Polymorphisms in the and genes of the SUMO-conjugating system and breast cancer risk

Transcription

1 Polymorphisms in the and genes of the SUMO-onjugating system and breast aner risk Thomas Dünnebier, Justo Lorenzo Bermejo, Susanne Haas, Hans-Peter Fisher, Christiane B. Pierl, Christina Justenhoven, Hiltrud Brauh, Christian Baish, Mihael Gilbert, Volker Harth, et al. To ite this version: Thomas Dünnebier, Justo Lorenzo Bermejo, Susanne Haas, Hans-Peter Fisher, Christiane B. Pierl, et al.. Polymorphisms in the and genes of the SUMO-onjugating system and breast aner risk. Breast Caner Researh and Treatment, Springer Verlag, 2009, 121 (1, pp < /s y>. <hal > HAL Id: hal Submitted on 20 May 2010 HAL is a multi-disiplinary open aess arhive for the deposit and dissemination of sientifi researh douments, whether they are published or not. The douments may ome from teahing and researh institutions in Frane or abroad, or from publi or private researh enters. L arhive ouverte pluridisiplinaire HAL, est destinée au dépôt et à la diffusion de douments sientifiques de niveau reherhe, publiés ou non, émanant des établissements d enseignement et de reherhe français ou étrangers, des laboratoires publis ou privés.

2 Polymorphisms in the UBC9 and PIAS3 genes of the SUMO-onjugating system and breast aner risk Thomas Dünnebier 1, Justo Lorenzo Bermejo 2, Susanne Haas 3, Hans-Peter Fisher 3, Christiane B. Pierl 4, Christina Justenhoven 5, Hiltrud Brauh 5, Christian Baish 6, Mihael Gilbert 1, Volker Harth 4,6, Anne Spikenheuer 4, Sylvia Rabstein 4, Beate Pesh 4, Thomas Brüning 4, Yon-Dshun Ko 6 and Ute Hamann 1* 1 Moleular Genetis of Breast Caner, Deutshes Krebsforshungszentrum, Heidelberg, Germany 2 Institute of Medial Biometry and Informatis, University Hospital Heidelberg, Heidelberg, Germany 3 Institute of Pathology, Medial Faulty of the University of Bonn, Bonn, Germany 4 BGFA-Forshungsinstitut für Arbeitsmedizin der Deutshen Gesetzlihen Unfall-versiherung, Ruhr University Bohum, Bohum, Germany 5 Dr. Margarete Fisher-Bosh-Institute of Clinial Pharmaology, Stuttgart, and University of Tübingen, Germany 6 Department of Internal Mediine, Evangelishe Kliniken Bonn ggmbh, Johanniter Krankenhaus, Bonn, Germany * Address for orrespondene and ontat Ute Hamann, PhD, Professor Deutshes Krebsforshungszentrum, Moleular Genetis of Breast Caner B055 Im Neuenheimer Feld 580, Heidelberg, Germany Tel.: 0049/6221/ Fax: 0049/6221/ u.hamann@dkfz-heidelberg.de

3 Abstrat SUMOylation onsists in the ovalent onjugation of small ubiquitin-related modifiers to target proteins. SUMOylation partiipates in proesses that are tightly linked to tumorigenesis and geneti variability in the SUMO-onjugating system may influene the development of breast aner. We reently reported that variation in the UBC9 gene enoding the SUMO-onjugating enzyme may affet the grade of breast tumors. Following omprehensive in silio analyses for detetion of putative funtional polymorphisms in 14 genes of the SUMO system we seleted one oding SNP in PIAS3 and seven tag SNPs in UBC9 for assoiation analyses. Results were based on 1,021 ases and 1,015 mathed ontrols from the population-based GENICA study. Odds ratios (OR and 95% onfidene intervals (CI were estimated by onditional logisti regression. To explore the assoiation with polymorphisms losely linked to the genotyped variants, multiple imputation based on HapMap data was applied. The study revealed assoiations of four UBC9 polymorphisms with risk of grade 1 tumors. Comparison of genotype and haplotype models indiated that the best representation of risk solely relied on rs under dominant penetrane. Women arrying the rare allele showed an inreased risk of grade 1 tumors ompared with ommon homozygotes (OR 1.87, 95% CI This effet appeared to be stronger in women with a family history of breast or ovarian aner. Imputation of polymorphisms in a 300 kb region around the genotyped polymorphisms identified no variants with stronger assoiations. Our findings suggest that geneti variation in UBC9 may affet the risk of grade 1 breast tumors. Key words: UBC9 and PIAS3 polymorphisms, SUMOylation, breast aner risk, tumor grade, multiple imputation

4 Introdution SUMOylation is an essential ellular proess, whih onsists in the ovalent onjugation of small ubiquitin-related modifiers (SUMO-1, 2 and 3 to target proteins [1]. This kind of posttranslational modifiation an hange and regulate the funtion of a protein by governing protein-protein and protein-dna interations. Thereby, SUMO an hange the loalization, ativity or stability of its substrates. SUMOylation is a multistep proess involving three lasses of enzymes alled E1, E2 and E3 [2]. First, mature SUMO is ativated by a SUMO-ativating enzyme omplex omposed of the E1 proteins AOS1 and UBA2. Next, SUMO is transferred from the E1 heterodimer to the SUMO-onjugating enzyme UBC9 (UBE2I, whih represents the only E2 omponent of the SUMO system. Finally, UBC9 transfers SUMO to a lysine residue of the substrate resulting in isopeptide bond formation. This last step is failitated by E3 SUMO ligases suh as the protein inhibitors of ativated STAT (PIAS family proteins that onfer substrate speifiity [1]. Due to the existene of SUMO-deonjugating enzymes, alled sentrinspeifi proteases (SENPs, SUMOylation is a reversible and highly dynami proess. This lass of enzymes also atalyses the proteolyti maturation of the SUMO preursor. SUMO ontrols multiple events inluding transription, nuleo-ytoplasmi traffiking and mitoti hromosome segregation [3, 4]. Further, it plays a pivotal role in the maintenane of genome integrity by regulating DNA repliation, repair and reombination [5]. There is inreasing evidene that SUMO is involved in tumorigenesis. Many tumor suppressors and onoproteins, suh as PML, WRN, BLM, -JUN, -FOS, TP53 and MDM2, are targets of SUMO [6-13]. In a mouse xenograft model using breast aner ells, it was shown that inreased ativity of UBC9 promotes tumor growth and low ativity deelerates it [14]. Moreover, several studies have desribed an up-regulation of enzymes involved in SUMO onjugation or deonjugation in a variety of malignanies [14-18]. In breast aner speimens a higher expression of UBC9 [18] and PIAS3 [16] has been reported. In addition, SUMO regulates

5 the ativity of several nulear hormone reeptors inluding estrogen reeptor alpha (ER, progesterone reeptor (PR and androgen reeptor [19], whih play a entral role in the development of hormone-driven breast tumors [20]. SUMOylation of ER is stimulated by PIAS1 and PIAS3 in a ligand-dependent manner and inreases the transriptional ativity of the reeptor [21]. SUMO is also onjugated to o-regulators of ER, thereby modulating their ability to interat with the nulear reeptor and to ativate transription [22-24]. Reently, we showed that geneti variation in UBC9 is assoiated with the histologial grade of breast tumors and is a useful marker for breast aner prognosis [25]. Due to the lose onnetion of SUMO with tumorigenesis, it is oneivable that geneti variability in genes of the SUMO-onjugating system also affets breast aner suseptibility. The present study addresses this hypothesis. Based on omprehensive in silio analyses for detetion of putative funtional polymorphisms in 14 genes of the SUMO-onjugating system (AOS1, UBA2, UBC9, PIAS1, PIAS2, PIAS3, PIAS4, SENP1, SENP2, SENP3, SENP5, SUMO1, SUMO2 and SUMO3 we seleted a oding polymorphism in PIAS3 and seven tag SNPs in UBC9 for assoiation analyses. Assoiations with overall breast aner risk and risks by tumor subtypes were assessed in 1,021 breast aner ases and 1,015 population-based ontrols from the German GENICA study. Materials and methods Study population The GENICA study partiipants of the population-based breast aner ase-ontrol study from the Greater Bonn Region, Germany, were reruited between 08/2000 and 9/2004 as previously desribed [26-28]. In brief, 1,143 inident breast aner ases and 1,155 population ontrols, mathed in 5-year lasses, partiipated in the study. The GENICA study was approved by the Ethi s Committee of the University of Bonn and all study partiipants gave written informed onsent. Cases and ontrols were eligible if they were of Cauasian ethniity, urrent residents of

6 the study region and below 80 years of age. Among the reruited individuals, DNA samples were available for 1,021 (89% breast aner ases and 1,015 (88% ontrols. Information on known and potential risk fators was olleted for all partiipants via in-person interviews. The response rate was 88% for ases and 67% for ontrols. Charateristis of the study population regarding potential breast aner risk fators inluded age at diagnosis, menopausal status (premenopausal, postmenopausal, family history of at least one first degree relative with breast or ovarian aner (yes, no, use of oral ontraeptives (OC (never, >0 to <5, 5 to <10, 10 years, use of hormone therapy (HT (never, >0 to <10, 10 years, body mass index (BMI (<20, 20 to <25, 25 to <30, 30 kg/m 2 and smoking status (never, former, urrent (Table 1. Information on linial and histopathologial tumor harateristis was olleted and inluded histology (dutal, lobular, dutolobular, histologial grade (G1, G2, G3, tumor size (T1, T2, T3, T4, lymph node status (N0, N 1, ER status (positive, negative, PR status (positive, negative and HER2 status (positive, negative (Table 2. Tumor grade was determined aording to the Nottingham Criteria, whih omprises formation of tubuli, nulear pleomorphism and mitoti rate. Searh for putative funtional single nuleotide polymorphisms (SNPs by in silio analyses To identify potential promoter regions, the upstream sequenes of the AOS1, UBA2, UBC9, PIAS1, PIAS2, PIAS3, PIAS4, SENP1, SENP2, SENP3, SENP5, SUMO1, SUMO2 and SUMO3 genes were analysed by the web-tool PromoterSweep [29]. The analysed sequenes enompassed the 3 kb upstream region of the transription start site (TSS, the region between the TSS and the translation start and the 200 bp downstream region of the translation start. The latter DNA segment was inluded in the analyses to failitate the automati identifiation of orthologs. In ase of more than one possible TSS, the most upstream TSS was used for defining the 3 kb upstream region. All sequene and TSS data were obtained from the database Ensembl (version 54. PromoterSweep employs a ombination of algorithms inluding promoter database

7 searhes, profile matrix searhes based on known transription fator binding sites (TFBs and de novo motif disovery by omparing orthologous promoter regions. The integration of different methods leads to an improved predition auray ompared to single method tools. Identified promoter motifs fulfilled three riteria: they (1 showed homology to a promoter region annotated in a promoter database, (2 fitted to a profile matrix of a transription fator and (3 appeared to be onserved among orthologous promoter regions. Predited TFBs were sreened for polymorphisms with a minor allele frequeny (MAF of more than 1% in the CEU HapMap population of European anestry [30]. Also non-synonymous oding SNPs in all 14 genes were analyzed for their potential effet on protein funtion using the web-tools Polyphen [31] and Panther [32, 33]. DNA isolation and genotyping Genomi DNA was extrated from heparinized blood samples (Puregene TM, Gentra Systems, In., Mineapolis, USA [27]. Genotyping of UBC9 polymorphisms (rs , rs , rs909915, rs , rs761059, rs and rs8063 was performed by TaqMan alleli disrimination as previously desribed [25]. Genotyping of the PIAS3 polymorphism (rs was performed by PCRbased restrition fragment length (RFLP analysis using the forward primer 5 -TGC ACC CAG CCT CAG ATT G-3 and the reverse primer 5 -GGA TCT CAT CAC AAT CTG ATC AG-3 (mismath is underlined, and the restrition enzyme Hpy188 III. Amplified DNA fragments were digested with 2.5 U Hpy188 III (New England Biolabs, Frankfurt, Germany, separated on a 3% agarose gel ontaining ethidium bromide (Sigma-Aldrih, Steinheim, Germany and sored by UV visualization. Fragment sizes are 255 bp and 23 bp for the C allele and 278 bp for the G allele.

8 Overall all rates ranged from 96.4% to 99.7%. Conordane rates of 135 dupliate samples (6.6% were equal or greater than 99%. The distribution of genotypes in the ontrol group was onsistent with Hardy-Weinberg equilibrium evaluated by Fisher s exat test. Single SNP analyses Our study had a 90% power to detet an OR of 1.34 (α = 0.05, two-sided test, dominant model, the lowest minor allele frequeny of the seven investigated UBC9 single nuleotide polymorphisms (SNPs was 11%. For PIAS3, the power was 80% to detet an OR of 1.75 given a minor allele frequeny of 2% (α = 0.05, two-sided test, dominant model. Assoiations between eah SNP and breast aner risk were analysed by logisti regression onditional on age in 5-year groups using the software pakage R (version Risks were adjusted for potential breast aner risk fators, whih inluded menopausal status, family history of breast or ovarian aner, OC use, HT use, BMI and smoking status. P values of Wald tests for individual SNPs 0.05 were onsidered statistially signifiant. Genotype speifi risks were alulated as odds ratios (OR and assoiated 95% onfidene intervals (CI. Assoiations between genotypes and risk of breast aner subtypes were explored upon stratifiation of ases by histologial grade, tumor histology, tumor size, lymph node status, ER, PR and HER2 status. Subgroups of ases were ompared to all ontrols. Only strata with a size greater than five were analysed. Due to our previous results [25], we primarily aimed to investigate possible assoiations with the risk of tumors stratified by grade. Other stratified analyses were performed in the sense of an exploratory testing. Global probability values for assoiations between genotypes and grade 1 tumor risk were orreted for multiple omparisons by permutation of the ase-ontrol status of eah individual (10,000 iterations [34] onsidering thirty-two assoiations (any, 1, 2 and 3 grades x 8 SNPs.

9 To identify subgroups of women at risk, we further investigated possible interation effets of eah SNP and the epidemiologial variables menopausal status, family history of breast or ovarian aner, use of OC, use of HT, BMI and smoking. Model seletion for the assoiation of risk of grade 1 breast tumors and ombinations of SNPs in UBC9 The relationship between genotype and grade 1 tumor risk was investigated based on four different penetrane models for rs and the genetially linked SNPs rs and rs (reessive, dominant, additive and three-genotype model. In all models, information on epidemiologial risk fators was inluded as ovariates (menopausal status, family history of breast or ovarian aner, use of OC, use of HT, BMI and smoking. The best model was seleted based on likelihood ratio tests using a ovariates-only model as referene. The linked SNP rs was not onsidered in this analysis beause the homozygous genotype of the rare allele was not represented in grade 1 tumor patients. To analyze the possible effet of ombinations of SNPs, we inferred haplotypes and haplogenotypes (diplotypes using the haplo.em funtion in the R pakage haplo.stats [35]. Sine the investigated polymorphisms were in linkage disequilibrium, the inferene of haplotypes should inrease the statistial power in omparison with an evaluation of possible additive or multipliative interations. Single SNP models were ompared with diplotype models based on likelihood ratio tests, trying to optimize the goodness of fit of the model (large likelihood and keeping the model as simple as possible (few degrees of freedom. Epidemiologial risk fators were inluded in any model. Model omparisons were limited to those haplogenotypes represented in both grade 1 tumor ases and ontrols. Imputation of genotypes

10 There is evidene that geneti assoiation studies may benefit from ombining information aross SNP markers and by exploiting existing atalogues of variation [36]. We imputed genotypes based on HapMap data to investigate geneti assoiations at a finer grid of loations aross the genome (detet possible assoiations with geneti variants that were not genotyped in our study. Imputation relied on inferene of haplotypes by means of the expetationmaximisation (EM algorithm in the presene of partially missing data. In brief, missing alleles were exluded from the alulation of allele frequenies. In the E-step, frequenies of partially missing genotypes were updated looping through all possible genotypes. In the M-step, all existing haplotypes that have alleles idential to the non-missing alleles of this haplotype were updated. The ertainty of imputation of genotypes using HapMap data was evaluated by rossvalidation and it was represented by minus the logarithm of the probability value (Pval for Cohen s Kappa between the true and the imputed genotypes. Seletion of variants for subsequent analysis of assoiation relied on the visual inspetion of reombination rates and imputation auraies in the ± 500 kb region around the genotyped SNPs. Unertainty in the imputed genotypes was taken into aount in the onditional ordinal logisti regression by bootstrapping from the multinomial distribution of the expeted genotypes given the observed, diretly genotyped variants (1,000 repliates. The p values referred to a three-genotype model. Results Seletion of SNPs in genes of the SUMO-onjugating system for assoiation studies To identify variants in 14 genes of the SUMO onjugating system that may influene the expression or funtion of the enoded proteins, we performed omprehensive in silio analyses using HapMap data [30]. The genes searhed for funtional SNPs enode the E1 omponents AOS1 and UBA2, the E2 protein UBC9, the E3 PIAS family members PIAS1, the splie variants PIASx and PIASx, PIAS3 and PIASy, the SUMO-speifi proteases SENP1, SENP2, SENP3

11 and SENP5 and the modifiers SUMO-1, SUMO-2 and SUMO-3. The upstream sequenes of these genes were analyzed by a novel in silio tool, whih employs a ombination of different methods to predit potential promoter regions and transription fators binding to them. None of the urrently known polymorphisms with a MAF of more than 1% in the European HapMap population was loated in predited transription fator binding sites. Sreening of these genes for non-synonymous oding polymorphisms revealed one SNP in PIAS3 (rs , MAF=0.02 resulting in an amino aid substitution of serine to ysteine at position 390 (Ensembl Protein ID ENSP that was predited to be of funtional relevane. For assoiation analyses, we seleted the putative funtional SNP in PIAS3 and seven tag SNPs (rs , rs , rs909915, rs , rs761059, rs and rs8063 in the UBC9 gene and the region 10,000 base pairs upstream of its transription start as previously desribed [25]. Assoiations of SNPs in UBC9 and PIAS3 with overall breast aner risk and risk by tumor subtypes We analyzed the eight polymorphisms in the UBC9 and PIAS3 genes within the GENICA study population. No assoiation with overall breast aner risk was observed (Table 3, any-grade. Frequenies of UBC9 haplotypes enompassing SNPs in strong linkage disequilibrium (rs , rs , rs and rs [25] did not differ signifiantly between ases and ontrols (data not shown. Stratifiation of ases by histologial grade, tumor histology, tumor size, lymph node status, ER, PR and HER2 status revealed assoiations with tumor grade. Four SNPs in UBC9 were signifiantly assoiated with the risk of grade 1 breast tumors (rs , global p=0.01; rs , global p=0.04; rs , global p=0.05; rs8063, global p=0.05 (Table 3.

12 Model seletion for single SNPs based on likelihood ratio tests revealed that a dominant model for rs was the best representation of the assoiation between UBC9 genotype and risk of grade 1 breast aner (Supplementary Table 1. Sine the four UBC9 SNPs rs , rs , rs and rs are in linkage disequilibrium, we investigated whether the risk of grade 1 tumors was affeted by ombinations of SNPs rather than by single polymorphisms. The dominant model based on rs was augmented by additional polymorphisms and likelihood ratio tests showed a non signifiant improvement of the goodness of fit. Model omparisons were limited to haplogenotypes represented in both grade 1 tumor ases and ontrols. Based on the dominant model and onditional logisti regression, women with the rare allele of UBC9 rs had a higher risk of grade 1 breast tumors than women homozygous for the ommon allele (OR 1.87, 95% CI The effet was more pronouned when ases had a family history of breast or ovarian aner (14 women affeted by grade 1 tumors with a family history, OR 2.18, 95% CI , however was statistially not signifiant. To orret for multiple testing (any, 1, 2 and 3 grades x 8 SNPs permutation analysis was performed. After orretion, assoiations between UBC9 polymorphisms and grade 1 breast aner risk did not remain statistially signifiant. Imputation of untyped SNPs flanking the genotyped SNPs in UBC9 Multiple imputation based on HapMap data was applied to investigate if rs or untyped SNPs in its proximity were responsible for the assoiation with grade 1 tumor risk. Visual inspetion of reombination rates in a 500 kb region entered on the genotyped SNPs, together with the results on ertainty of imputation, suggested the seletion of a 300 kb region omprising 210 SNPs, whih showed heterozygosity in HapMap (Supplementary Figure 1. Conditional logisti regression based on genotyped and imputed SNPs indiated that rs shows the

13 strongest assoiation with risk of grade 1 breast aner (Figure 1A. rs showed no signifiant assoiation with risk of grade 2, grade 3 and any-grade breast tumors (Figures 1B, 1C and 1D. Assoiation between SNPs and breast aner risk by epidemiologial parameters To identify subgroups of women at risk, we further investigated possible interation effets of the genotyped polymorphisms and menopausal status, family history of breast or ovarian aner, OC use, HT use, BMI and smoking status. No statistially signifiant interations were found. Disussion There is inreasing evidene of a ruial role of SUMO modifiation in tumorigenesis. Therefore, we hypothesized that variation in genes of the SUMO-onjugating system may affet the risk of breast aner. In an initial step we sreened 14 genes involved in SUMO modifiation for putative funtional polymorphisms in the promoter and oding regions by performing omprehensive in silio analyses. We obtained evidene for one funtional polymorphism in the oding region of the SUMO ligase gene PIAS3. In addition, we seleted seven tag SNPs overing the omplete promoter and gene region of UBC9, whih enodes a key enzyme of SUMOylation. All SNPs were analyzed for assoiations with breast aner risk in the population-based aseontrol study GENICA. None of the investigated polymorphisms seemed to alter the overall risk of breast aner. However, stratifiation of patients by tumor grade revealed that four SNPs in UBC9 (rs , rs , rs and rs8063 were assoiated with the risk of grade 1 tumors. The best assoiation model inluded rs under dominant penetrane, and this model was not improved by inlusion of additional SNPs in linkage disequilibrium. Women arrying the rare allele of rs had a risk of grade 1 tumors 1.9 times higher than women homozygous for

14 the ommon allele. The identifiation of three additional assoiation signals in the region adds onsisteny to this finding. Individual variants are expeted to show stronger effets in familial, genetially enrihed ases [37]. Although not signifiant, we found that arriers with a family history of breast or ovarian aner had a 2.2 times inreased risk of grade 1 tumors, thus further supporting the biologial relevane of rs variation. Results from multiple imputation onfirmed that rs showed the strongest assoiation with the risk of grade 1 tumors. Thus, the identifiation of multiple assoiations in the region, the stronger effet for familial ases and the imputation results suggest a ontribution of rs to the risk of grade 1 breast tumors. Multiple imputation has been shown to failitate the detetion of ausal variants that have not been diretly genotyped in the study [36]. We applied imputation tehniques followed by onditional logisti regression to assess if rs , or rather a linked SNP in its proximity, was responsible for the observed assoiation. Imputation of genotypes relied on the assumption of similar patterns of geneti linkage in the GENICA population and in the publi repository HapMap. In this study, none of the imputed SNPs in the investigated gene region showed a signal of assoiation stronger than that of rs Although the ausal variant is yet unidentified, the result suggests that rs or a losely linked SNP may be responsible for the observed assoiation with risk of grade 1 breast tumors. The ausal variant ould be a rarer SNP lose to rs , whih was not genotyped or imputed in our study. As yet, funtional studies on rs are laking. The polymorphism is loated 14.8 kb upstream of the UBC9 translation start and disrupts a potential binding site for the transription fator SP1 [25]. In onsequene, the rare allele may result in a lower UBC9 expression. To date, however, data on UBC9 expression levels supporting this hypothesis are not available. One previous study using an in silio approah to predit putative genes on the short arm of hromosome 16 suggested an open reading frame enompassing rs [38]. Thus, we

15 annot exlude the possibility that this polymorphism influenes the funtion of a yet unidentified protein. Many tumor suppressors and onoproteins have been identified as SUMO substrates emphasizing SUMO s ruial role in proesses that are tightly linked to anerogenesis. Although SUMO modifiation seems to be highly regulated, both spatially and temporally, it is oneivable that expression levels of UBC9 influene the balane between SUMO onjugation and deonjugation and thus the funtion of target proteins. Several SUMO targets are involved in ell differentiation, proliferation and ell yle ontrol [39], suh as -JUN, -FOS [40], p53 [41, 42] and ER [43], providing a possible link between SUMOylation and tumor grade. Two reent studies have reported a link between dereased SUMO onjugation and redued growth of tumor ells. The first study used a mouse xenograft model and breast aner ells to show that dereased UBC9 ativity slows down tumor growth in vivo [14]. Yang and Pashen reported that bloking SUMO-2 and 3 onjugation altered the expression of genes involved in ell proliferation and differentiation and resulted in redued growth of neuroblastoma ells in vitro [44]. Thus, the assumption of rs or a losely linked SNP attenuating the expression of UBC9 would be in line with its effet of favouring the development of slowly growing grade 1 tumors. Moreover, the inreased risk of grade 1 tumors assoiated with the rare allele of rs is in agreement with our previous results of a higher frequeny of this allele in patients with low grade tumors [25]. It is important to underline that our previous and the present study differ with respet to study design, ase-only and ase-ontrol study, respetively and analyzed different events. For example, in the former study, we showed a higher frequeny of the rare rs TT genotype (14.3% in grade 1 ases ompared to grade 2 (8.5% and grade 3 (4.1% ases. In the present study we additionally reported genotype frequenies in healthy ontrols. The present findings indiate that women affeted by grade 1 breast tumors showed a higher TT genotype frequeny (14.9% than grade 2 (8.6% and grade 3 (4.2% ases as well as

16 unaffeted ontrols (6.8%, whih refers to an inreased risk of grade 1 breast aner among TT arriers. In summary, in this study on geneti variation in the SUMO-onjugating system, we found assoiations between four UBC9 SNPs (rs , rs , rs and rs8063 and the risk of grade 1 breast tumors. The strongest assoiation was observed for rs and the effet appeared to be stronger in familial ases. A model seletion strategy and multiple imputation indiated that, among genotyped and losely linked variants analyzed in the International HapMap Projet, rs showed the most signifiant ontribution to risk. Our data suggest that UBC9 variation influenes the risk of grade 1 breast tumors. Repliation studies in independent populations should be arried out to onfirm this result. Aknowledgements We are indebted to all women partiipating in the GENICA study. We gratefully aknowledge support by interviewers as well as physiians and pathologists of the study region. We thank Axel Benner for his ontribution to the statistial analysis and Agnes Hotz-Wagenblatt as well as Karl-Heinz Glatting for their support in using the software PromoterSweep. Further gratitude goes to Antje Seidel-Renkert for expert tehnial assistane. This work was supported by the Federal Ministry of Eduation and Researh (BMBF Germany grants 01KW9976/8, 01KW9975/5, 01KW9977/0 and 01KW0114, the Deutshes Krebsforshungszentrum, Heidelberg, the Robert Bosh Foundation of Medial Researh, Stuttgart, BGFA-Forshungsinstitut für Arbeitsmedizin der Deutshen Gesetzlihen Unfallversiherung, Bohum, and the Department of Internal Mediine, Evangelishe Kliniken Bonn ggmbh, Johanniter Krankenhaus, Bonn, Germany. Competing interests: The authors delare that they have no ompeting interests.

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20 Figure legends Figure 1. Assoiation between risk of breast aner and genotyped (blak irles or imputed (grey irles UBC9 polymorphisms. The plot shows the -log 10 p values for a three-genotype model versus a model, whih does not inlude individual genotypes. Risk of grade 1 (A, grade 2 (B, grade 3 (C and any-grade (D breast tumors were evaluated. Multiple imputation was based on HapMap data and the alled SNPs rs , rs , rs and rs (blak irles. Assoiation of rs with grade 1 tumor risk ould not be assessed beause the homozygous genotype of the rare allele was not represented in grade 1 tumor patients. Table 1 Charateristis of the GENICA study population Charateristi Cases n (% Controls n (% Age (years ( ( ( ( ( ( ( ( ( ( ( ( ( (16.3 Menopausal status Premenopausal 249 ( (23.5 Postmenopausal 758 ( (76.5 Breast or ovarian aner in No 860 ( (91.3 first-degree relatives Yes 161 ( (8.7 OC use (years Never 372 ( (36.3 >0 to <5 180 ( ( to < ( ( ( (33.6 HT use (years Never 506 ( (50.2 >0 to < ( ( ( (21.1 BMI (kg/m 2 <20 90 ( ( to < ( ( to < ( ( ( (14.4 Smoking Never 586 ( (54.7 Former 192 ( (21.2 Current 242 ( (24.1

21 OC: oral ontraeptive, HT: hormone therapy, BMI: body mass index

22 Table 2 Histopathologial parameters of the inident breast tumors of the GENICA ases Tumor parameter Cases n (% Histology Dutal 634 (69.5 Lobular 177 (19.4 Dutolobular 101 (11.1 Histologial grade G1 77 (8.2 G2 567 (60.4 G3 295 (31.4 Tumor size T1 582 (61.9 T2 289 (30.7 T3 30 (3.2 T4 39 (4.1 Nodal status N0 602 (63.8 N1 342 (36.2 ER status Positive 755 (77.8 Negative 216 (22.2 PR status Positive 678 (70.0 Negative 291 (30.0 HER2 status Positive 189 (27.7 Negative 493 (72.3 ER: estrogen reeptor, PR: progesterone reeptor

23 Table 3 Odds ratios for breast aner risk by tumor grade Gene/S NP Geno - type Cont rols n (% Cas es n (% Any-grade Grade 1 Grade 2 Grade 3 Glo Glo Glo a Cas a Cas a a OR adj bal OR es adj bal OR es adj bal OR Cases adj (95% p (95% p (95% p (95% n n n (% CI valu CI valu CI valu CI e b (% e b (% e b Glo bal p valu e b UBC9 rs CC 526 (52.9 C>T CT 400 (40.2 TT 68 ( ( ( (7.8 ne 1.01 ( ( ( ( (14. 9 ne 1.71 ( ( ( ( (8.6 ne 1.01 ( ( ( ( (4.2 ne 0.95 ( ( rs AA 385 (38.8 A>G AG 469 (47.2 GG 139 ( ( ( (15. 1 ne 0.99 ( ( ( ( (21. 6 ne 1.72 ( ( ( ( (15. 0 ne 1.01 ( ( ( ( (12.0 ne 0.91 ( ( rs CC 784 (79.0 C>T CT 195 (19.6 TT 14 (1.4 CT+ TT 209 ( ( ( ( (20. 8 ne 0.97 ( ( ( ( ( ( (20. 3 ne 0.87 ( ( ( (1.6 ne 0.99 ( ( ( ( (2.8 ne 0.90 ( ( rs GG 691 (69.5 G>C GC 279 (28.1 CC 24 ( ( ( (2.8 ne 0.96 ( ( ( ( (8.1 ne 1.56 ( ( ( ( (2.3 ne 0.97 ( ( ( ( (2.1 ne 0.83 ( ( rs GG 338 (34.6 G>A GA 477 (48.9 AA 161 ( ( ( (17. 6 ne 1.01 ( ( ( ( (14. 7 ne 0.79 ( ( ( ( (17. 0 ne 1.14 ( ( ( ( (19.3 ne 0.88 ( ( rs GG 688 (69.6 G>A GA 264 ( ( (30. 2 ne 1.08 ( ( (34. 2 AA ne 368 ( (30. 2 ne 1.11 ( ( ( ne 1.07 (

24 GA+ AA (3.7 ( (30.4 (33. 2 ( ( ( (2.7 ( ( ( (3.2 ( rs8063 GG 495 (50.3 G>A GA 409 (41.6 AA 80 ( ( ( (8.8 ne 0.96 ( ( ( ( (12. 7 ne 1.53 ( ( ( ( (8.2 ne 0.94 ( ( ( ( (8.1 ne 0.87 ( ( PIAS3 rs CC 950 (95.7 C>G CG 42 (4.2 GG 1 (0.1 CG+ GG 43 ( ( (4.8 0 ( (4.8 ne 1.03 ( ( (5.5 0 (0.0 4 (5.5 ne 1.37 ( ( (5.7 0 ( (5.7 ne 1.15 ( ( (3.2 0 (0.0 9 (3.2 ne 0.71 ( a b Odds ratio onditional on age in 5-year groups adjusted for menopausal status, family history of breast or ovarian aner, use of oral ontraeptives, use of hormone therapy, body mass index and smoking. P value of testing the null hypothesis of no assoiation between SNP and risk of any-grade, grade 1, grade 2 and grade 3 breast tumors based on onditional logisti regression, not orreted for multiple omparisons. Statistially signifiant results are given in bold. Not analyzed due to small numbers of ases and/or ontrols.