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A variant associated with nicotine dependence, lung cancer and peripheral arterial disease
Author: Thorgeir E. Thorgeirsson
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"LETTERS A variant associated with nicotine dependence, lung cancer and peripheral arterial disease Thorgeir E. Thorgeirsson 1 *, Frank Geller 1 *, Patrick Sulem 1 *, Thorunn Rafnar 1 *, Anna Wiste 1,2 , Kristinn P. Magnusson 1 , Andrei Manolescu 1 , Gudmar Thorleifsson 1 , Hreinn Stefansson 1 , Andres Ingason 1 , Simon N. Stacey 1 , Jon T. Bergthorsson 1 , Steinunn Thorlacius 1 , Julius Gudmundsson 1 , Thorlakur Jonsson 1 , Margret Jakobsdottir 1 , Jona Saemundsdottir 1 , Olof Olafsdottir 1 , Larus J. Gudmundsson 1 , Gyda Bjornsdottir 1 , Kristleifur Kristjansson 1 , Halla Skuladottir 3 , Helgi J. Isaksson 4 , Tomas Gudbjartsson 5 , Gregory T. Jones 8 , ThomasMueller 9 ,AndersGottsa�ter 10 ,AndreaFlex 11 ,KatjaK.H.Aben 12,13 ,FemmiedeVegt 12 ,PeterF.A.Mulders 14 , Dolores Isla 15 , Maria J. Vidal 15 , Laura Asin 16 , Berta Saez 17 , Laura Murillo 18 , Thorsteinn Blondal 19 , Halldor Kolbeinsson 6 , Jon G. Stefansson 6 , Ingunn Hansdottir 20 , Valgerdur Runarsdottir 20 , Roberto Pola 11,21 , Bengt Lindblad 10 , Andre M. van Rij 8 , Benjamin Dieplinger 9 , Meinhard Haltmayer 9 , Jose I. Mayordomo 15,16,17 , Lambertus A. Kiemeney 12,13,14 , Stefan E. Matthiasson 22 , Hogni Oskarsson 23 , Thorarinn Tyrfingsson 20 , Daniel F. Gudbjartsson 1 , Jeffrey R. Gulcher 1 , Steinn Jonsson 7 , Unnur Thorsteinsdottir 1,22 , Augustine Kong 1 & Kari Stefansson 1,22 Smoking is a leading cause of preventable death, causing about 5 million premature deaths worldwide each year 1,2 . Evidence for genetic influence on smoking behaviour and nicotine dependence (ND) 3?8 has prompted a search for susceptibility genes. Further- more, assessing the impact of sequence variants on smoking- related diseases is important to public health 9,10 . Smoking is the major risk factor for lung cancer (LC) 11?14 and is one of the main risk factors for peripheral arterial disease (PAD) 15?17 . Here we identify a common variant in the nicotinic acetylcholine receptor gene cluster on chromosome 15q24 with an effect on smoking quantity, ND and the risk of two smoking-related diseases in populations of European descent. The variant has an effect on thenumberofcigarettessmokedperdayinoursampleofsmokers. The same variant was associated with ND in a previous genome- wide association study that used low-quantity smokers as controls 18,19 , and with a similar approach we observe a highly sig- nificant association with ND. A comparison of cases of LC and PAD with population controls each showed that the variant con- fers risk of LC and PAD. The findings provide a case study of a gene?environment interaction 20 , highlighting the role of nicotine addiction in the pathology of other serious diseases. To perform a genome-wide association (GWA) study of smoking quantity (SQ), we used questionnaire data limited to basic questions on smoking behaviour that were available for a large number of lifetimesmokers.TheGWAscancomprises10,995Icelandicsmokers whohadbeenassayedwithInfiniumHumanHap300SNPchipsfrom Illumina. A set of 306,207 single-nucleotide polymorphisms (SNPs) fulfilling our quality criteria was tested. We focused on cigarette smoking, with SQ reported as cigarettes per day. All SQ data were clustered into categories (see Supplementary Information) and we refer to them as ?SQ levels?. The SQ levels were 0 (1?10 cigarettes per day), 1 (11?20), 2 (21?30) and 3 (31 or more). Each increment represents an increase in SQ of 10 cigarettes per day. Allele T of the SNP rs1051730 was most strongly associated with SQ, and the association washighlysignificant (P55310 216 ).TheSNPiswithin the CHRNA3 gene in a linkage disequilibrium block also containing two other genes,CHRNA5and CHRNB4, that encode nicotinic acetyl- choline receptors (ref. 18). Six other SNPs on chromosome 15q24 passed the threshold of genome-wide significance (P,2310 27 ), but they are all correlated with rs1051730 (r 2 50.14?0.93). After correction for rs1051730, none of these six SNPs showed a P value below 10 23 (see Supplementary Table 1). A quantile?quantile plot for the GWA scan (see Supplementary Fig. 1a) shows the observed excess of signals, whereas a quantile?quantile plot after removing 182 markers located within 1 megabase of rs1051730 is consistent with noise (seeSupplementaryFig. 1b), illustratingthat all ofthestrongest signals standing out in the first plot are located on chromosome 15q24. An additional 2,950 smokers from Iceland were genotyped for rs1051730, giving a total of 13,945 smokers(Table 1) with a mean variant frequency of 34.7%, which is not significantly different from thefrequency of34.4% observedin4,203individualswhoweregeno- typed and who reported never having smoked (odds ratio (OR) 1.01, 95% confidence interval (CI) 0.96?1.07, P50.60). Indeed, the fre- quency of the variant in the 3,627 low-quantity smokers (10 or fewer cigarettes per day) is significantly less than that in those who do not smoke (OR 0.83, 95% CI 0.78?0.90, P54.5310 27 ). The increase in *These authors contributed equally to this work. 1 deCODE Genetics, 101 Reykjavik, Iceland. 2 Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA 30322, USA. 3 Department of Medical Oncology, 4 Department of Pathology, 5 Department of Cardiothoracic Surgery, 6 Department of General Adult Psychiatry, and 7 Department of Medicine, Landspitali University Hospital, 101 Reykjavik, Iceland. 8 Department of Surgery, Dunedin School of Medicine, University of Otago, Dunedin 9054, New Zealand. 9 Department of Laboratory Medicine, Konventhospital BarmherzigeBrueder,4020Linz,Austria. 10 CenterforVascularDiseases,UniversityHospital,MAS,20502Malmo�,Sweden. 11 LaboratoryofVascularBiologyandGenetics,A.Gemelli University Hospital, Universita Cattolica del Sacro Cuore School of Medicine, 00168 Rome, Italy. 12 Department of Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands. 13 Comprehensive Cancer Center East, 6501 BG Nijmegen, The Netherlands. 14 Department of Urology, Radboud University Nijmegen Medical Centre, 6525 GA Nijmegen, The Netherlands. 15 Division of Medical Oncology, University Hospital, 5009 Zaragoza, Spain. 16 Nanotechnology Institute of Aragon, 5009 Zaragoza, Spain. 17 Health Science Institute, 5009 Zaragoza, Spain. 18 Division of Medical Oncology, Hospital Reina Sofia, 31500 Tudela, Spain. 19 Department of Lung Disease, Primary Health Care Centre, 101 Reykjavik, Iceland. 20 Vogur SAA Addiction Treatment Center, 112 Reykjavik, Iceland. 21 OASI Institute for Research and Care (IRCCS), 94018 Troina (EN), Italy. 22 School of Medicine, University of Iceland, 101 Reykjavik, Iceland. 23 Therapeia, 101 Reykjavik, Iceland. Vol 452|3 April 2008|doi:10.1038/nature06846 638 Nature Publishing Group�2008 frequency between levels varies, and the largest increase (4.5%) is observed between the lowest levels (0 and 1), whereas the increase between the highest levels (2 and 3) is only 1.1%. In the context of a case-control LC study, an additional 523 smokers from Spain and 1,375smokersfromTheNetherlandswere genotyped.Weperformed multiple regression analyses of SQ data from the three countries, with adjustment for sex and year of birth (Table 2). Results from Spain and The Netherlands combined gave an estimated increase of 0.074 SQ units (P50.012) for each copy of the variant, which is not significantly different (P50.45) from the estimate of 0.098 SQ units (P510 218 ) based on the Icelandic data. Combining all results, each copy of the variant was estimated to increase SQ level by 0.095 units (P56310 220 ), which corresponds to about one cigarette perday. A recent GWA study reported an association between SQ and SNP rs6495308 (P56.9310 25 , correlation between this SNP and rs1051730, r 2 50.18 in the HapMap project) for about 7,500 indivi- duals from two study groups, as well as association between SQ and rs1317286 (P52.6310 26 , r 2 50.90 to rs1051730 in the HapMap project) in a candidate gene study based on 1,740 heavy smokers (more than 25 cigarettes per day) and 6,200 low-quantity smokers (fewer than 5 cigarettes per day) 21 . Sex and year of birth are also strongly associated with SQ (Table 2). However, neither the interaction between variant and sex nor that between variant and year of birth is significant, indicating that the effect of the variant is similar for both sexes and is robust to population-wide changes in smoking habits over time. The phenotypic variance explained by the variant was highest in Iceland, amounting to 0.7%. Association of the same variant with ND was previously reported in a candidate gene study involving 3,713 SNPs 18 . We assessed the association with ND, defined as a score of 4 or higher on the FagerstromTestforNicotineDependence(FTND) 22 orendorsement of at least three of the seven Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) criteria (see Supplemen- tary Information). The variant is associated with ND in Iceland in a subsetof2,394smokersfromtheSQstudytestedagainstboth28,455 population controls (OR 1.17, 95% CI 1.10?1.25, P53.3310 26 ) and 3,506 low-quantity smokers (OR 1.40, 95% CI 1.29?1.52, P57310 215 ). The latter OR of 1.40 is comparable to the results ofthecandidategenestudy 18 .Theyusednon-NDsmokersascontrols (that is, individuals who had smoked but had an FTND score of 0), and reported association with rs1051730 (OR 1.3, P510 23 ). rs1051730 is in strong linkage disequilibrium with rs16969968 (r 2 50.90 in the HapMap project), which was highlighted in the previous study with a similar result (OR 1.3, P56310 24 ) 18 . Dependence on nicotine drives repeated self-administration of nic- otine 23?25 andhighSQisastrongsignofNDandoneofthecriteriafor a diagnosis of ND (SQ is included in the FTND scale). This, together withthefactthattheNDsubjectsarepartoftheSQstudy,meansthat the associations with ND and SQ cannot be considered independent results. Both the FTND and the DSM-IV scales include many items that are not based on SQ, and their total scores are measures of ND severity. In our ND group, positive scores on most items in both scales show a trend towards higher frequency of the variant, as does the total score on both the FTND and DSM-IV scales. Thus, the frequency of the variant increases with addiction severity, and is 46.8% and 43.8% for the highest deciles of FTND and DSM-IV, respectively (see Supplementary Table 2a, b). ND is believed to be the main reason for continued smoking. To explore the frequency of the variant in the context of the ability to quit smoking, we investi- gateddifferencesbetween6,388currentand6,687pastsmokersfrom the SQ analysis by a logistic regression model adjusting for sex and year of birth. The variant was associated with current smoking with an OR of 1.07 (95% CI 1.01?1.13, P50.015) (see Supplementary Table 3), and the effect is similar when corrected for SQ (OR 1.06, 95% CI 1.00?1.12, P50.036), indicating that carriers of the variant are less likely to quit smoking. Smokingisamajorriskfactorformanydiseases,andwedecidedto study the effect of the variant on LC and PAD risk directly. The LC study was based on 1,024 cases and 32,244 controls from Iceland, Spain and The Netherlands (Table 3); the PAD study was based on 2,738 cases and 29,964 controls from five caucasian populations (Iceland, New Zealand, Austria, Sweden and Italy) (Table 3). The results for LC and PAD (Table 4) represent the overall effect on LC and PAD including indirect effects through SQ and ND. Significant association was observed with LC for both the Icelandic data (OR 1.27, P54.1 310 25 ) and the data for Spain and The Netherlands combined (OR 1.39, P56.6310 25 ). These two estimates are not significantly different from each other (P50.34), and combining results from all three groups gave an OR of 1.31 (95% CI 1.19? 1.44, P51.5310 28 ). There is no significant difference in frequency of the variant between histological types of LC, which is not surpris- ing given the small number of cases per group (see Supplementary Table 4). Association with PAD was found both in the Icelandic data (OR1.18,P55.3 310 25 )andinthedatafortheforeignpopulations combined (OR 1.23, P55.9310 24 ). These two estimates are not significantly different from each other (P50.57), and combining results from all five groups gave an OR of 1.19 (95% CI 1.12?1.27, P51.4310 27 ). Table 1 | Genotype status and SQ level of 13,945 Icelandic smokers Parameter Genotype of rs1051730 Total n (frequency) Frequency of T allele GG GT TT Cigarettes per day (SQ level) 1 to 10 (0) 1,743 1,558 326 3,627 (0.260) 0.305 11 to 20 (1) 2,727 2,865 810 6,402 (0.459) 0.350 21 to 30 (2) 1,145 1,416 427 2,988 (0.214) 0.380 31 and more (3) 341 448 139 928 (0.067) 0.391 All levels (frequency) 5,956 (0.427) 6,287 (0.451) 1,702 (0.122) 13,945 (1.000) 0.347 Mean SQ level (mean6s.d.) 1.0160.85 1.1260.86 1.2260.85 1.0960.86 Table 2 | Multiple regression of SQ level as a function of rs1051730 genotype, sex and year of birth Copies of T allele Sex (male) P for year of birth (categorical) P for interactions: allele3sex, allele3age Study group n Estimate (95%CI) P Estimate (95%CI) P Iceland 13,945 0.098 (0.076?0.120) 10 218 0.411 (0.383?0.438) ,10 216 ,10 216 0.53, 0.85 Spain 523 0.061 (20.059?0.180) 0.32 0.504 (0.290?0.718) ,10 25 0.006 0.80, 0.76 The Netherlands 1,375 0.078 (0.012?0.145) 0.021 0.326 (0.225?0.427) ,10 29 ,10 24 0.68, 0.27 Foreign combined 1,898 0.074 (0.016?0.132) 0.012 NA ?? ? All combined 15,771 0.095 (0.075?0.115) 6310 220 NA MultipleregressionofSQlevelonalleleT,sexandyearofbirth,givingadjustedvaluesforeachexplanatoryvariableadjustingfortheothers.Forthetestsofinteraction,theinteractiontermsinvolving the variant were individually added to the initial model. NA, not available. NATURE|Vol 452|3 April 2008 LETTERS 639 Nature Publishing Group�2008 Genotypic ORs for LC, PAD and ND did not deviate significantly fromthoseobtainedforthemultiplicativemodel(seeSupplementary Table5),andnosignificantdifferencesintheORsbetweensexeswere observed (see Supplementary Table 6). According to our estimates for Icelandic LC patients, the correla- tionbetweenSQandLCisconsistentwithnumbersreportedinother studies 26,27 . Combining these estimates with our estimate of the asso- ciation of the variant with SQ, the expected OR between the variant and LC is only about 1.05 in Iceland (see Supplementary Information), which is well below the direct OR estimate for LC of 1.27 (95% CI 1.13?1.43). A similar indirect estimate for PAD is 1.04, whichagainissubstantiallylowerthantheobserveddirectestimateof 1.18 (95% CI 1.09?1.27). It is not surprising that the ORs for LC and PAD cannot be explained by the effect of the variant on SQ alone, because the involvement of both SQ and the duration of smoking in LC and PAD was established in previous studies 15,28 . The SQ data for mostindividualswerederivedfromasinglepointintimeandcannot be expected to cover all aspects of smoking behaviour affected by the variant and relevant to LC and PAD. An effect on other aspects of smoking behaviour, in particular smoking duration, is likely to account for the observed difference between the indirect and direct estimates of the LC and PAD risks. An alternative possibility is that the variant directly confers risk of LC and PAD, for example by increasing the vulnerability to tobacco smoke. Thus, we have unequivocally demonstrated a correlation between, on the one hand, a sequence variant in the cluster of genes on chro- mosome 15that encode nicotinic acetylcholine receptors and,onthe other,SQandND.Thevariantdoesnotinfluencesmokinginitiation; however, among smokers, carriers of the variant smoke more than non-carriers and have higher rates of ND. This variant was reported in a previous study of 1,050 ND cases and 879 controls who smoked and had an FTND score of 0 (refs 18, 19) and the authors concluded thatthevariantcontributestoND(ref.18).Thisconclusionisputon firm ground by the highly significant OR of 1.40 (P57310 215 ) for NDcomparedwithlow-quantitysmokers(tenorfewercigarettesper day). The direct measurement of the risk of LC and PAD revealed genome-wide significant associations with allelic ORs of 1.31 and 1.19, respectively. This demonstrates that a sequence variant assoc- iatedwithND,abraindisorder,confersriskoflungandcardiovascu- lar diseases through an effect on behaviour, which is an example of activegene?environmentcorrelation 20 inthepathogenesisofdisease. A calculation of the population attributable risk for the variant gives 18% for LC and 10% for PAD. Although these population attri- butablerisksareatbestapproximatefiguresgiventhecomplexinter- playbetweenthevariant,smoking,andsmoking-relateddiseases,itis likely that the variant accounts for a substantial fraction of PAD and LC cases and the associated morbidity and mortality. Theresults of the study described here showthat it is important to keep in mind, while attempting to shed light on the role of nature versus nurture in the pathogenesis of common or complex disease, that variants in the sequence of our genome influence not only how we respond to our environment but also our tendency to seek or avoidenvironment. Thelinebetweennatureandnurture istherefore sometimes conspicuously absent. METHODS SUMMARY Subjects. Written informed consent was obtained from all subjects in the seven participating populations (Iceland, Spain, The Netherlands, Sweden, Italy, Austria and New Zealand; see Table 3). Inclusion in the study required the availability of genotypes from either GWA studies performed at deCODE Table 3 | Demographics, age and phenotype breakdown Study group Cases Controls n Male/female Age (years) n Male/female Age (years) Smoking phenotypes Cigarettes per day available Iceland 13,945 6,134/7,811 58.7617.8 Spain 523 354/169 54.0616.3 The Netherlands 1,375 762/613 61.5610.2 ND (Iceland) 2,394 800/1,594 48.1611.028,455 12,600/15,855 58.7621.8 Never smokers (Iceland) 4,203 1,273/2,930 55.4621.6 LC Iceland 665 346/319 69.7611.128,752 12,174/16,578 56.8621.5 Spain 269 238/31 64.9611.6 The Netherlands 90 71/19 68.569.5 PAD Iceland 1,503 926/577 74.2610.628,752 12,174/16,578 56.8621.5 New Zealand 441 251/189 70.669.6 435 248/187 68.266.4 Austria 457 322/135 68.4611.0 403 284/119 67.3610.7 Sweden 172 92/80 77.569.9 140 64/76 67.961.5 Italy 165 111/54 73.069.3 234 162/72 72.666.4 Ages are shown as means6s.d. Table 4 | Association of rs1051730 allele T with LC and PAD Controls Cases Study group n Frequency n Frequency OR (95%CI) P LC Iceland 28,752 0.342 665 0.398 1.27 (1.13?1.43) 4.1310 25 Spain 1,474 0.390 269 0.483 1.46 (1.22?1.76) 5.4310 25 The Netherlands 2,018 0.314 90 0.350 1.18 (0.86?1.61) 0.31 Foreign combined 3,492 ? 359 ? 1.38 (1.18?1.62) 6.6310 25 All combined 32,244 ? 1,024 ? 1.31 (1.19?1.44) 1.5310 28 PAD Iceland 28,752 0.342 1,503 0.379 1.18 (1.09?1.27) 5.3310 25 New Zealand 435 0.274 441 0.337 1.35 (1.10?1.65) 0.0041 Austria 403 0.352 457 0.395 1.20 (0.99?1.46) 0.068 Sweden 140 0.304 172 0.331 1.14 (0.81?1.60) 0.46 Italy 234 0.378 165 0.412 1.15 (0.86?1.54) 0.33 Foreign combined 1,212 ? 1,235 ? 1.23 (1.09?1.39) 5.9310 24 All combined 29,964 ? 2,738 ? 1.19 (1.12?1.27) 1.4310 27 LETTERS NATURE|Vol 452|3 April 2008 640 Nature Publishing Group�2008 geneticsorfollow-upgenotypingofrs1051730inadditionalsubjects.Allsubjects were of European descent. For details on recruitment periods and diagnostic criteria used for the ND, LC and PAD groups, see Methods and Supplementary Information. Association analysis. For the genome-wide study of SQ the significance thres- hold was set at 2 310 27 , which is about 0.05 divided by 306,207, the number of SNPs passing quality control. Regressing SQ level as a quantitative variable on the number of copies of the allele carried (0/1/2), a likelihood ratio x 2 statistic was used for testing. Evaluation of statistical significance took the relatedness of the individuals into account by dividing the x 2 statistic by a correction factor based either on the method of genomic control 29 or on a simulation procedure using the known genealogy that we had previously employed 30 (see Supple- mentary Information). The variant did not correlate with sex or year of birth in the controls; the association analyses were therefore not adjusted for these factors. 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Grant,S.F.etal.Variantoftranscriptionfactor7-like2(TCF7L2)geneconfersrisk of type 2 diabetes. Nature Genet. 38, 320?323 (2006). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank the participants in the genetic studies whose contributionsmadethisworkpossible,thestaffatNoatun(deCODE?srecruitment centre),andpersonnelatdeCODE?scorefacilities.Thisworkwassupportedinpart byfundsfromtheNationalInstituteofDrugAbuseandtheEuropeanCommission. Author Contributions T.E.T., F.G., P.S., and T.R. contributed equally to this work. T.E.T., F.G., P.S, T.R., A.W., D.F.G., A.K. and K.S. wrote the first draft of the paper. Ha.S., H.J.I., T.G. and S.J. recruited and diagnosed the Icelandic lung cancer patients. S.E.M. recruited and diagnosed the Icelandic peripheral arterial disease patients. T.B., H.K., J.G.S., I.H, V.R., H.O., T.T. and S.J. recruited and diagnosed nicotine addiction subjects. K.K.H.A., F.d.V., P.F.A.M. and L.A.K. recruited and diagnosedthesubjectsfromTheNetherlands.D.I.,M.J.V.,L.A.,B.S.,L.M.andJ.I.M. recruited and diagnosed the Spanish subjects. G.T.J. and A.M.v.R. recruited and diagnosed the subjects from New Zealand. T.M., B.P. and M.H. recruited and diagnosed subjects from Austria. A.G. and B.L. recruited and diagnosed subjects from Sweden. A.F. and R.P. recruited and diagnosed subjects from Italy. A.W., A.I., S.N.S., J.T.B., S.T., J.G., M.J., J.S., O.O. and S.N.S. performed genotyping and experimental work. L.J.G., G.B. and K.K. incorporated phenotypic data into a databaseandanalysedit.T.E.T.,F.G.,P.S.,T.R.,A.W.,K.P.M.,A.M.,G.T.,D.F.G.and A.K. analysed the data. T.E.T., F.G., P.S., T.R., K.P.M., Hr.S., T.J., J.I.M., L.K., H.O., T.T., J.R.G., S.J., D.G., U.T., A.K. and K.S. planned, supervised and coordinated the work. All authors contributed to the final version of the paper. Author Information The authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/nature. Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to T.E.T. (thorgeir@decode.is) or K.S. (kari.stefansson@decode.is). NATURE|Vol 452|3 April 2008 LETTERS 641 Nature Publishing Group�2008 METHODS Icelandic subjects. For all studies involving Icelandic subjects, the study proto- cols were approved by the National Bioethics Committee (NBC) and the Data Protection Authority (DPA) of Iceland. The DPA encrypted all personal identi- fiers associated with information or blood samples with the use of a third-party encryption system 31 . In all, the Icelandic study involves 10,995 subjects with information on SQ available in the GWA, an additional 2,950 subjects with information on SQ, and 4,203 never-smokers. In the studies of LC and PAD, 665 and 1,503 patients, respectively, and 28,752 population controls were used (for details see Table 3). Smoking. All Icelandic subjects in the study of smoking-related phenotypes, including Icelandic population controls, were originally recruited for different genetic studies conducted over 11 years (1996?2007) at deCODE Genetics, and information on the number of cigarettes smoked per day was available from questionnaires. The information on cigarettes smoked per day was categorized into SQ levels and used as a quantitative variable. Detailed information on SQ was also available for the foreignLC populations(Supplementary Information), but not for the foreign PAD populations. Nicotinedependence.ForasubsetoftheIcelandicsmokers,informationonthe criteria used to diagnose ND was available from ongoing studies of ND and anxiety/depression 32 .Weexcludedindividualswithdiagnosesofothersubstance dependence or abuse, giving a total of 2,394 ND subjects. A score of 4 or higher on the FTND 22 , or endorsement of three or more DSM criteria, were used to assign affected status for ND. Additional information on the Icelandic smoking and ND study group is available in the Supplementary Information. Lung cancer. Iceland: recruitment was initiated in the year 1998 with a nation- widelistfromtheIcelandicCancerRegistry(ICR).About1,265LCpatientswere alive during the period of recruitment, and 665 participated in the project. Information in the ICR includes year and age at diagnosis, year of death, SNOMED (Systematized Nomenclature of Medicine) code and ICD-10 (International Statistical Classification of Diseases and Related Health Problems, 10th revision) classification. Histological and cytological verification was available for 647 cases; the remaining 18 cases were diagnosed clinically. The Netherlands: the 90 patients and 2,018 controls were identified retro- spectively through three different ongoing studies on genetic risk factors of disease. All three study protocols were approved by the Institutional Review Board of the Radboud University Nijmegen Medical Centre. Spain: patients were recruited from the Oncology Department of Zaragoza Hospital, from June 2006 to June 2007, and of 330 patients who were invited to participate, 292 enrolled (88%). Clinical information including age at onset and histology were collected from medical records. The 1,474 control individuals were approached at Zaragoza University Hospital. Study protocols were approved by the Institutional Review Board of Zaragoza University Hospital. PAD. Iceland: patients have been recruited over the past nine years, as part of a genetic study at deCODE, from a registry of individuals diagnosed with PAD at the major hospital in Reykjavik, the Landspitali University Hospital, during the years 1983?2006. Diagnosis was confirmed by vascular imaging or segmental pressure measurements. Austria: patients and controls were recruited through the Linz Peripheral Arterial Disease (LIPAD) study during 2000 to 2002, at the Department of Surgery, St John of God Hospital. Of the patients admitted for evaluation of suspected or definite PAD, all patients with chronic atherosclerotic occlusive disease of the lower extremities associated with typical symptoms?such as claudication or leg pain on exertion, rest pain, or minor or major tissue loss? were includedon thebasis of the final clinicaldiagnosisestablishedby attending vascular surgeons. The diagnosis was verified by interview, physical examina- tion, noninvasive techniques, and angiography 33 . All control subjects were patients at the same hospital and fulfilled the following criteria: no clinical indication of PAD by history and physical examination, and systolic brachial bloodpressureequaltoorlessthanthebloodpressureineachoftherightandleft anterior tibial and posterior tibial arteries (that is, ankle brachial index$1.0) 33 . Smoking status was assessed as described in ref. 34. Sweden: patients and controls were recruited at the Department of Vascular Diseases at Malmo� University Hospital, a single referral centre for all patients with critical limb ischaemia in the three southernmost health-care districts in Sweden (723,750 inhabitants in 2001). The diagnosis of critical limb ischaemia was made in accordance with Trans-Atlantic Inter-Society Consensus scientific criteria 35 of ulceration, gangrene, or rest pain caused by PAD proved by ankle pressure (less than 50 to 70mmHg), reduced toe pressure (less than 30 to 50mmHg)orreducedtranscutaneousoxygentension.Diagnosiswasconfirmed by an experienced vascular surgery consultant. The control group consisted of healthy individuals without symptomatic PAD included in a health screening programme for a preventive medicine project 36 . Italy: patients and controls were recruited from subjects admitted to the Department of Medicine of the A. Gemelli University hospital of Rome, from 2000 to 2001. Inclusion criteria for the PAD group were Caucasian origin and presenceofPAD,diagnosedinaccordancewithestablishedcriteria 37 .Allpatients had an ankle/arm pressure index lower than 0.8 and were at Fontaine?s stage II, with intermittent claudication and no rest pain or trophic lesions. Inclusion criteria for the control group were caucasian origin, absence of PAD and CAD and no relationship to cases. Additional exclusion criteria from the study were tumours, chronic inflammatory diseases, and autoimmune diseases 38 . New Zealand: patients were recruited from the Otago?Southland region, and PAD was confirmed by an ankle brachial index of less than 0.7, pulse volume recordingsandangiography/ultrasoundimaging.Thecontrolgroupconsistedof elderly individuals with no history of vascular disease from the same geograph- icalregion.ControlswereasymptomaticforPADandhadanklebrachialindexes of more than 1. An abdominal ultrasound scan excluded concurrent abdominal aortic aneurysm from both the PAD and control groups, and Anglo-European ancestry was required for inclusion. Genotyping. All 10,995 samples in the GWA study of SQ were genotyped with genotyping systems and specialized software (Human Hap300 and Human Hap300-duo1 Bead Arrays; Illumina) 39 . rs1051730 was genotyped with a Centaurus assay (Nanogen) for 8,566 Icelandic samples and all samples in the foreignstudygroups.Informationonthegenotypingandqualitycontrolisgiven in the Supplementary Information. 31. Gulcher, J. R., Kristjansson, K., Gudbjartsson, H. & Stefansson, K. Protection of privacybythird-partyencryptioningeneticresearchinIceland.Eur.J.Hum.Genet. 8, 739?742 (2000). 32. Thorgeirsson, T. E. et al. Anxiety with panic disorder linked to chromosome 9q in Iceland. Am. J. Hum. Genet. 72, 1221?1230 (2003). 33. Mueller, T. et al. Factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations are not associated with chroniclimbischemia:theLinzPeripheralArterialDisease(LIPAD)study.J.Vasc. Surg. 41, 808?815 (2005). 34. Rutherford, R. B. et al. Recommended standards for reports dealing with lower extremity ischemia: revised version. J. Vasc. Surg. 26, 517?538 (1997). 35. Dormandy, J. A. & Rutherford, R. B. Management of peripheral arterial disease (PAD). TASC Working Group. TransAtlantic Inter-Society Consensus (TASC). J. Vasc. Surg. 31, S1?S296 (2000). 36. Barani, J., Nilsson, J. A., Mattiasson, I., Lindblad, B. & Gottsater, A. Inflammatory mediators are associated with 1-year mortality in critical limb ischemia. J. Vasc. Surg. 42, 75?80 (2005). 37. Anonymous.. Suggested standards for reports dealing with lower extremity ischemia.PreparedbytheAdHocCommitteeonReportingStandards,Societyfor Vascular Surgery/North American Chapter, International Society for Cardiovascular Surgery. J. Vasc. Surg. 4, 80?94 (1986). 38. Flex, A. et al. The2174 G/C polymorphism of the interleukin-6 gene promoter is associated with peripheral artery occlusive disease. Eur. J. Vasc. Endovasc. Surg. 24, 264?268 (2002). 39. Barrett, J. C. & Cardon, L. R. Evaluating coverage of genome-wide association studies. Nature Genet. 38, 659?662 (2006). doi:10.1038/nature06846 Nature Publishing Group�2008 "
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