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2005;14:1539-1544. Published online June 7, 2005.Cancer Epidemiol Biomarkers PrevJiali Han, Graham A. Colditz, Jun S. Liu, et al.Cancer

, Sun Exposure, and Risk of SkinXPDGenetic Variation in

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Genetic Variation in XPD, Sun Exposure, and Riskof Skin Cancer

Jiali Han,1,5 Graham A. Colditz,1,2,4 Jun S. Liu,3,6 and David J. Hunter 1,2,5

1Channing Laboratory, Department of Medicine, Brigham and Womens Hospital and Harvard Medical School; 2Department ofEpidemiology, 3Department of Biostatistics, 4Harvard Center for Cancer Prevention, and 5Program in Molecular andGenetic Epidemiology, Harvard School of Public Health, Boston, Massachusetts and 6Department of Statistics,

Harvard University, Cambridge, Massachusetts

Abstract

The XPD gene is involved in the nucleotide excisionrepair pathway removing DNA photoproducts inducedby UV radiation. Genetic variation in XPD may exert asubtle effect on DNA repair capacity. We assessed theassociations between two common nonsynonymous poly-morphisms (Asp312Asn and Lys751Gln) with skin cancerrisk in a nested case-control study within the NursesHealth Study (219 melanoma, 286 squamous cell carci-noma, 300 basal cell carcinoma, and 874 controls) alongwith exploratory analysis on the haplotype structure of

the XPD gene. There were inverse associations betweenthe Lys751Gln and Asp312Asn polymorphisms and the risks

of melanoma and squamous cell carcinoma. No associationwas observed between these two polymorphisms andbasal cell carcinoma risk. We also observed that theassociation of the 751Gln allele with melanoma risk wasmodified by lifetime severe sunburns, cumulative sunexposure with a bathing suit, and constitutional suscepti-bility score (P for interaction = 0.03, 0.04, and 0.02respectively). Similar interactions were also observed forthe Asp312Asn. Our data suggest these two XPD non-synonymous polymorphisms may be associated with skin

cancer risk, especially for melanoma. (Cancer EpidemiolBiomarkers Prev 2005;14(6):153944)

Introduction

Skin cancer is the most common neoplasm in Caucasians inthe United States. The genotoxic effect of sunlight exposurehas been clearly shown in the etiology of both melanoma andnonmelanocytic skin cancer (1-3). One important defensemechanism against skin cancer is the ability to repair DNAdamage induced by UV light. It has been suggested thatreduced DNA repair capacity (DRC) is a susceptibility factorpredisposing individuals to skin cancer (4-7). The predomi-

nant form of UV-induced DNA damage is DNA photo-products caused by the direct absorption of UVB by DNA.Cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimi-done photoproducts constitute the two major DNA photo-products (2). DNA photoproducts are mainly removed bythe nucleotide excision repair (NER). The NER is a versatilerepair system to remove a variety of bulky, helix-distortinglesions, including UV photoproducts and bulky adducts (8, 9).Individuals with xeroderma pigmentosum, deficient in theNER, have a >1,000-fold increased risk of skin cancer.

Human XPD maps to chromosome 19q13.3 and spans f54kb. It comprises 23 exons and is 761 amino acids in length. TheXPD gene encodes an ATP-dependent DNA helicase involvedin the NER and in basal transcription as part of thetranscription factor TFIIH. Disruption of the mouse Xpd gene

results in preimplantation lethality (10). Mutations in the XPDgene lead to NER defects (11) and three clinical syndromes,Cockayne syndrome, xeroderma pigmentosum, and tricho-thiodystrophy, depending on the location of the mutation (9,12). In addition, the XPD and p53 proteins can interact witheach other to modulate apoptosis and the NER. The p53 binds

and modulates the helicase activity of the TFIIH, and the repairof UV-induced dimers was attenuated in Li-Fraumeni syn-drome cells (heterozygote p53 mutant; ref. 13). A deficiency inp53-mediated apoptosis was reported in XPD lymphoblastoidcell lines and fibroblasts from xeroderma pigmentosumpatients with germ line mutations in the XPD gene (14, 15).

We evaluated two common nonsynonymous XPD poly-morphisms (Asp312Asn and Lys751Gln) in relation to skin

cancer risk in a nested case-control study within the NursesHealth Study along with exploratory analysis on the haplotypestructure of the XPD gene. We further investigated thehypothesis that XPD genetic variants modify the associationsof sunlight-related risk factors with skin cancer risk.

Materials and Methods

Study Population. The Nurses Health Study was estab-lished in 1976, when 121,700 female registered nurses betweenages 30 and 55 years completed a self-administered question-naire on their medical histories and baseline health-relatedexposures. Updated information has been obtained by ques-tionnaires every 2 years. Between 1989 and 1990, bloodsamples were collected from 32,826 of the cohort members.Eligible cases in this study consisted of women with incidentskin cancer from the subcohort who gave a blood specimen,including squamous cell carcinoma (SCC) and basal cellcarcinoma (BCC) cases with a diagnosis anytime after bloodcollection up to June 1, 1998 and melanoma cases (includingin situ cases) up to June 1, 2000 with no previously diagnosedskin cancer. All available pathologically confirmed melanomaand SCC cases and 300 self-reported BCC cases randomlyselected from f2,600 available self-reported BCC cases wereincluded. The validity of self-report of BCC is high in thismedically sophisticated population (90%; ref. 16). All the SCCand BCC cases had no history of melanoma diagnosis. Acommon control series (case/control, 1:1) was randomly

selected from participants who gave a blood sample and werefree of diagnosed skin cancer up to and including the

Cancer Epidemiology, Biomarkers & Prevention 15

Cancer Epidemiol Biomarkers Prev 2005;14(6). June 2005

Received 11/16/04; revised 3/22/05; accepted 4/7/05.

Grant support: NIH grants CA97746 and CA87969 and Harvard Specialized Programs ofResearch Excellence in Skin Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

Requests for reprints: Jiali Han, Channing Laboratory, Harvard Medical School, 181Longwood Avenue, Boston, MA 02115. Phone: 617-525-2098; Fax: 617-525-2008.

E-mail: [email protected] D 2005 American Association for Cancer Research.


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questionnaire cycle in which the case was diagnosed. Onecontrol was matched to each case by year of birth (F1 year)and self-reported race (Caucasian, Asian, Hispanic, AfricanAmerican, and unknown). More than 95% of cases andcontrols were Caucasian. At the time we selected cases andcontrols, 47 cases and 69 controls were deceased. To obtainadditional information by supplementary questionnaires, werandomly selected a second matched living control when thefirst control was deceased and collected supplementaryquestionnaires from these second living controls. The nestedcase-control study consisted of 219 melanoma cases (including77 in situ cases), 286 SCC cases, 300 BCC cases, and 874matched controls. We mailed to 758 living cases and 804 livingcontrols a supplementary questionnaire on lifetime sunexposure and other skin cancer risk factors; 695 casesresponded, 15 cases refused to participate, and 48 cases didnot respond after three mailings (participation rate, 92%).Among controls, 713 responded, 9 refused, and 82 did notrespond (participation rate, 89%). The study protocol wasapproved by the Committee on Use of Human Subjects of theBrigham and Womens Hospital (Boston, MA).

Exposure Data. Information regarding skin cancer riskfactors was obtained from the prospective biennial question-naires and the retrospective supplementary questionnaire.

Information on natural hair color and childhood and adolescenttendency to sunburn or tan was asked in the 1982 prospectivequestionnaire (ethnic group in the 1992 questionnaire). Theretrospective supplementary questionnaire consisted of ques-tions in three major areas: (a) pigmentation, constitutional, andsusceptibility factors; (b) history of residence (states andtowns), sun exposure habits, and severe sunburns at differentages; and (c) family history of skin cancer (father, mother, andsiblings). In addition, the 11 states of residence of cohortmembers at baseline were grouped into three regions:Northeast (Connecticut, Massachusetts, Maryland, New Jersey,New York, and Pennsylvania), North Central (Michigan andOhio), and West and South (California, Texas, and Florida).

To estimate sunlight exposure for each subject, a UVdatabase for 50 U.S. states was developed. The database usedreports from the Climatic Atlas of the United States, whichreported mean daily solar radiation (in Langleys) at theearths surface for weather stations around the country (17).The records of average annual solar radiation for January and

July were extrac ted to repres ent winter and summerradiation, respectively. The mean solar radiation for eachresidence was derived from the UV values measured at thenearest weather station, and both summer and winterradiation indices were developed for each residence. Acumulative lifetime sun exposure was developed by combin-ing the UV database and the information obtained from thesupplementary questionnaires. Questions about sun exposurewhile wearing a bathing suit were used to define acumulative lifetime intermittent (recreational) sun exposure

variable for this behavior.Single Nucleotide Polymorphism Identification. There are

two common nonsynonymous polymorphisms in XPD(Asp312Asn and Lys751Gln). Because these two single nucleo-tide polymorphisms (SNP) are not in complete linkagedisequilibrium in the database and have potentially functionalrelevance, we genotyped both of them in our study. The XPDgene was resequenced by the National Institute of Environ-mental Health Sciences Environmental Genome Project atthe University of Washington (http://egp.gs.washington.edu/data/ercc2/) on a subset of 90 samples from the NIH DNAPolymorphism Discovery Resource (18). Among the 136polymorphisms identified across the genomic region of theXPD gene, we selected 86 polymorphisms with allele

frequency over 1%. Based on these 86 polymorphisms, sevencommon haplotypes (>2% frequency) were inferred by the

partition-ligation expectation-maximization algorithm of Qinet al. (19). The threshold of 2% was set to ascertain potentialcommon (>5%) Caucasian-specific haplotypes from this mixedpopulation (20). Five haplotype-tagging SNPs (htSNP) wereselected by the BEST algorithm (21) to tag these seven commonhaplotypes and were genotyped in this study.

Laboratory Assays. Genotyping was done by the 5Vnucleaseassay (TaqMan), using the ABI PRISM 7900HT SequenceDetection System (Applied Biosystems, Foster City, CA), in

384-well format. TaqMan primers and probes were designedusing the Primer Express Oligo Design software version 2.0(ABI PRISM). Laboratory personnel were blinded to case-control status and blinded quality-control samples wereinserted to validate genotyping procedures; concordance forthe blinded samples was 100%. Primers, probes, and con-ditions for genotyping assays are available on request.

Statistical Methods. To avoid potential population stratifi-cation, we excluded one Asian melanoma case and one control,one Hispanic SCC case and two controls, and one AfricanAmerican control. We used a m2 test to assess whether the XPDgenotypes were in Hardy-Weinberg equilibrium and todetermine Ps for differences in haplotype frequencies betweencases and controls. Unconditional logistic regression was

employed to calculate odds ratio (OR) and 95% confidenceinterval (95% CI) to assess the risk of skin cancer for XPDgenotypes among all women. A test for trend was calculatedacross the three genotypes for each polymorphism. We used alikelihood ratio test to evaluate heterogeneity in the effects of theXPD genotypes on different types of skin cancer in polytomouslogistic regression models (22). To summarize multiple varia-

bles, we constructed a multivariate confounder score to create aconstitutional susceptibility score for skin cancer (23). Briefly,we applied the logistic regression coefficients from a multivar-iate model, including age, race, natural skin color, natural haircolor, child or adolescent tendency to burn, and the number ofpalpably raised moles on arms, to each individuals values forthe latter four of these variables and summed the values tocompute a susceptibility risk score in the logit scale. We used

median value of this score among controls to define womenwith low and high constitutional susceptibility. The number ofsevere lifetime sunburns and cumulative sun exposure with a

bathing suit were categorized into tertiles based on thedistribution of controls.

To evaluate interactions between the environmental expo-sures and the XPD genotypes, we modeled them as ordinalvariables to test significance of a single multiplicativeinteraction term. We modeled the genotype into three levels(homozygous wild-type, heterozygotes, and homozygousvariants). For environmental risk factors, we modeled thenumber of lifetime severe sunburns into three levels, cumu-lative sun exposure with a bathing suit into three levels, andconstitutional susceptibility score as a dichotomous variable.All statistical tests were two sided.

Results

Descriptive Characteristics of Cases and Controls. Detaileddescription of characteristics of cases and controls will bereported elsewhere.7 In brief, the mean age at diagnosis ofmelanoma cases was 63.4 years and that of SCC and BCC caseswas 64.7 and 64.0 years, respectively. Women in the West andSouth regions were more likely to be diagnosed with SCC orBCC compared with those in Northeast. Cases of each type ofskin cancer were more likely to have used sunlamps or

7

Han et al. Risk factors of skin cancer: a nested case-control study within theNurses Health Study, in preparation.

XPD Gene, Sun Exposure, and Skin Cancer40



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attended tanning salons. A family history of skin cancer was arisk factor for the three types of skin cancer. Cases of each typeof skin cancer were more likely to have had higher cumulativesun exposure with a bathing suit, more lifetime severesunburns that blistered, and higher constitutional susceptibil-ity risk score (Table 1).

Associations of XPD Asp312Asn and Lys751Gln with SkinCancer Risk. The genotype distributions of the two poly-morphisms were in Hardy-Weinberg equilibrium among

controls. The two polymorphisms were in linkage disequilib-rium in controls (DV = 0.77, P = 8.7 1084; r2 = 0.50, P = 8.4 1043). Genotype concordance between the two polymor-phisms was 76.0% in controls, which is consistent with aprevious report (24). The linkage disequilibrium statistics andgenotype concordance in each case series was similar to thosein controls. Inverse associations of the 751Gln and 312Asnalleles with the risks of melanoma and SCC were observed(Table 2). No association was observed between these twopolymorphisms and BCC risk. There was no significantheterogeneity in the main effect of each polymorphism onthe three types of skin cancer (Table 2). We also evaluated thecombined effect of both polymorphisms. We grouped ourpopulation into three genotype categories, double wild-type

(carriage of both Asp/Asp and Lys/Lys genotypes), doublehomozygous variants (carriage of both Asn/Asn and Gln/Glngenotypes), and others. The results of this combined analysiswere similar to the results of the individual polymorphismanalysis (Table 2).

Interactions between XPD Polymorphisms and RiskFactors on Melanoma Risk. We evaluated gene-environmentinteractions between the two nonsynonymous polymorphismsand the number of lifetime severe sunburns, cumulative sunexposure while wearing a bathing suit, and constitutionalsusceptibility score on skin cancer risk (Table 3). An interactionwas observed between the number of lifetime severe sunburnsand the Lys751Gln polymorphism on melanoma risk (P forinteraction = 0.03; Table 3). The number of lifetime severesunburns was significantly associated with an increased risk ofmelanoma among women with the 751Lys/Lys genotype (z5versus never, OR, 3.26; 95% CI, 1.39-7.63), and this excess riskwas attenuated among those who carried the Gln allele. Thesignificantly inverse association of the 751Gln allele withmelanoma risk was limited to women with five or morelifetime sunburns (P for trend = 0.02), whereas no association

between the 751Gln allele and melanoma risk was observedamong women who had four or fewer lifetime sunburns. Asimilar interaction pattern was also seen between the numberof lifetime severe sunburns and the polymorphism Asp312Asnon melanoma risk (P for interaction = 0.03).

We observed a similar interaction pattern betweencumulative sun exposure with a bathing suit and the

Lys751Gln polymorphism on melanoma risk (Table 3).Cumulative sun exposure with a bathing suit was signifi-cantly associated with an increased risk of melanoma amongwomen with the 751Lys/Lys genotype (highest tertile versuslowest tertile, OR, 4.84; 95% CI, 2.16-10.82), which wasattenuated among those who carried the Gln allele. Weobserved an inverse association of the 751Gln allele withmelanoma risk among the women in the highest tertile ofsun exposure with a bathing suit (P for trend = 0.06) but notamong those in the lowest or intermediate tertile (P forinteraction = 0.04). No significant interaction was observed

between the Asp312Asn polymorphism and cumulative sunexposure with a bathing suit on melanoma risk.

We also observed interactions between the constitutionalsusceptibility score and both polymorphisms Lys751Gln andAsp312Asn on melanoma risk (Table 3). A significantly inversetrend between the 751Gln allele and melanoma risk (P fortrend =0.008) was only seen among women with low susceptibilityscore but not among those with high score (P for interaction =0.02). A similar interaction pattern was observed for theAsp312Asn polymorphism (P for interaction = 0.08).

We also evaluated the combined effect of both polymor-phisms in the gene-environment interactions. Compared withthe individual polymorphism, similar patterns of interactions

were observed when we assessed the interactions of threegenotype groups (double wild-type, double homozygousvariants, and others) with history of sunburns (P forinteraction = 0.007), cumulative sun exposure while wearinga bathing suit (P for interaction = 0.11), and constitutionalsusceptibility score (P for interaction = 0.14).

There was no significant difference in the Lys751Gln andAsp312Asn genotype distributions in different categories ofexposures among controls (i.e., the interactions we observedwere not driven by controls). In addition, we did not observeany interaction between the genotypes and geographic regionon melanoma risk. No significant interactions were observed

between the two polymorphisms and the above exposure riskfactors on SCC or BCC risk.

XPD Haplotypes and Skin Cancer Risk. We did explor-atory analysis of the XPD haplotype structure. There appearstwo haplotype blocks in the XPD gene based on theresequencing data on 90 samples from the EnvironmentalGenome Project. We evaluated the long-range haplotype acrossthe two blocks. Five htSNPs were selected, two htSNPs in thefirst block (G1305C and G7988A), one in the second block(T19938C), and two in the linker region between the two blocks(T12733G and C12799T). Nine common inferred haplotypeswith allele frequency >5% in controls accounted for 83.3% ofthe alleles of controls in the present study (Table 4). Weobserved that three different common haplotypes were lesscommon in the cases of melanoma, SCC, and BCC thancontrols, respectively.

Table 1. Characteristics of skin cancer cases and controls in the nested case-control study

Characteristics Controls(n = 870)

Melanoma cases(n = 218)

SCC cases(n = 285)

BCC cases(n = 300)

Age at diagnosis (mean, years) 64.5 63.4 64.7 64.0Geographic region at baseline (%)

Northeast 55.2 58.0 51.7 49.3North central 23.4 16.9 17.1 20.3West and South 21.4 25.1 31.1 30.3

Sunlamp use or tanning salon attendance (%) 10.0 19.2 14.3 14.7Family history of skin cancer (%) 25.1 36.5 35.7 42.7Highest tertile of cumulative sun exposure

with a bathing suit (%)33.4 53.3 46.1 42.6

No. lifetime severe sunburns (mean) 5.4 9.6 7.8 8.2Median above constitutional susceptibility

risk score (%)

49.9 75.8 72.0 68.3




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Discussion

In this nested case-control study, we observed that the XPD751Gln allele was associated with decreased risks of melanomaand SCC along with finding that the effect of the 751Gln allele onmelanoma risk was modified by the number of lifetime severesunburns, cumulative sun exposure while wearing a bathingsuit, and constitutional susceptibility score. Similar main effect

and interactions were also observed for the Asp312

Asn inmelanoma risk. The nested case-control design, high follow-uprate, and high response rate for the retrospective supplemen-tary questionnaire strengthen the validity of this study.

Several studies have examined the functional significance ofthe two XPD nonsynonymous polymorphisms Asp312Asn andLys751Gln using DNA adducts and UV-induced photoprod-ucts, which are mainly repaired by the NER. The 312Asn and751Gln alleles were associated with decreased DRC for removalof UV photoproducts (25) and benzopyrene diol epoxide-DNAadducts (26) by host cell reactivation assays. Hemminki et al.(27) reported that the combined 312Asn/Asn and 751Gln/Glngenotype was associated with reduced DRC of UV-inducedcyclobutane dimers in human skin in situ and the 751Gln allelewas associated with reduced repair among individuals ages

z50 years. Consistent with these observations, a positiveassociation was observed of the 312Asn or 751Gln allele withincreased aromatic DNA adduct level in peripheral lympho-cytes (28). Seker et al. (29) reported that the 312Asn allele wasassociated with enhanced apoptotic response to UV. Theseauthors did not detect any effect of the two polymorphisms inin vitro p53 binding or DRC. Nevertheless, the increased UV-induced apoptosis may reflect slightly impaired DRC (i.e., thesubtle reduction in DRC may cause the accumulation of excessDNA damage and in turn trigger apoptosis). It was reportedthat unrepaired UV-induced damage on the transcribed strand

blocks transcription, triggers the accumulation of p53, andinduces apoptosis (30).

We observed that the 751Gln allele was associated with a

decreased risk of melanoma, and this decreased risk was moreapparent among women with five or more severe lifetime

sunburns or those in the highest tertile of cumulative sunexposure with a bathing suit. Similar interactions were alsoobserved for the 312Asn allele. A suggestive positive associa-tion of the variant allele with melanoma risk was observedamong women who never had severe blistering sunburns orwere in low category of sun exposure. Given previous datasuggesting the reduced DRC of the 751Gln and 312Asn alleles,our data showed that the positive association of the variant

allele and melanoma risk occurred in the context of low levelsof DNA damage. However, when challenged by an over-whelmingly high dose of exposure measured as five or moresunburns or high level of cumulative sun exposure with a

bathing suit, melanocytes with impaired DRC may accumulateexcess DNA damage, inducing apoptosis and in turn decreas-ing the risk of melanoma.

We also observed a significant interaction between theconstitutional susceptibility score and the 312Asn and 751Glnvariants. A significant association of the 751Gln or 312Asn allelewith melanoma risk was seen among women with lowscore butnot among those with high score. In addition, we also observeda decreased risk of SCC associated with the 751Gln allele.

Although melanocytes are less sensitive to apoptosis thankeratinocytes because of the lower levels of cell cycle and

proliferation and the higher content of antiapoptotic proteins(31, 32), we observed that the 312Asn and 751Gln allele wereassociated with a decreased risk of melanoma potentially dueto apoptosis. As suggested by the previous functional data(29), the two 312Asn and 751Gln alleles, which are in linkagedisequilibrium, may enhance the cellular apoptotic response toUV exposure. This is consistent with our data that thedecreased risk was stronger among women with high sunexposure as measured by lifetime sunburns and cumulativesun exposure while wearing a bathing suit.

We did exploratory analysis of XPD long-range haplotype.Across two haplotype blocks, we inferred nine commonhaplotypes (>5% allele frequency) from five htSNPs. We usedthe data on 90 multiethnic samples from the Environmental

Genome Project to infer haplotype structures. Two recentstudies of haplotype variation in different ethnic groups (33, 34)

Table 2. XPD genotypes and skin cancer risk

Genotype Controls Melanoma Cases SCC Cases BCC Cases

OR* ORc OR* ORc OR* ORc

G7988A (Asp312Asn)Asp/Asp 342 88 1.00 1.00 128 1.00 1.00 104 1.00 1.00Asp/Asn 373 99 1.03 (0.75-1.43) 1.02 (0.72-1.44) 115 0.82 (0.62-1.10) 0.80 (0.59-1.09) 149 1.32 (0.98-1.76) 1.26 (0.93-1.71)Asn/Asn 121 19 0.61 (0.36-1.05) 0.65 (0.37-1.15) 37 0.82 (0.54-1.24) 0.81 (0.52-1.26) 32 0.86 (0.55-1.34) 0.88 (0.55-1.41)P for trend 0.19 0.27 0.21 0.19 0.84 0.87

Heterogeneityb

0.27A20337C (Lys751Gln)

Lys/Lys 295 81 1.00 1.00 126 1.00 1.00 98 1.00 1.00Lys/Gln 415 99 0.88 (0.63-1.23) 0.89 (0.62-1.26) 112 0.63 (0.47-0.85) 0.62 (0.45-0.85) 141 1.03 (0.77-1.39) 1.00 (0.73-1.37)Gln/Gln 134 23 0.63 (0.38-1.05) 0.67 (0.39-1.15) 42 0.73 (0.49-1.10) 0.73 (0.48-1.12) 47 1.06 (0.70-1.59) 1.06 (0.69-1.62)P for trend 0.09 0.16 0.02 0.03 0.78 0.84Heterogeneityb 0.07

Combined effect of Asp312Asn and Lys751GlnAsp/Asp + Lys/Lys 239 66 1.00 1.00 101 1.00 1.00 80 1.00 1.00Others 487 115 0.85 (0.60-1.20) 0.86 (0.59-1.24) 144 0.70 (0.52-0.94) 0.68 (0.49-0.93) 169 1.04 (0.76-1.41) 1.03 (0.75-1.43)Asn/Asn + Gln/Gln 88 15 0.62 (0.34-1.15) 0.69 (0.36-1.32) 30 0.81 (0.50-1.30) 0.81 (0.49-1.34) 25 0.84 (0.50-1.41) 0.86 (0.50-1.47)P for trend 0.12 0.23 0.09 0.10 0.70 0.75Heterogeneityb 0.39

NOTE: The number of participants does not sum to total women because of missing data on genotype.*Unconditional logistic regression adjusted for the matching variables: age and race (Caucasian or unknown).cUnconditional logistic regression adjusted for the matching variables, constitutional susceptibility score, family history of skin cancer, the number of lifetime severesunburns which blistered (0, 1-5, 6-11, or >11), sunlamp use or tanning salon attendance (yes/no), cumulative sun exposure while wearing a bathing suit, andgeographic region.bLikelihood ratio test to evaluate heterogeneity in the effects of the XPD genotypes on different types of skin cancer in polytomous logistic regression models adjustedfor variables in the second multivariate model.




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reported substantial conservation of haplotypes among eth-nicities with the fewest population-specific common haplo-types in Caucasians. In addition, using the BEST algorithm to

tag the haplotypes of 105 genes, Sebastiani et al. showed thatan average of 95% (and in most cases 100%) of the htSNPs in

the European American sample is a subset of the htSNPs of theAfrican American sample (21). These suggest that use of thedata from the Environmental Genome Project to infer

haplotype in our study of mainly Caucasian individuals isappropriate to define common Caucasian haplotypes. We

Table 3. Interaction between XPD polymorphisms and risk factors on melanoma risk

Cases/controls OR (95% CI) Cases/controls OR (95% CI) Cases/controls OR (95% CI) P for trend

Lys751Gln

Lys/Lys Lys/Gln Gln/Gln

Lifetime severe sunburns*Never 9/84 1.00 12/105 1.13 (0.44-2.94) 6/33 1.73 (0.54-5.57) 0.461-4 16/75 1.67 (0.67-4.16) 22/115 1.54 (0.65-3.65) 6/28 2.03 (0.63-6.53) 0.86z

5 37/63 3.26 (1.39-7.63) 42/104 2.23 (0.98-5.06) 7/36 1.07 (0.35-3.27) 0.02P for interaction = 0.03

Cumulative sun exposure with a bathing suitc

Low 10/88 1.00 20/97 1.82 (0.78-4.24) 5/35 1.53 (0.47-5.00) 0.24Intermediate 17/74 1.92 (0.80-4.59) 18/110 1.43 (0.61-3.39) 6/36 1.42 (0.46-4.38) 0.41High 39/66 4.84 (2.16-10.82) 42/118 3.12 (1.43-6.82) 8/31 2.13 (0.73-6.23) 0.06

P for interaction = 0.04

Constitutional susceptibility scoreb

Low 25/139 1.00 23/217 0.50 (0.27-0.94) 3/67 0.23 (0.07-0.81) 0.008High 56/156 1.82 (1.04-3.20) 76/198 2.03 (1.19-3.46) 20/67 1.69 (0.84-3.39) 0.92


Asp312Asn

Asp/Asp Asp/Asn Asn/Asn

Lifetime severe sunburn*Never 9/96 1.00 12/100 1.40 (0.54-3.63) 6/26 2.59 (0.78-8.59) 0.151-4 20/89 2.11 (0.88-5.07) 21/98 1.97 (0.83-4.69) 5/31 1.52 (0.45-5.16) 0.63z5 38/77 3.17 (1.37-7.31) 47/92 3.17 (1.41-7.15) 4/32 0.84 (0.23-3.09) 0.07






Low 22/171 1.00 24/187 0.83 (0.44-1.57) 2/68 0.21 (0.05-0.91) 0.06High 66/171 2.82 (1.61-4.95) 75/186 3.09 (1.78-5.36) 17/53 2.64 (1.26-5.57) 0.88


Lys751

Gln + Asp312

Asn

312Asp/Asp + 751Lys/Lys Others 312Asn/Asn + 751Gln/Gln

Lifetime severe sunburns*Never 5/69 1.00 15/128 1.83 (0.61-5.46) 5/20 3.51 (0.85-14.57) 0.131-4 13/61 2.72 (0.88-8.42) 27/134 2.51 (0.89-7.04) 4/17 3.34 (0.76-14.76) 0.89z5 31/52 5.25 (1.82-15.14) 50/117 3.65 (1.34-9.99) 3/23 1.25 (0.26-6.00) 0.03






Low 17/115 1.00 29/249 0.68 (0.35-1.31) 2/47 0.26 (0.06-1.20) 0.08High 49/124 2.46 (1.29-4.70) 86/238 2.30 (1.27-4.18) 13/41 2.31 (0.99-5.41) 0.88


NOTE: The number of participants does not sum to total women because of missing data on genotype.*Unconditional logistic regression adjusted for the matching variables, constitutional susceptibility score, family history of skin cancer, sunlamp use or tanning salonattendance (yes/no), cumulative sun exposure with a bathing suit (tertile), and geographic region.cUnconditional logistic regression adjusted for the matching variables, constitutional susceptibility score, family history of skin cancer, the number of lifetime severesunburns which blistered (0, 1-5, 6-11, >11), sunlamp use or tanning salon attendance (yes/no), and geographic region.bUnconditional logistic regression adjusted for the matching variables, family history of skin cancer, the number of lifetime severe sunburns which blistered (0, 1-5, 6-11, >11), sunlamp use or tanning salon attendance (yes/no), cumulative sun exposure with a bathing suit (tertile), and geographic region.




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observed that three different common haplotypes were lessfrequent in the cases of melanoma, SCC, and BCC risk thancontrols, respectively. Block-specific haplotype analysis isunder investigation to better understand the associations of

the polymorphisms in the regulatory regions of the XPD gene.In summary, this is the first report of interactions betweentwo XPD nonsynonymous polymorphisms (Asp312Asn andLys751Gln) and sun exposure on skin cancer risk. We observedan inverse association of the XPD 751Gln allele on melanomaand SCC risk and effect modification of the 751Gln allele on sunexposure related factors for melanoma risk along with thesimilar findings for the Asp312Asn. Additional functional dataon XPD polymorphisms are warranted to elucidate the role ofthese variants in the development of skin cancer.

AcknowledgmentsWe thank Dr. Hardeep Ranu and Craig Labadie for their laboratoryassistance, Rong Chen for her programming support, Dr. David Coxfor his program to calculate linkage disequilibrium statistics, and theparticipants in the Nurses Health Study for their dedication andcommitment.

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Table 4. Frequencies of inferred XPD haplotypes in cases and controls

SNP Allele frequency

5 22 40 41 76 Melanomacases (%),n = 348

P SCCcases (%),n = 458

P BCCcases (%),n = 460

P Commoncontrols (%),n = 1,384

1 1 0 0 0 0 9.2 0.02 16.5 0.13 13.9 0.80 13.42 0 0 0 0 0 9.5 0.82 10.5 0.37 7.1 0.18 9.03 0 0 1 0 0 10.0 0.15 6.5 0.52 8.9 0.32 7.4

4 0 1 0 0 0 8.3 0.11 6.9 0.01 10.7 0.84 11.15 1 0 1 1 0 8.6 0.44 6.5 0.58 8.9 0.29 7.36 1 0 1 0 0 21.9 0.09 18.1 0.85 18.2 0.80 17.77 0 1 1 0 1 5.2 0.88 5.5 0.94 5.9 0.67 5.48 0 1 1 0 0 6.0 0.83 7.1 0.30 5.5 0.93 5.79 1 1 0 1 0 5.6 0.64 6.8 0.69 3.4 0.01 6.3

NOTE: Haplotype frequencies from the observed genotypes were estimated using partition-ligation expectation-maximization. The Ps for differences in haplotypefrequencies between cases and controls were determined by the two-sample proportion test incorporating SE from partition-ligation expectation-maximization output.0, common allele; 1, rare allele.



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