{"paper_id":"73d652bc-07b2-4a6d-a163-1b9c34cfe1ec","body_text":"Int J Mol Epidemiol Genet 2013;4(4):193-206\nwww.ijmeg.org /ISSN:1948-1756/IJMEG1309001\nOriginal Article\nFine mapping of variants associated with endometriosis \nin the WNT4 region on chromosome 1p36\nHien TT Luong1, Jodie N Painter1, Konstantin Shakhbazov2, Brett Chapman1, Anjali K Henders1, Joseph E \nPowell2, Dale R Nyholt1, Grant W Montgomery1\n1Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, \nQueensland, Australia; 2University of Queensland Diamantina Institute, University of Queensland, Princess Alexan-\ndra Hospital, Brisbane, Queensland, Australia\nReceived September 6, 2013; Accepted October 15, 2013; Epub November 28, 2013; Published December 15, \n2013\nAbstract: Genome-wide association studies show strong evidence of association with endometriosis for markers on \nchromosome 1p36 spanning the potential candidate genes WNT4, CDC42 and LINC00339. WNT4 is involved in \ndevelopment of the uterus, and the expression of CDC42 and LINC00339 are altered in women with endometriosis. \nWe conducted fine mapping to examine the role of coding variants in WNT4 and CDC42 and determine the key SNPs \nwith strongest evidence of association in this region. We identified rare coding variants in WNT4 and CDC42 pres-\nent only in endometriosis cases. The frequencies were low and cannot account for the common signal associated \nwith increased risk of endometriosis. Genotypes for five common SNPs in the region of chromosome 1p36 show \nstronger association signals when compared with rs7521902 reported in published genome scans. Of these, three \nSNPs rs12404660, rs3820282, and rs55938609 were located in DNA sequences with potential functional roles \nincluding overlap with transcription factor binding sites for FOXA1, FOXA2, ESR1, and ESR2. Functional studies will \nbe required to identify the gene or genes implicated in endometriosis risk. \nKeywords: Endometriosis, WNT4, CDC42, chromosome 1p36, rare variants, common variants\nIntroduction\nLarge gene mapping studies in endometriosis \nhave consistently identified strong association \nwith disease risk for markers in the region close \nto wingless-type MMTV integration site family, \nmember 4 (WNT4) on chromosome 1 [1-4]. The \nsingle nucleotide marker with the strongest sig-\nnal rs7521902 identified in the original studies \n[1, 2] is located approximately 20 kb upstream \nfrom the transcription start site for WNT4. This \nis an interesting region as rs7521902 is also \nimplicated in risk for ovarian cancer [5], bone \nmineral density and risk of fracture [6]. \nSubsequent studies in endometriosis cases \nand controls re-analysed data for a higher den-\nsity of SNPs across this region with genotypes \nestimated by imputation [3, 4]. Results provide \nstrong evidence for replication of association in \nthe region of WNT4 with the highest signals \nlocated within the WNT4 genomic sequence. \nHowever, the region of strong association \nextends across adjacent genes and spans the \nregion including the long intergenic non-protein \ncoding RNA LINC00339, cell division cycle 42 \n(CDC42) and WNT4 [3, 4].\nWNT4 is a strong candidate for functional \nchanges increasing risk for endometriosis and \novarian cancer. Expression of WNT4 is critical \nfor development of the female reproductive \ntract [7, 8]. WNT4 and other WNT family mem -\nbers are expressed in human endometrium dur-\ning both the proliferative and secretory phases \n[9] and expression is up-regulated by oestrogen \nin an oestrogen receptor independent manner \n[10]. However, adjacent genes within the DNA \nblock showing association include CDC42 and \nLINC00339, both implicated in endometriosis. \nCDC42 is a small GTPase of the Rho-subfamily, \nwhich regulates signalling pathways that con -\ntrol diverse cellular functions including cell mor-\nphology, migration, endocytosis and cell cycle \nprogression. The gene is expressed in the endo-\n\nWNT4 region and endometriosis risk\n194 Int J Mol Epidemiol Genet 2013;4(4):193-206\nmetrium and is reported to be differentially \nexpressed in endometriosis [11]. LINC00339 \n(also known as HSPC157) is differentially \nexpressed in endometriosis lesions versus \nautologous uterine endometrium [12]. Both \nCDC42 and LINC00339 must also be consid -\nered as candidates for the action of genetic \nvariants on endometriosis risk.\nEffects of individual SNPs on disease risk are \nsmall and determining the specific genes and \npathways affecting disease risk is the best \napproach to understand mechanisms leading \nto endometriosis. The aims of this study were to \ngenotype SNPs in the exons of WNT4 and \nCDC42 to determine whether the association \nsignal could be explained by coding variants, \nand to genotype common non-coding variants \npreviously implicated from imputed data to \nconfirm results for the best SNPs located in the \nregion of WNT4. We also conducted in silico  \nanalyses to evaluate possible functional roles \nof our top 50 non-coding variants across the \nregion.\nMaterials and methods\nThe coding regions of WNT4 were screened for \nvariants in 100 unrelated individuals chosen \nfrom 100 case-dense families drawn from the \nQIMR Berghofer Medical Research Institute \ndataset [13]. They include 15 families with at \nleast four affected sisters, 71 families with \nthree affected sisters and 14 families with at \nleast two sisters and two other relatives diag -\nnosed with endometriosis. Cases for genotyp -\ning were one sample per family chosen as the \ncase with the most severe stage of disease in \neach family.\nCoding variants were genotyped in samples \nfrom 958 endometriosis cases with a family \nhistory drawn from our Australian study of \nendometriosis [13, 14]. All cases had surgically \nconfirmed endometriosis and disease severity \nincluding four-stages (I-IV) was assessed using \nthe revised American Fertility Society (rAFS) \nclassification system [15]. The control group \nincluded 959 unrelated controls drawn from \nwomen recruited for a study of twins who self-\nreported that they had never been diagnosed \nwith endometriosis [14]. After removing indi -\nviduals in case families with non-Caucasian \nancestry revealed by our GWA studies [16], 930 \ncases and 959 controls were included in our \nanalyses. \nEthical approvals\nStudy protocols were reviewed and approved by \nthe QIMR Human Research Ethics Committee. \nParticipation was voluntary and each partici -\npant gave written informed consent. \nHigh Resolution Melt (HRM) assay and se-\nquencing\nWe screened for coding variants in the exons of \nWNT4 in DNA from 100 endometriosis cases \nusing a High Resolution Melting (HRM) tech -\nnique [17]. WNT4 PCR primers were designed \nto amplify the 5’ and 3’UTRs, non-coding and \ncoding exons, including at least 50 bp of intron-\nic sequence either side of each exon to cover \nintron-exon boundaries using the Primer 3.0 \nprogram [18]. PCR products were screened on \na Rotor-Gene 6000 Real-time Rotary Analyser \n(Corbett Research, QIAGEN, Hilden, Germany). \nMelting curves were analysed using the Rotor-\nGene 6000 analysis software v 1.7 (Corbett \nResearch). Samples showing patterns different \nfrom wild-type were sequenced using BigDye \n3.0 terminator chemistry (Applied Biosystems).\nWe also searched for coding variants in both \nWNT4 and CDC42 using public databases with \nresults from extensive exome sequencing \nincluding dbSNP (http://www.ncbi.nlm.nih.gov/\nprojects/SNP/), 1000 Genomes (http://brow-  \nser.1000genomes.org/), the NHLBI GO Exome \nSequencing Project (ESP, http://evs.gs.wash -\nington.edu/EVS/), and the exome chip genotyp-\ning array design (http://genome.sph.umich.\nedu/wiki/Exome_Chip_Design). \nGenotyping\nTwenty seven coding variants in WNT4, five cod-\ning and four potential functional variants (locat-\ned in the promoter area and splice sites) in \nCDC42, and six key imputed SNPs identified \nfrom the GWA studies [3] were genotyped using \nSequenom MassARRAY technology in the 930 \ncases and 959 controls. Genotyping assays \nwere designed using standard procedures and \nSNPs were typed using iPLEX™ chemistry on a \nMALDI-TOF Mass Spectrometer (Sequenom \nInc., San Diego, CA, USA) [14].\nData analysis\nPLINK software was used to analyse SNP data \nfor quality control measures and to test for \n\nWNT4 region and endometriosis risk\n195 Int J Mol Epidemiol Genet 2013;4(4):193-206\nTable 1. Variants identified in WNT4 during re-sequencing of 100 endometriosis cases including the minor allele frequencies (MAF) observed in \npublic databases and in the cases\nSNP name Location (Hg19) Role Nucleotide variant Amino acid change MAF MAF in 100 cases sequenced Functional changes predicted\nrs115547783 22456050 Intronic c.236 + 59C/G 0.008# 0.015\nNovel* 22456184 Exonic c.161C/T p.Tyr80His Not_obs 0.005 No\nrs34228276 22446768 Exonic c.243A/G p.Pro277Pro 0.025# 0.015\nNovel 22446536 3’UTR c.473 T/-in-del Not_obs 0.005 micRNA binding sites\n#Minor allele frequency (MAF) estimate from phase 1 data from the 1000 Genome Project. *This variant was novel at the time of sequencing, but subsequently reported by Exome. \nSequencing Project (ESP) (release ESP6500 data_20 June 2012). Not_obs: Polymorphisms were not observed.\nTable 2. Results for tests of association for WNT4 and CDC42 variants genotyped in 930 endometriosis cases and 959 controls\nVariants Nucleotide variant Role MAF in cases MAF in controls P value OR Functional prediction*\nWNT4 variants\nIn-del novel variant T/-in-del 3’UTR 0.001 0 0.242 NA micRNA binding sites\nExon 2 novel variant c.161C/T Non-synonymous 0.001 0 0.489 NA No damage prediction\nrs12067696 A/G Synonymous 0.001 0 0.492 NA Splicing (ESE & abolish domain)\nConservation: +++\nrs34228276 A/G Synonymous 0.018 0.023 0.302 0.78 (0.50-1.23) Conservation: +++\nrs115547783 C/G Intronic 0.016 0.015 0.894 1.07 (0.64-1.81) NA\nCDC42 variants\nrs191653816 C/T promoter 0.001 0 0.492 NA TF binding sites\nrs16860621 A/G promoter 0.125 0.130 0.708 0.96 (0.79-1.17) TF binding sites\nrs17837976 C/T Intronic, splice site 0.002 0.005 0.180 0.41 (0.13-1.32) Conservation: +++\n*Functional prediction using: miRBase and MicroInspector program for micRNA binding site prediction, SIFT and Polyphen program for amino acid change prediction, HaploReg \nprogram for TF binding site prediction and ‘snp funtion prediction’ program.\n\nWNT4 region and endometriosis risk\n196 Int J Mol Epidemiol Genet 2013;4(4):193-206\nassociation between SNPs and endometriosis \nrisk. Call rates were all greater than 95% and all \nSNPs were in Hardy-Weinberg equilibrium (P > \n0.05). Association tests for endometriosis risk \nwere performed in PLINK using the standard \nchi-square (--assoc) test for common variants \nand Fisher’s exact (--fisher) test for low frequen-\ncy variants [19]. \nTo assess the combined contribution of multi -\nple rare variants towards endometriosis risk, \nmultiple logistic regression was performed. \nBriefly, genotypes for each SNP were re-coded \nto allele dosages using PLINK’s --recodeA \noption (i.e., additive recoding of the number of \nminor alleles). Multiple logistic regression anal-\nysis using the glm function in the R statistical \npackage [20] compared the model with multi -\nple SNPs to the model with zero SNPs to pro -\nvide a multiple degree of freedom test for \nassociation.\nBioinformatics analysis\nWe performed bioinformatic analyses on a 150 \nKb region of chromosome 1 across the CDC42 \nand WNT4 genes. Linkage disequilibrium (LD) \nbetween SNPs and the presence of haplotype \nblocks was determined using the Haploview \nprogram [21]. Potential effects of non-synony -\nmous (coding) variants were predicted using \nthe Sorting Intolerant from Tolerant (SIFT) pro -\ngram [22 , 23] Polyphen [24], the PANTHER \nPSEC classification system [25] and the predic-\ntion of pathological mutations program (PMut) \n[26]. SNPs in the 3’UTR were screened for their \npotential to change microRNA binding sites \nusing the miRBase [27 , 28], miRDB [29], and \nMicroInspector databases [30].\nHigh throughput functional assays have been \nconducted by the international ENCODE project \nto identify functional regions of the genome \noutside of gene coding regions. The ENCODE \ndata includes areas of open chromatin identi -\nfied using DNaseI hypersensitivity (HS), For-  \nmaldehyde-Assisted Isolation of Regulatory \nElements (FAIRE), and Chromatin immunopre -\ncipitation (ChIP) experiments and the locations \nof functional regulatory elements including pro-\nmoters, enhancers, silencers, insulators, locus \ncontrol regions and novel elements [31-33]. \nSeveral programs are available to search the \nENCODE data. We used HaploReg version 1 \n[34] to search for SNPs with functional annota-\ntions in high LD (r 2 > 0.8) to our most strongly \nassociated SNPs and the RegulomeDB pro -\ngram [35] to rank potential functional roles for \nSNPs based on their location across the entire \n150 Kb region. The scoring system for Regu-  \nlomeDB ranges from 1-6 with the strongest evi-\ndence for functional roles as scores 1  (a-f). A \nscore of 2 includes evidence of transcription \nfactor (TF) binding and DNase footprint signals. \nScores of 1 require additional evidence of eff-  \nects of the SNP on specific gene expression. \nResults\nSearch for rare and novel variants in WNT4 \nFive coding exons (exons 1-5), the 5’ and \n3’UTRs, and the intron/exon boundaries for \nWNT4 were screened by HRM assay. Four vari -\nants were detected amongst 100 endometrio -\nsis cases using HRM including two known vari -\nants and two novel variants (Table 1). One SNP \nrs34228276 is a synonymous A to G change \ncoding for the amino acid proline at position \n277 and was seen at a similar frequency in con-\ntrols (Table 2). The other one, rs115547783 is \nin intron 2. A novel non-synonymous T to C \nchange in exon 2 was predicted to change an \namino acid from tyrosine to histidine at position \n80 (p.Tyr80His). This was detected in one endo-\nmetriosis patient in heterozygous form. The \nsecond novel variant was an insertion of a T \nbase in the non-coding region of the 3’UTR (six \nbase pairs downstream of the termination \ncodon) and found in another patient in hetero -\nzygous form ( Table 1 ). All members of both \nfamilies for whom DNA samples were available, \nincluding the proband individuals, were then \nscreened via HRM and/or sequencing for the \npresence of the relevant variants. The exon 2 \nvariant was observed in two affected sisters \nand the father of the proband, but an affected \naunt did not carry the variant ( Figure 1A). The \n3’UTR insertion/deletion (in-del) T in the sec -\nond family was present in all available affected \nsisters and their father (Figure 1B). These vari-\nants were not detected in other cases or in any \ncontrol samples by Sequenom genotyping \n(Table 2).\nBoth novel variants were examined for poten -\ntial functional effects in silico . There was no \npredicted effect of the p.Tyr80His substitution \nin exon 2 (corresponding to the variant \n\nWNT4 region and endometriosis risk\n197 Int J Mol Epidemiol Genet 2013;4(4):193-206\nrs115547783) using the Polyphen and SIFT \nprograms and the PANTHER PSEC classifica -\ntion system. The novel in-del variant located in \nthe 3’UTR is predicted to be in a binding site of \nmicroRNA-4767-5p (miRBase) and was predict-\ned to change a binding site from hsa-miR-3151 \nto hsa-miR-4507 by MicroInspector (Table 2).\nGenotyping known coding variants in WNT4 \nand CDC42\nWe genotyped 36 coding variants in WNT4 and \nCDC42 documented in public databases (Table \n3), in addition to the novel variants identified by \nHRM. Genotyping of these variants in our set of \n930 endometriosis cases and 959 controls \nrevealed only 3 of 27 WNT4 variants and 3 of 9 \nvariants in CDC42 were polymorphic in our \nsample (Tables 2 and 3, Figures 2 and 3). Two \nvariants, rs12067696 in WNT4 and \nrs191653816 in CDC42, were found only in \ncases and not in controls, although the differ -\nences in allele frequencies were not significant \n(P = 0.492). Frequencies for these variants \nwere <0.001 and they were not observed in the \n100 cases screened by HRM. The WNT4 syn -\nonymous variant rs12067696 is conserved \nthrough mammalian and vertebrate species \nand predicted to be an exonic splicing enhanc -\ner (ESE) by the SNP Function Prediction \n(FuncPred) program. The SNP rs191653816 in \nthe CDC42 promoter was located in the binding \nsites of 22 TFs by HaploReg (Table 2). \nThere was no evidence that individual coding \nvariants contributed to endometriosis risk, but \nseveral rare variants were detected only in \ncases. We therefore analysed data for the six \nvariants listed in Table 2 using multiple logistic \nregression and found no significant evidence \nfor combined effects on association with endo -\nmetriosis risk (x6\n2\n = 6.973; P = 0.323).\nCommon variants in WNT4 \nWe previously reported significant association \nacross the CDC42-WNT4 region with the stron -\ngest signal from genotypes estimated by impu -\ntation. To confirm imputation results we geno -\ntyped the five top imputed SNPs together with \nrs7521902, the top SNP from the original GWA \nstudies, in our 930 cases and 959 controls. \nOur genotyping results showed high concor -\ndance between genotyped and imputed data \n(98-100%) and confirmed stronger evidence for \nassociation with SNPs located in intron 1 of \nWNT4 ( Table 4 ) than for rs7521902 located \n~20 kb upstream of WNT4.\nThe five SNPs are in high LD and together the \nrisk alleles form a single risk haplotype (P = 7 \nx10-4) with a similar estimate for disease risk as \nthe individual SNPs. \nFunctional annotation for WNT4 common vari-\nants\nFunctional annotation of our top SNPs revealed \nlimited evidence for a functional role for the \nsentinel SNP rs61768001 (defined as the SNP \nwith best association signal in a multi-SNP \nanalysis) or for the original genotyped SNP \nrs7521902. Analysis of 50 SNPs (with stron -\ngest evidence of association from the GWAS \ndata) across the 150 Kb region using \nRegulomeDB identified seven variants in strong \nLD (r2 > 0.8) with rs61768001 and two SNPs in \nhigh LD with rs7521902 with evidence of pre -\ndicted functional roles ( Table 5, Figures 3 and \n4I). The SNP with the best score (2b) was \nrs12404660 (r 2 = 0.84 with rs61768001; \nTable 5, Figure 4II and 4III). SNP rs12404660 \nis located in an area of histone protein \nH3K4me1 chromatin modification associated \nwith transcription enhancer sequences, open \nchromatin, altered regulatory motifs for the \ntranscription factor YY1 and a binding site for \nthe transcription factor CTCF, all of which are \nsuggestive of regulatory potential. Two addi -\ntional SNPs may also have functional roles as \npredicted by the HaploReg, and JASPAR core \nprograms. SNP rs3820282, located in intron 1 \nof WNT4, (r2 = 0.94 to rs61768001), is predict-\ned to lie in a conserved region within regulatory \nFigure 1. Pedigrees of endometriosis cases carrying \nnovel variants in the WNT4 gene: (A) Pedigree of the \nendometriosis family with a novel non-synonymous \nvariant in exon 2 (p.Tyr80His) and (B) Pedigree of an \nendometriosis family with a novel insertion/deletion \nvariant in 3’UTR (in-del T). The proband individual \nscreened by HRM is marked with an asterisk. \n\nWNT4 region and endometriosis risk\n198 Int J Mol Epidemiol Genet 2013;4(4):193-206\nmotifs bound by the transcription factors oes -\ntrogen receptor 1 (ESR1) and oestrogen recep-\ntor 2 (ESR2). No TF binding sites were predicted \nat the original G allele of this variant. However, \nthe change to the minor A allele introduced \npotential regulatory sites for ESR1 and ESR2 \nidentified by HaploReg and JASPAR (Figure 4III, \nTable 5). The SNP rs55938609, in high LD with \nrs61768001 (r2 = 0.98, Figure 5), is located in \na region of histone protein H3K4Me2 modifica-\nTable 3. Known coding variants in WNT4 and CDC42 identified from dbSNP, 1000 Genomes (1000G), \nExome Sequencing Project (ESP), Exome Chips manifest (EC), and COSMIC project\nGene Variants Location  \n(Hg19) Source Role MAF Nucleotide \nvariant\nAmino acid  \nchange\nWNT4 rs112942159 22446645 dbSNP, 1000G Synonymous Not_obs T/C p.318Ala/Ala\nWNT4 rs112452625 22446690 dbSNP, 1000G Synonymous 0.003# A/G p.303Ile/Ile\nWNT4 rs145169034 22446782 dbSNP, 1000G Synonymous 0.001# G/A p.273Leu/Leu\nWNT4 rs201963772 22446798 ESP5400 release Non-synonymous Not_obs T/A p.267Asp/Glu\nWNT4 rs140080433 22446860 dbSNP, 1000G Non-synonymous Not_obs A/G p.247Arg/Cys\nWNT4 rs41441349 22446902 dbSNP, 1000G Non-synonymous 0.002# A/G p.233Ala/Thr\nWNT4 rs140262773 22446925 dbSNP, 1000G Non-synonymous Not_obs A/G p.225Pro/Leu\nWNT4 rs193047338 22446929 dbSNP, 1000G Non-synonymous 0.001# T/C p.224Val/Met\nWNT4 rs121908650 22446952 dbSNP, 1000G Non-synonymous Not_obs G/A p.216Glu/Gly\nWNT4 rs138491414 22447774 dbSNP, 1000G Non-synonymous Not_obs G/C p.173Arg/Pro\nWNT4 rs12067696 22447821 dbSNP, 1000G Synonymous 0.017# A/G p.157Asp/Asp\nWNT4 rs139165736 22447940 dbSNP, 1000G Non-synonymous 0.002# C/T p.148Gln/Arg\nWNT4 rs121908651 22448042 dbSNP, 1000G Non-synonymous Not_obs T/C p.114Ala/Val\nWNT4 rs139045509 22448044 dbSNP, 1000G Synonymous Not_obs A/G p.113Tyr/Tyr\nWNT4 rs16826648 22456146 dbSNP, 1000G Synonymous 0.010# A/G p.92Leu/Leu\nWNT4 rs121908652 22456175 dbSNP, 1000G Non-synonymous Not_obs T/C p.83Arg/Trp\nWNT4 rs34611251 22456205 dbSNP, 1000G Frame shift Not_obs G/- p.73Leu/Trp\nWNT4 rs144407094 22456326 dbSNP, 1000G Synonymous 0.001# T/C p.32Ser/Ser\nWNT4 rs121908653 22469381 dbSNP, 1000G Non-synonymous Not_obs C/T p.12Leu/Pro\nWNT4 unknown 22446572 ESP Non-synonymous 0.0001§ A/G p.343Trp/Arg\nWNT4 unknown 22446857 ESP Non-synonymous 0.0002§ A/G p.248Cys/Arg\nWNT4 unknown 22446859 ESP Non-synonymous 0.0001§ T/C p.247His/Arg\nWNT4 unknown 22447712 ESP Non-synonymous 0.0001§ T/C p.194Ser/Gly\nWNT4 unknown 22456127 ESP Non-synonymous 0.0001§ T/C p.99Ser/Gly\nWNT4 unknown 22456174 ESP Non-synonymous 0.0001§ T/C p.83Gln/Arg\nWNT4 unknown 22446566 EC Non-synonymous 0.0007§ G/C\nWNT4 unknown 22446856 EC Non-synonymous 0.0003§ T/C\nCDC42 unknown 22412982 ESP Non-synonymous 0.0001§ C/G p.77Leu/Val\nCDC42 COSM46468 22405060 1000G, COSMIC Non-synonymous Not_obs T/C p.30Ser/Leu\nCDC42 rs142108830 22413282 ESP, dbSNP Non-synonymous Not_obs G/A p.137Ile/Val\nCDC42 COSM260019 22417991 COSMIC Non-synonymous Not_obs A/G p.557Arg/His\nCDC42 COSM229859 22405006 COSMIC Non-synonymous Not_obs T/G p.12Gly/Val\nCDC42 rs16860621 22379360 dbSNP, 1000G promoter 0.146# A/G\nCDC42 rs16860623 22379375 dbSNP, 1000G promoter Not_obs T/G\nCDC42 rs191653816 22379314 dbSNP, 1000G promoter 0.001# T/C\nCDC42 rs17837976 22408212 dbSNP, 1000G Intron, splice site 0.014# C/T\n§MAF estimate from Exome Sequencing Project. #MAF estimate from phase 1 data from 1000 Genomes. COSMIC, Catalogue of \nSomatic Mutations in Cancer.\n\nWNT4 region and endometriosis risk\n199 Int J Mol Epidemiol Genet 2013;4(4):193-206\ntion, associated with both promoters and enh-  \nancers (Figure 4III), and potential binding sites \nfor the transcription factors forkhead box A1 \n(FOXA1) and A2 (FOXA2) (Figure 4III, Table 5). \nDiscussion\nGWAS for endometriosis report strong associa -\ntions for disease risk with markers at chromo -\nsome 1p36, a region which spans potential \ncandidate genes WNT4, CDC42 and LINC00339 \n[1-4]. To further investigate the WNT4 locus we \nfirst sequenced the WNT4 coding regions and \nidentified two novel variants. One variant was a \nnon-synonymous variant coding for an amino \nacid substitution p.Tyr80His with no predicted \ndeleterious effect from in silico analysis. The \nsecond variant was a 3’UTR insertion/deletion \npredicted to be in a micro-RNA binding site for \nmiR-4767-5p and to change a binding site for \nmiR-3151, associated with melanoma [36] and \nchildhood acute lymphoblastic leukemia [37]. \nBoth novel variants were seen only in the fami -\nlies in which they were first detected. \nDuring the project, data on coding variants \nfrom sequencing ~12,000 individuals were \nreleased from the Exome Sequencing Project \n(ESP6500 data, Jun 2012) reducing the need \nfor further sequencing. For example the exome \ndata included our novel p.Tyr80His variant \n(rs115547783) with a minor allele frequency \n(MAF) of 0.00012 in the European American \npopulation. Genotyping all coding variants in \nFigure 2. Rare coding variants in WNT4 identified by High Resolution Melt analysis or from public databases and \ngenotyped in 930 cases and 959 controls.\nFigure 3. Rare coding variants in CDC42 identified by High Resolution Melt analysis or from public databases and \ngenotyped in 930 cases and 959 controls.\n\nWNT4 region and endometriosis risk\n200 Int J Mol Epidemiol Genet 2013;4(4):193-206\nTable 4. Results for association tests for imputed common variants in WNT4 genotyped in 930 endometriosis cases and 959 controls\nVariants Location (Hg19) Role N_variant MAF MAF in cases MAF in controls P value OR\nrs61768001 22465820 Intron 1-2 C/T 0.234# 0.202 0.156 2.002 x10-4 1.38 (1.16-1.63)\nrs12037376 22462111 Intron 1-2 A/G 0.224# 0.201 0.156 2.735 x10-4 1.37 (1.15-1.61)\nrs56318008 22470407 Promoter T/C 0.220# 0.190 0.147 3.860 x10-4 1.36 (1.15-1.62)\nrs55938609 22470451 Promoter C/G 0.230# 0.190 0.148 4.730 x10-4 1.36 (1.14-1.61)\nrs7412010 22436446 3’ near gene C/G 0.186* 0.203 0.160 7.696 x10-4 1.33 (1.13-1.57)\nrs7521902 22490724 Genomic A/C 0.235* 0.267 0.241 0.067 1.15 (0.99-1.33)\n*MAF estimate from HapMap CEU population (HapMap data release 28; phase 1, 2 & 3; August 10). #MAF estimate from phase 1 data from 1000 Genomes.\nTable 5. Functional regulation annotation for common SNPs in high LD (r2 ≥ 0.8) with rs61768001 and rs7521902 using HaploReg and Regu-\nlomeDB\nVariants Location (hg19) SNP score* Cons DNAseI HS peak (N) TFs binding Motifs changed Gene Genomic position\nCommon SNPs in high LD with rs61768001\nrs12038474 22403357 - SEF-1 CDC42 intronic \nrs7412010 22436446 - HES1 7.4 kb 3’ of WNT4 intergenic\nrs12404660 22458794 2b 45 CTCF YY1# WNT4 intronic\nrs12037376 22462111 5 6 SEF-1, WNT4 intronic\nrs61768001 22465820 5 9 Gabpa# WNT4 intronic\nrs3820282 22468215 5 +++ 33 Esr2, Esr1 WNT4 intronic\nrs56318008 22470407 5 34 Gfi1b 887 bp 5’ of WNT4 promoter\nrs55938609 22470451 4 31 FOXA1, FOXA2 931 bp 5’ of WNT4 promoter\nCommon SNPs in high LD with rs7521902\nrs72478520 22489567 4 11 IKZF1# 20 kb 5’ of WNT4 intergenic\nrs7521902 22490724 5 1 20 kb 5’ of WNT4 intergenic\nrs3920498 22492887 5 5 Smad3, TP53# 23 kb 5’ of WNT4 Intergenic\n*Scores from RegulomeDB. Prediction for SNP with score = 2b: TF binding + any motif + DNase Footprint + DNase peak. Prediction for SNP with score = 4: TF binding and DNase \npeak. Prediction for SNP with score = 5: TF binding or DNase peak. #Identified by various methods in a number of cell types (http://regulome.stanford.edu/). N is number of cell \ntypes in which DNase peaks marked.\n\nWNT4 region and endometriosis risk\n201 Int J Mol Epidemiol Genet 2013;4(4):193-206\nWNT4 and CDC42 reported in public databases \nidentified only 6 variants that were polymorphic \nin our sample. Two of these variants were rare, \nhad predicted functional effects and were \nfound only in cases. Tested either individually \nor together, the six rare variants did not provide \nFigure 4. ENCODE annotation of WNT4 GWA and imputed variants in endometriosis. (I) Overview of the region of \nchromosome 1 including the CDC42 and WNT4 genes. Genotyped markers are shown by vertical lines with results \n(log-10 P values) for all endometriosis cases in black and stage B endometriosis cases in brown. Peaks for histone \nmodification marks included in the ENCODE data [39]  are shown for H3K4Me1 on 7 cell types (Tier 1 and Tier 2). \nThe ENCODE data also shows areas of open chromatin as revealed by DNaseI, FAIRE and ChIP experiments in Tier \n1 and Tier 2 cell types indicated by solid blocks (Regions showing peaks defined by both DNase I and FAIRE assays \nare in black, highly significant regions with combinations of peaks from these assays are in blue. Less significant \nregions identified by only DNase I HS as peaks are in green). The peak signals of open chromatin by FAIRE for normal \nendometrial tissue are shown in the Endometrium FAIRE Pk track. (II) Regions of interest around nine imputed SNPs. \nThree SNPs with the strongest association signals are located in regions of open chromatin as shown by DNaseI \nsignals in the ENCODE data. (III) A zoomed-in image for the three top SNPs; (A) rs12404660, (B) rs3820282, and \n(C) rs55938609 (purple vertical lines) and functional annotation by ENCODE. Regulatory protein bound regions and \nhistone modifications indicated by ChIP-seq of H3K4m2 in a subset of Tier 1 and 2 cell types (in greyscale light (low-\nest) to dark (highest)). The level of conservation across the region is shown for mammals. (A) SNP rs12404660 lies \nin histone modification area for H3K4Me1, open chromatin location by DNaseI, FAIRE, and ChIP assays, transcrip -\ntion factors CTCF by ChIP and sequence motifs of transcription factor YY1 by footprinting assays (B) rs3820282 is \nlocated within sequence motifs for transcription factors ESR1 and ESR2 and (C) rs55938609 lies within sequence \nbound by the transcription factors FOXA1 and FOXA2.\n\nWNT4 region and endometriosis risk\n202 Int J Mol Epidemiol Genet 2013;4(4):193-206\nevidence for association with disease risk in \nour case-control sample. Our results demon -\nstrate no evidence that rare exonic or promoter \nregion variants in WNT4 or CDC42 were associ-\nated with disease risk. \nOur case-control sample for this study has only \n80% power to detect a genotype relative risk \n(GRR) of 1.4 at an allele frequency of 0.25 [14] \nand very low power to identify association with \nrare SNPs with low MAF unless the GRR was \nmuch greater (e.g. a GRR of at least 1.7 at an \nallele frequency of 0.05 is required for 80% \npower [14]). We chose familial cases for initial \ngenotyping because the frequencies of alleles \ncontributing to disease are generally enriched \nin these designs [38]. We have a further 4000 \ncases available for genotyping to increase \npower, but concluded that while effects of \nsome rare variants contributing to disease risk \nin carriers cannot be excluded, the evidence \ndid not justify further genotyping. All coding \nvariants were rare and the association signal in \nthis region cannot be explained by coding vari -\nants in either WNT4 or CDC42. \nAnalysis with imputed SNPs previously report -\ned genome-wide significant association for a \nnumber of common SNPs close to WNT4 and \nCDC42 [3]. Genotyping key common SNPs \ndemonstrated strong concordance (>98%) \nbetween observed and imputed genotypes and \nconfirmed previous suggestions that the \nstrongest association signals lie in the \nnon-coding regions around WNT4. \nHaplotype analyses of our common SNPs \nspanning the region show a single com -\nmon risk haplotype and this result sug -\ngests the observed association signal at \nthis locus is not being driven by a rare \ncausal variant in either the coding or non-\ncoding regions. The association signal is \nmost likely due to functional effects of one \nor more of the common non-coding SNPs \nin this haplotype block. \nUp to 88% of variants associated with \ncommon diseases are found within introns \nand intergenic regions [39] and it is likely \nthat causal variants function by altering \ngene regulation. To annotate potential \nfunctional roles of our top variants, we \nscreened 50 SNPs with the lowest P val -\nues from imputed data [3]. Three variants, \nrs12404660, rs3820282, and rs55938-  \n609, all in high LD with our top SNP \nFigure 5. Linkage disequilibrium (r 2) between genotyped and \nimputed common variants across the region of WNT4 and \nCDC42. \nrs61768001, had the best evidence for poten -\ntial functional effects in the ENCODE data as \nidentified by the HaploReg and RegulomeDB \nprograms [35]. All three variants are located in \nvalidated and significant regions of open chro -\nmatin with combinations of DNaseI HS and \nFAIRE peaks in multiple cell types ( Figure 4III) \n[39]. The three variants were also located in \nbinding sites for TFs or altered regulatory \nmotifs. SNP rs12404660 is located at a bind -\ning site for the transcription factor CTCF, and \nthe regulatory motif for YY1, TFs which function \nas enhancer blockers, transcriptional repres -\nsors and activators, and initiators of transcrip -\ntion [40-43]. \nSNP rs55938609 is located at a predicted TF \nbinding site for FOXA1 in T-47D cells and FOXA2 \nin HepG2 cells in the ENCODE data. FOXA1 \nmediates oestrogen receptor (ER) function and \nis a critical factor for interactions between ER \nand chromatin [44]. FOXA1 and H3K4me2 \ninteract at enhancers to increase gene tran -\nscription [45]. The minor A allele at rs3820282 \nmay alter regulatory motifs for oestrogen recep-\ntor (ESR1) identified in various cell types by \nfootprinting experiments [35] and ESR2 [34]. \nESR1 and ESR2 are involved in differentiation \nand proliferation of cells in target tissues \nincluding ovaries, uterus, and mammary gland \n[46-48]. Regulatory elements at these sites \n\nWNT4 region and endometriosis risk\n203 Int J Mol Epidemiol Genet 2013;4(4):193-206\noccur in multiple cell types, but functional stud-\nies of potential regulatory sites must be carried \nout in relevant target tissues to determine any \nrole in endometriosis risk. \nIn conclusion, we identified coding variants in \nWNT4 and CDC42 present only in women with \nendometriosis, but these variants were rare \nand there was no evidence for association with \nincreased disease risk. Genotyping key com -\nmon non-coding variants confirmed imputation \nresults showing a strong association signal cov-\nering the chromosome region that includes \nLINC00339, CDC42 and WNT4. The strong \nassociation signal is not explained by coding \nvariants in WNT4 or CDC42 and our genetic \ndata cannot determine which of the genes in \nthis region are affected. Functional studies on \ngene expression and gene regulation in rele -\nvant target tissues will be necessary to identify \nthe gene or genes responsible for increased \nendometriosis risk. SNPs located in sites with \npotential functional roles in binding of tran -\nscription factors FOXA1, FOXA2, ESR1 and \nESR2 indicate potential targets to prioritise for \nfuture functional experiments to understand \nthe molecular pathophysiology of endome-  \ntriosis.\nAcknowledgements\nWe thank L. Wallace, B. Haddon, D. Smyth, H. \nBeeby, and O. Zheng for project and database \nmanagement and sample processing. We \nthank Brisbane gynaecologist D.T. O’Connor for \nconfirmation of diagnosis and staging of dis -\nease from clinical records of many cases, \nincluding 251 in these analyses. We acknowl -\nedge with appreciation all the women who par -\nticipated in the QIMR and OXEGENE studies. \nWe thank Endometriosis Associations for sup -\nporting the study recruitment and the many \nhospital directors and staff, gynaecologists, \ngeneral practitioners and pathology services in \nAustralia who provided assistance with confir -\nmation of diagnoses. We thank S. Nicolaides \nand the Queensland Medical Laboratory for pro \nbono collection and delivery of blood samples \nand other pathology services for assistance \nwith blood collection. The study was supported \nby grants from the National Health and Medical \nResearch Council (NHMRC) of Australia \n(241944, 339462, 389927, 389875, 389891, \n389892, 389938, 443036, 442915, 442981, \n496610, 496739 552485, 552498, and \n1026033), the Cooperative Research Centre \nfor Discovery of Genes for Common Human Dis- \neases (CRC), Cerylid Biosciences (Melbourne) \nand donations from N. Hawkins and S. Hawkins. \nD.R.N. was supported by the NHMRC Fellowship \n(339462 and 613674) and the ARC Future \nFellowship (FT0991022) schemes and G.W.M. \nwas supported by the NHMRC Fellowships \nScheme (339446, 619667).\nDisclosure of conflict of interest\nNone.\nAddress correspondence to:  Dr. Grant W Mont-  \ngomery, Department of Genetics and Computational \nBiology, QIMR Berghofer Medical Research Institute, \nBrisbane, Queensland, Australia. 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