiredto stabilize the primer strand. G295R is three-residues away fromanother XP-V mutation F290S (Fig. 4). The G295R mutation in-troduces a long sidechain where there is no space for even a methylsidechain like Ala and most certainly alters the thumb domain struc-ture if not unfolding it. The reducing protein amount and catalyticactivity are predicted as observed by Western analysis. If the mutantprotein has residual catalytic activity, it would be more error pronewhen carrying out TLS bypassing CPDs because of reduced stabilityof DNA primer. This may explain that this patient developed 24melanomas and numerous carcinomas in her life.

XP853VI patient has homozygous R93P mutation. Arg93 inter-acts with the phosphate of the template strand (at the –2 position)and also links the finger and LF domains together to form the molec-ular splint to keep the template strand in the normal B-form evenin the presence of a UV lesion. In addition R93 is in the middle ofan alpha helix, and R93P mutation would disrupt the helix, so theprotein should be less stable, which is exactly what is observed byWestern analysis. The function of UV lesion bypass is almost com-pletely lost and this may explain the very early and severe symptomsobserved in this patient. Two previously reported XP-V mutationsR111H and R361S [Broughton et al., 2002] are near R93P in thethree-dimensional structure (Fig. 4) and likely have similar delete-rious effects on DNA template binding by Polη. These three POLHmutations may define a hotspot of disease-causing mutations.

XP843VI patient is homozygous of V266D. The mutation is some-what removed from the DNA binding interface and the active site.The protein would be less stable, less active, and less accurate thanthe wild-type, but the structural and functional defects of V266Dare not expected to be as severe as R93P. Interestingly, the in silicoanalysis suggests that the effect of this missense mutation is less dele-terious than R93P and G295R (Supp. Table S2). This is correlatedwith relatively mild symptoms experienced by this patient. V266Dis located in the thumb domain as is G295R. V266D is very close tothe previously identified XPV mutations G263V [Broughton et al.,2002] and A264P [Biertumpfel et al., 2010]. These three mutationsfound in different XPV families are located on the same alpha-helix (Fig. 4) and adjacent to two other XPV mutations, G295R andF290S. The cluster for these five mutations may indicate a hotspotdomain among XP-V patients (Fig. 4).

XP966VI and XP967VI, two sisters exhibit the in-frame deletionof V37 in the finger domain next to Q38, which is conserved amongall Polη homologs and plays a role in the efficiency and accuracyof UV-lesion bypass. This Val37 deletion is the first and only pointmutation reported in the finger domain of Polη. The mutant proteinis most likely not as efficient and as accurate as WT in bypassing UVlesions. Moreover, the mutant protein is less stable as observed byWestern. Indeed, a shorter half-life of the mutated protein as com-pared with WT was found in the presence of cycloheximide (Fig. 2D

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and E). Again the mutation may not abolish the TLS ability entirely;therefore the siblings both have mild symptoms taking into consid-eration their ages of 72 and 86 years, probably the oldest XP variants.

XP872VI patient has two homozygous point mutations that arenot located on the active site. Because of the point mutation on theendogenous termination codon, the protein is 8 amino acids longerprobably causing partial degradation as the lengthened protein isalmost undetectable by Western. However, the residual protein maybe functional. Her clinical phenotype has been classified as mildtaking into account her relatively young age and her number oftumors.

The same mutation identified in cells from the two sistersXP606VI and XP737VI affects the splice site of the intron 1 (Ta-ble 1). However, the mRNA encodes a normal Polη protein. ByWestern blot analysis, we were able to detect Polη but at very lowlevel (<5%). This observation can be enlightened by the strong re-duction of the mRNA level in these cells [Faili et al, 2009]. It isinteresting to note that even if the amount of Polη protein seemsto be similar in these two individuals, one patient (XP606VI) hadmultiple malignant skin lesions, whereas none has been reportedfor her sister (XP737VI). This difference could be explained bydifferent sunlight exposure between the two sisters as indicated bythe patients themselves.

Of the 29 mutations reported in Table 1, 20 resulted in truncatedproducts because of stop codons, frameshifts, or exon deletions. Asexpected, the Polη protein is undetectable in the cell extracts fromthese individuals by using an antibody recognizing the C-ter part ofthe polymerase (Fig. 2C and D) but in some cases a stable shorterform was observed by using antibody recognizing the N-ter part ofthe polymerase (Supp. Fig. S1). All these patients are identical interms of Polη deficiency but their phenotypes are quite different,since about half of them exhibit mild/medium symptoms and abouthalf very aggressive symptoms (see Table 1 and Supp. Table S1).

ConclusionCorrelation between clinical features and the types of POLH mu-

tations is difficult to make, at least in cases of absent or truncatedprotein. Among 23 XP-V patients the median age for the first skincancer was 22.7 years. Only three patients did not develop ma-lignant tumors at 32, 44, and 46 years old. The XP961VI patient(Fig. 1F) lived all her life in Congo at the equator level with a lotof sun exposure, although we classified her as mild syndrome. Theabsence of tumors should be partly due to her black skin showingclearly the major protection role of melanin even in the XP back-ground. We reported the same high protection in XP-C patientsliving in Mayotte in the Indian Ocean [Cartault et al., 2011]. Thealready-described white-skinned XP737VI living in the middle ofFrance (with medium sun exposure) that she considered herself aslow exposure as compared with her younger sister (XP606VI) whodeveloped five melanomas and one SCC. Finally, XP968VI did notdevelop any tumors at the age of 32 years, whereas her brother(XP888VI) with the same truncating mutation developed severalcarcinomas and melanoma. These different patients clearly demon-strate an absence of direct correlation between the lack of full-lengthPolη protein and a given picture of clinical features. It appears thatthe clinical course of XP-V patients, including cancer incidence, isbetter correlated with total lifetime sun exposure more than withthe type of truncating germinal POLH mutations. Other geneticfactors such as modifier genes for melanoma or carcinoma suscep-tibility are being studied to determine if they can partly explain thisdiscrepancy.

These data, deduced from the largest cohort of published XP vari-ants, should be taken into consideration by clinicians in explainingclearly to XP-V patients to accept a full protection against sun expo-sure to live with no or few cancers. Early protection is also importantwhen possible because most of XP-V patients are used to live nor-mally for sun-exposure until the age of 20. Clinicians should suggestthe diagnosis of XP-V as early as possible, even in the absence ofconsanguinity or family history of XP-V, and even in populationswhere we usually do not suspect such a diagnosis.

Acknowledgments

We are thankful to Mrs. Daniele Pham who carried out the UDS and post-UV survival in the presence of caffeine, to Mrs. Jacqueline Guymarho forcell cultures and to Dr. Brigitte Bressac for the sequencing of CDKN2A-CDK4-p14 genes. The authors are indebted to the help of the followingclinicians: Dr. Raoul Triller, Pr. Maurice Mimoun, Dr. Francoise Boitier forproviding clinical photographs of their patients, and Dr. Christine Chiaveriniand Prof. Marie-Francoise Avril for providing clinical data and photographsof her patients. The authors are thankful to Prof. Alan Lehmann (SussexUniversity, UK) with whom we started the analysis of our first XP-V patients.I (A.S.) would like very much to thank Dr. Julia Lopez and Dr. Johan T.den Dunnen, from the LOVD database, for their help in curating our list ofPOLH mutants with much efficiency.

Patient consents: Obtained.Disclosure statement: The authors declare no conflict of interest.

References

Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, WiderG, Peter M, Lehmann AR, Hofmann K, Dikic I. 2005. Ubiquitin-binding domainsin Y-family polymerases regulate translesion synthesis. Science 310:1821–1824.

Bienko M, Green CM, Sabbioneda S, Crosetto N, Matic I, Hibbert RG, Begovic T, NiimiA, Mann M, Lehmann AR, Dikic I. 2010. Regulation of translesion synthesis DNApolymerase eta by monoubiquitination. Mol Cell 37:396–407.

Biertumpfel C, Zhao Y, Kondo Y, Ramon-Maiques S, Gregory M, Lee JY, MasutaniC, Lehmann AR, Hanaoka F, Yang W. 2010. Structure and mechanism of humanDNA polymerase eta. Nature 465:1044–1048.

Bradford PT, Goldstein AM, Tamura D, Khan SG, Ueda T, Boyle J, Oh KS, Imoto K,Inui H, Moriwaki SI, Emmert S, Pike KM, et al. 2011. Cancer and neurologicdegeneration in xeroderma pigmentosum: long term follow-up characterises therole of DNA repair. J Med Genet 48:168–176.

Broughton BC, Cordonnier A, Kleijer WJ, Jaspers NGJ, Fawcett H, Raams A, GarritsenVH, Stary A, Avril MF, Boudsocq F, Masutani F, Hanaoka F, Fuchs RP, SarasinA, Lehmann A. 2002. Molecular analysis of mutations in DNA polymerase eta inxeroderma pigmentosum variant patients. Proc Natl Acad Sci USA 99:815–820.

Cartault F, Nava C, Malbrunot AC, Munier P, Hebert JC, N’Guyen P, Djeridi N, PariaudP, Pariaud J, Dupuy A, Austerlitz F, Sarasin A. 2011. A new XPC gene splicingmutation has lead to the highest worldwide prevalence of xeroderma pigmentosumin black Mahori patients. DNA Repair (Amst) 10:577–585.

Chimenti MS, Khangulov VS, Robinson AC, Heroux A, Majumdar A, Schlessman JL,Garcıa-Moreno B. 2012. Structural reorganization triggered by charging of Lysresidues in the hydrophobic interior of a protein. Structure 20:1071–1085.

Cleaver JE. 1968. Defective repair replication of DNA in xeroderma pigmentosum.Nature 218:652–656.

Despras E, Daboussi F, Hyrien O, Marheineke K, Kannouche P. 2010. ATR/Chk1pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells. Hum Mol Genet 19:1690–1701.

Faili A, Aoufouchi S, Weller S, Vuillier F, Stary A, Sarasin A, Reynaud CA, WeillJC. 2004. DNA polymerase eta is involved in hypermutation occurring duringimmunoglobulin class switch recombination. J Exp Med 199:265–270.

Faili A, Stary A, Delbos F, Weller S, Aoufouchi S, Sarasin A, weill JC, Reynaud CA. 2009.A backup role of DNA polymerase kappa in Ig gene hypermutation only takes placein the complete absence of DNA polymerase eta. J Immunol 182:6353–6359.

Giglia-Mari G, Sarasin A. 2003. TP53 mutations in human cancers. Human Mutat21:217–228.

Gueranger Q, Stary A, Aoufouchi S, Faili A, Sarasin A, Reynaud CA, Weill JC. 2008. Roleof DNA polymerases η, ι and ζ in UV-resistance and UV-induced mutagenesis ina human cell line. DNA Repair 7:1551–1562.

HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014 127

Hoeijmakers JH. 2001. Genome maintenance mechanisms for preventing cancer. Na-ture 411:366–374.

Inui H, Oh KS, Nadem C, Ueda T, Khan SG, Metin A, Guzukara E, Emmert S,Slor H, Busch DB, Baker CC, DiGiovanna JJ, Tamura D, et al. 2008. Xerodermapigmentosum-variant patients from America, Europe and Asia. J Invest Dermatol128:2055–2068.

Johnson RE, Kondratick CM, Prakash S, Prakash L. 1999. hRAD30 mutations in thevariant form of xeroderma pigmentosum. Science 285:263–265.

Kannouche P, Stary A. 2003. Xeroderma pigmentosum variant and error-prone DNApolymerases. Biochimie 85:1123–1132.

Kannouche P, Broughton BC, Volker M, Hanaoka F, Mullenders LH, LehmannAR. 2001. Domain structure, localization, and function of DNA polymeraseeta, defective in xeroderma pigmentosum variant cells. Genes Dev 15:158–172.

Kannouche P, Wing J, Lehmann AR. 2004. Interaction of human DNA polymerase etawith monoubiquitinated PCNA: a possible mechanism for the polymerase switchin response to DNA damage. Mol Cell 14:491–500.

Lehmann AR, Kirk-Bell S, Arlett CF, Paterson MC, Lohman PH, de Weerd-Kastelein EA,Bootsma D. 1975. Xeroderma pigmentosum cells with normal levels of excisionrepair have a defect in DNA synthesis after UV-irradiation. Proc Natl Acad SciUSA 72:219–223.

Masaki T, Ono R, Tanioka M, Funasaka Y, Nagano T, Moriwaki S, Nishigori C. 2008.Four types of possible founder mutations are responsible for 87% of Japanesepatients with xeroderma pigmentosum variant type. J Dermatol Sci 52:144–148.

Masutani C, Kusumoto R, Yamada A, Dohmae N, Yokoi M, Yuasa M, Araki M, IwaiS, Takio K, Hanaoka, F. 1999a. The XPV (xeroderma pigmentosum variant) geneencodes human DNA polymerase eta. Nature 399:700–704.

Masutani C, Araki M, Yamada A, Kusumoto R, Nogimori T, Maekawa T, Iwai S,Hanaoka F. 1999b. Xeroderma pigmentosum variant (XP-V) correcting proteinfrom HeLa cells has a thymine dimer bypass DNA polymerase activity. EMBO J18:3491–3501.

Sarasin A, Hanawalt PC. 1980. Replication of ultraviolet-irradiated SV40 in monkeykidney cells. J Mol Biol 138:299–319.

Sarasin A, Blanchet-Bardon C, Renault G, Lehmann A, Arlett C, Dumez Y. 1992.Prenatal diagnosis in a subset of trichothiodystrophy patients defective in DNArepair. Br J Dermatol 127:485–491.

Soufir N, Ged C, Bourillon A, Austerlitz F, Chemin C, Stary A, Armier J, Pham D,Khadir K, Roume J, Hadj-Rabia S, Bouadjar B, et al. 2010. A prevalent mutationwith founder effect in xeroderma pigmentosum group C from North Africa. JInvest Dermatol 130:1537–1542.

Stary A, Sarasin A. 2002. The genetics of the hereditary xeroderma pigmentosumsyndrome. Biochimie 84:49–60.

Stary A, Kannouche P, Lehmann AR, Sarasin A. 2003. Role of DNA polymerase eta inthe UV mutation spectrum in human cells. J Biol Chem 278:18767–18775.

Tanioka M, Masaki T, Ono R, Nagano T, Otoshi-Honda E, Matsumura Y, TakigawaM, Inui H, Miyachi Y, Moriwaki S, Nishigori C. 2007. Molecular analysis of DNApolymerase eta gene in Japanese patients diagnosed as xeroderma pigmentosumvariant type. J Invest Dermatol 127:1745–1751.

Tayeb T, Laure B, Sury F, Lorette G, Goga D. 2011. Facial resurfacing with split-thicknessskin grafts in xeroderma pigmentosum variant. J Cranio-Maxil Surg 39:496–498.

Yoon JH, Prakash L, Prakash S. 2009. Highly error-free role of DNA polymerase etain the replicative bypass of UV-induced pyrimidine dimers in mouse and humancells. Proc Natl Acad Sci USA 106:18219–18224.

128 HUMAN MUTATION, Vol. 35, No. 1, 117–128, 2014