Patent Application: US-201113640845-A

Abstract:
the present invention is based on the identification of a number of genetic markers that are associated with susceptibility to paget &# 39 ; s disease of bone . this invention provides details of markers and nucleotide sequences as well as associated proteins / peptides and / or compositions and methods , for use in treating , preventing and / or detecting / diagnosing pdb and / or a susceptibility / predisposition thereto .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 : the first two component of multidimensional scaling analysis of ibs sharing matrix of all case ( red diamonds ) and control ( blue squares ) samples including hapmap project samples of european ( ceu ; green triangles ), asian ( chb + jpt ; cyan plus sign ), and african ( yri ; purple circles ) population . samples located outside the main ceu cluster ( greyed , n = 22 ) were excluded before testing for association with paget &# 39 ; s disease of bone . fig2 : a q - q plot showing the distribution of expected compared to observed − log 10 p for the association test results using stratified cochran - mantel - haenszel test before ( red squares ; λ = 1 . 096 ) and after genomic control adjustment ( blue diamonds ; λ = 1 . 0004 ). the green triangles represent the distribution of − log 10 p after removal of genome wide significant and correlated snps from chr1 , chr10 , and chr18 . fig3 : detection of loci conferring susceptibility to pdb by genome - wide association . manhattan plot of association test results from the discovery cohort showing chromosomal positions of the 294 , 633 snps passing quality control plotted against genomic control - adjusted − log 10 p . association with pdb was tested using stratified cmh tests . the red horizontal line indicates the threshold for genome - wide significance ( p & lt ; 1 . 7 × 10 − 7 ). fig4 : details of loci associated with pdb . ( a - c ) association and ld plots of regions showing genome - wide significant association with pdb located on ( a ) 1p13 , ( b ) 10p13 and ( c ) 18q21 . the chromosomal positions ( based on ncbi human genome build 36 ) of the snps are plotted against genomic control - adjusted − log 10 p . genotyped snps are shown as red triangles and imputed snps are blue diamonds . the estimated recombination rates ( cm / mb ) from hapmap ceu release 22 are shown as gray lines ; the red horizontal line indicates the genome - wide significance threshold ( p & lt ; 1 . 7 × 10 − 7 ). genotyped snps were tested using stratified cmh tests ; imputed snps were tested using a regression analysis based on imputed allelic dosage and adjusting for population clusters . snps reaching genome - wide significance are indicated with red text . ld plots for the indicated regions are based on hapmap ceu release 22 showing ld blocks depicted for alleles with maf & gt ; 0 . 05 using the r 2 coloring scheme of haploview 37 . the blue arrows indicate known genes in the region ; possible recombination hot spots (& gt ; 20 cm / mb ) are shown as green arrows on the ld plots . fig5 : linkage disequilibrium between snp at each of the three candidate loci are shown as depicted in the hapmap ceu panel ( top panel ), the discovery sample ( middle panel ), and replication sample ( bottom panel ). ld values shown are r 2 determined using haploview v 4 . 1 . fig6 : loci for susceptibility to pdb detected by genome wide association study . manhattan plot of association test results of gwas stage data showing chromosomal position of 2 , 487 , 078 genotyped or imputed snps plotted against genomic - control adjusted − log 10 p . the red horizontal line represents the threshold for genome wide significance ( p & lt ; 5 × 10 − 8 ). fig7 : regional association plots of loci showing genome wide significant association with pdb . details of loci on chromosome ( a ) 7q33 , ( b ) 15q24 . 1 , ( c ) 8q22 . 3 and ( d ) 14q32 . 12 showing the chromosomal position ( based on ncbi human genome build 36 ) of snps in each region plotted against − log 10 p values . genotyped ( squares ) and imputed ( circles ) snps are colour - coded according to the extent of linkage disequilibrium with the snp showing the highest association signal ( represented as purple diamonds ) from each region in the combined analysis the estimated recombination rates ( cm / mb ) from hapmap ceu release 22 are shown as light blue lines and blue arrows represent known genes in each region . the associated regions were defined based on ld with the highest association signal ( r 2 & gt ; 0 . 2 ) within a window of 500 kb . fig8 : forest plots of overall effect size for snps associated with pdb risk from the identified loci on ( a ) 7q33 ( rs4294134 ), ( b ) 8q22 . 3 ( rs2458413 ), ( c ) 14q32 . 12 ( rs10498635 ), and ( d ) 15q24 . 1 ( rs5742915 ). the overall effect size was estimated using meta - analysis of the gwas sample and the six replication samples . the black squares represent the effect estimates for the individual cohorts and the horizontal lines represent the 95 % confidence interval of the estimates . the sizes of the squares are proportionate to the weight of the estimate . the diamonds and triangles represent the overall estimate under fixed effect and random effects model , respectively . odds ratio ( or ) and their 95 % confidence interval ( ci ), p - values , i 2 statistics , and p - values for heterogeneity ( p het ) are shown next to the overall effect estimate . the dotted vertical lines represent the overall fixed effect estimates . fig9 : cumulative contribution of genome - wide significant loci to the risk of pdb . risk allele scores defined by the seven loci associated with pdb risk is plotted against the odds ratio ( or ) for pdb . risk alleles were weighted according to their estimated effect size and weighted risk allele scores were divided into ten equal parts ( deciles ) using data from the replication cohorts . the or for pdb risk was calculated for each decile in reference to the fifth decile ( d5 ). vertical bars represent 95 % confidence intervals . fig1 : linkage disequilibrium patterns of newly identified or confirmed loci associated with pdb risk . linkage disequilibrium ( ld ) patterns of genomic regions associated with pdb risk on ( a ) 7q33 ; ( b ) 8q22 . 3 ; ( c ) 14q32 . 12 ; and ( d ) 15q24 . 1 . ld patterns were derived from the ceu hapmap release 22 data using haploview 4 . 2 . blue arrows represent genes in the region and the snp showing the lowest p - values for association with pdb are shown in red text . fig1 : replication of association for the previously identified loci at csf1 , optn and tnfrsf11a . forest plots of overall effect size for snps associated with pdb risk from the previously identified loci on ( a ) 1p13 . 3 ( rs10494112 ), ( b ) 10p13 ( rs1561570 ), and ( c ) 18q21 . 33 ( rs3018362 ). the overall effect size was estimated by meta - analysis of the gwas sample and the six replication cohorts . the black squares represent the effect estimates for the individual cohorts and the horizontal lines represent the 95 % confidence interval of the estimates . the sizes of the squares are proportionate to the weight of the estimate for each cohort . the diamonds and triangles represent the overall estimates under fixed effect and random effects models , respectively . odds ratio ( or ) and their 95 % confidence interval ( ci ), p - values , i 2 statistics , and p - values for heterogeneity ( p het ) are shown next to the overall effect estimate . the dotted vertical lines represent the overall fixed effect estimates . fig1 : quantile - quantile plot of association test results a q - q plot showing the distribution of expected compared to observed − log 10 , p for the association test results before ( red squares ; λ = 1 . 051 ) and after genomic control adjustment ( blue diamonds ; λ = 1 . 00 ). the green triangles represent the distribution of − log 10 p values after removal of genome wide significant and correlated snps from the seven loci associated with pdb . fig1 : population ancestry in the gwas sample . multidimensional scaling analysis of ibs sharing matrix of pdb cases ( purple diamonds ) and controls ( blue squares ) including hapmap project samples of european ( ceu ; yellow circles ), asian ( chb + jpt ; orange squares ), and african ( yri ; green diamonds ) population . samples located outside the main ceu cluster ( grey , n = 6 ) were excluded before testing for association with paget &# 39 ; s disease of bone . the genome - wide association study was conducted in a discovery sample of 750 cases of predominantly british descent with clinical and radiological evidence of pdb in whom mutations of the sqstm1 gene had been excluded by dna sequencing . these comprised subjects who had participated in the prism study ( n = 597 ), a randomized trial of two different treatment strategies for pdb 38 ; clinic - based subjects from the uk with sporadic pdb ( n = 55 ); and subjects with a family history of pdb derived from the uk ( n = 20 ), australia ( n = 66 ), new zealand ( n = 8 ) and italy ( n = 4 ). details of the 1 , 002 control subjects have previously been described 9 ; in brief , they comprised healthy subjects of scottish descent with no clinical evidence of pdb . for the replication study , we conducted genotyping in an additional 500 pdb cases without sqstm1 mutations who were diagnosed according to standard techniques . these comprised subjects with sporadic pdb who had been recruited from hospital clinics in the uk ( n = 226 ), italy ( n = 20 ) and spain ( n = 200 ); subjects with sporadic pdb who had participated in the prism study ( n = 43 ); and subjects with a positive family history of pdb who had been recruited from hospital clinics in australia ( n = 10 ) and the uk ( n = 1 ). the 535 replication controls comprised subjects from the uk who had been referred for investigation of osteoporosis but who had been found to have normal bone density on examination by dual energy x - ray absorptiometry ( n = 248 ), spouses of participants of the prism study who were not known to be affected by pdb ( n = 252 ) and clinic - based controls from spain ( n = 35 ). all study participants were of european descent . the studies were approved by ethics review committees at the relevant institutions and all participants provided informed consent . the discovery sample had 96 % power to detect disease - associated alleles with maf = 0 . 2 and a genotype relative risk of 1 . 6 , assuming a multiplicative model and a disease with population prevalence of 2 %. genotyping of pdb cases was performed at the genetics core of the wellcome trust clinical research facility using illumina humanhap300 - duo beadchip v2 . genotyping of the controls had been previously performed by illumina inc . using humanhap300 v1 and humanhap240s arrays 9 . genotypes for cases and controls were called using beadstudio v3 . 2 ( illumina , inc .) by following the manufacturer &# 39 ; s recommended protocol . genotype data for control subjects were provided after applying the quality - control measures described previously 9 . for the cases , we used a no - call threshold of 0 . 15 in beadstudio and quality - control metrics such as cluster separation , ab t mean ( the mean normalized theta values of the heterozygote cluster ) and ab r mean ( the mean normalized intensity of the heterozygote cluster ) to exclude badly performing snps . samples with a call rate of less than 90 % were excluded ( n = 30 ). the data were then subjected to further quality - control measures using plink 39 to exclude snps with a call rate of less than 95 %, those with hardy - weinberg equilibrium p values of less than 1 . 0 × 10 − 4 in controls and those with a minor allele frequency of less than 1 %. this left a total of 294 , 663 snps common to cases and controls with at least 95 % call rates in each set . samples with excess heterozygosity ( 1 case ), non - european ancestry ( 21 cases and 1 control ) and related subjects ( 6 cases ) were excluded before analysis , leaving a final total of 692 cases and 1 , 001 controls with an average (± standard deviation ) genotype call rate of 99 . 63 ± 1 . 0 . the genotype cluster plots for all snps showing association with pdb at p & lt ; 1 . 0 × 10 − 4 were visually inspected in beadstudio . population ancestry was determined using multidimensional scaling analysis of the ibs distances matrix of all individuals after combining genotype data from the hapmap project ( release 22 ) samples of european ( ceu ), asian ( chb and jpt ) and african ( yri ) ancestry . for this analysis , we first removed snps in areas of extended ld ( chr . 2 : positions 134 . 0 - 138 . 0 ; chr . 6 : 25 . 0 - 34 . 0 ; chr . 8 : 8 . 0 - 12 . 0 ; chr . 11 : 45 . 0 - 57 . 0 ) 40 and those with r 2 & gt ; 0 . 2 within a 150 - snp window . snps with call rate & lt ; 99 %, maf & lt ; 5 % and hardy - weinberg equilibrium p & lt ; 1 . 0 × 10 in cases or controls were also excluded , leaving a total of 63 , 528 snps . the genome - wide average ibs distances matrix for all pairs of individuals was then calculated based on the total 63 , 528 snps using plink and was then used for multidimensional scaling analysis . fig1 is a plot of the first two components of the multidimensional scaling analysis showing three clusters corresponding to the ceu , chb with jpt , and yri samples , with the majority of cases and controls located within the european ceu cluster . we identified 21 cases and 1 control as outliers from the ceu cluster , and these were excluded from further analysis . based on genome - wide ibs distance , we identified five identical pairs ( ibs distance & gt ; 99 %) and one related pair ( ibs distance & gt ; 85 %) of samples from the cases cohort ; the sample with the lowest call rate was excluded from each pair before further analysis . genotyping of replication samples was performed by sequenom using the massarray iplex platform . dna from cases and controls was distributed into 384 - well plates so that each plate had the same number of cases and controls to minimize genotyping bias due to variations between runs . we included 100 samples from stage 1 as a quality - control measure . the concordance rate between illumina and sequenom platforms was & gt ; 99 . 9 %. replication samples with call rate & lt ; 95 % were excluded ( 19 cases and 15 controls ), leaving a total of 481 cases and 520 controls with an average genotype call rate of 99 . 61 %. the call rate of all the genotyped snps was & gt ; 95 %. genotypes were imputed using mach 41 for untyped variants located within 2 . 0 mb of snps identified in stage 1 as having genome - wide significant association with pdb . the hapmap ceu genotype data from release 22 were used as a reference . to avoid spurious association caused by inaccurate imputation of snps located at both ends of the imputed segments , analysis was restricted to snps located in the middle 2 mb of each 4 - mb imputed segment . we used 200 rounds of markov chain iterations to estimate allele dosage and the most likely genotypes of individuals in the stage 1 data . imputation quality was assessed by estimating the correlation ( r 2 ) between imputed and true genotypes . snps with r & lt ; 0 . 3 were excluded before further analysis . analysis of imputed data was performed using logistic regression implemented in mach2dat 42 in which the imputed allelic dosage was used to account for uncertainty in imputed genotypes . statistical analyses were performed using plink ( version 1 . 07 ) 39 . in stage 1 , genotyped snps were tested for association with pdb using a stratified cmh test . samples were stratified based on their genome - wide ibs similarity so that individuals assigned to one cluster were not genetically different ( p & gt ; 0 . 001 , obtained from a pairwise population concordance test ). the quantile - quantile plot and genomic control factor ( λ ) were used to assess overdispersion of the test statistics and were calculated using the statistical package r version 2 . 7 . 2 ( see urls ) based on the 90 % least significant snps as described previously10 , stepwise logistic regression was used to test for independent effects of an individual snp , where the allelic dosage of the conditioning snp was entered as a covariate in the regression model along with the population clusters identified by ibs - sharing analysis described above in order to adjust for population substructure . haplotype analysis was performed by logistic regression , which looked at the presence or absence of the test haplotype and included the population clusters as a covariate in the model . haplotypes were phased using the expectation - maximization algorithm implemented in plink , and only haplotypes with a frequency of ≧ 1 % were analyzed . the cutoff point for genome - wide significance was set as p & lt ; 1 . 7 × 10 − 7 ( 0 . 05 / 294 , 663 total snps ) for stage 1 , and p & lt ; 3 × 10 − 3 ( 0 . 05 / 16 total snps ) for the replication stage . for the combined analysis , we set the threshold for significance as p & lt ; 5 × 10 − 8 as recently proposed 43 . the replication and combined datasets were analyzed as described above except that the replication dataset was considered as a separate cluster when population clusters were used in a stratified cmh test or as a covariate in logistic regression models . the population attributable risk ( par ) for markers showing association with pdb was calculated according to the following formula : where p is the frequency of the risk allele in controls and or is the risk allele odds ratio . the cumulative par was calculated as follows : where n is the number of variants and pari is the individual par for the i th snp . urls . r , http :// www . r - project . org /. in this study , we sought to identify genetic variants that predispose to pdb in individuals without sqstm1 mutations by using a genome - wide association approach . in the discovery population ( stage 1 ), we genotyped 750 cases and 1 , 002 controls 9 using illumina arrays . in the replication population ( stage 2 ), we genotyped the most significant snps identified from stage 1 in an independent set of 500 cases and 535 controls using the sequenom massarray iplex platform . details of the subjects used in the discovery and replication stages of the study are provided in the online methods . we used a multidimensional scaling analysis of an identity - by - state ( ibs ) sharing matrix of all individuals plus hapmap samples to assess population ancestry ( fig1 ). after applying quality - control measures and excluding subjects with non - european ancestry , genotypic data in the discovery group were available for 294 , 663 snps in 692 cases and 1 , 001 controls . association testing of the discovery stage data was performed using a stratified cochran - mantel - haenszel ( cmh ) test in which samples were stratified according to their genome - wide ibs - sharing similarity . a quantile - quantile plot comparing the observed and expected distributions of − log 10 of p found by the cmh test showed some evidence for inflation of the test statistics given the multiple nationalities of pdb cases ( genomic inflation factor 10 λ = 1 . 096 ; fig2 ). to exclude the possibility of false positive association due to hidden population substructure , the observed test statistics were corrected using the genomic control method 10 . six snps showed genome - wide significant associations with pdb after bonferroni correction for multiple testing ( fig3 ). three were located within a 14 - kb region of chromosome 1p13 . 3 ( rs10494112 , rs499345 and rs484959 ), one was located on chromosome 10p13 ( rs1561570 ) and two were located within a 22 - kb region of chromosome 18q21 . 33 ( rs2957128 and rs3018362 ; table 1 , fig4 and table 2 ). we then used genotype data from hapmap to impute snps located within 1 . 0 mb of the six snps which reached genome wide significance . association testing of imputed snps revealed several variants with genome - wide significance located close to the six genotyped significant snps identified from stage 1 ( fig4 ). the imputed snps showed a slightly stronger association signal than the genotyped markers due to the conservative nature of the stratified cmh test used to analyze the genotyped markers as compared to the regression methods used to test imputed markers . in total , 76 snps with p values of 1 × 10 − 4 or less were identified in the discovery dataset ( table 3 ). from these , we selected those snps with p & lt ; 1 . 0 × 10 − 6 and also those with p & lt ; 1 . 0 × 10 − 5 in which an additional snp within 50 kb attained a p value of 1 . 0 × 10 − 3 or less for further analysis in the replication group . following application of quality - control measures on the replication dataset , genotype data were obtained for 481 cases and 520 controls for the 16 selected snps ( table 4 ). eight snps showed significant association with pdb in the replication stage after correction for multiple testing ( p & lt ; 3 × 10 − 3 ), resulting in the identification of eight snps for which the p values attained genome - wide significance in the combined dataset ( table 4 ). the distribution of minor alleles and the direction of associations were similar in both the discovery and replication datasets ( table 1 and table 4 ). although all samples used in the replication stage were from individuals of european ancestry , confounding due to population substructure is possible given the multiple nationalities of the replication cohorts . to address this issue , we tested for association in replication samples from individuals of british descent only ( 256 cases and 488 controls ) using the cmh test . this yielded results that were qualitatively similar to those obtained from the entire replication cohort ( table 5 ). linkage disequilibrium ( ld ) patterns in the associated regions were also similar across the study samples and were similar to those observed in hapmap ceu samples ( fig5 ). furthermore , the distribution of allele frequencies for snps showing genome - wide significant association to pdb was broadly similar across individuals in this study when grouped by origin ( table 6 ), and the replicated hits were not located in genomic regions with known geographic variation among european populations 11 , 12 . this indicates that the associations reported here are unlikely to be confounded by population substructure . significant association with pdb was observed on chromosome 1p13 . 3 for three snps ( rs10494112 , rs499345 and rs484595 ); the strongest signal was with rs484959 ( combined p = 5 . 38 × 10 − 24 ; table 1 and table 2 ). these snps were weakly correlated ( r 2 & lt ; 0 . 36 ) with other genotyped snps and are located in a 14 - kb ld block 87 kb upstream of csf1 ( fig4 ). another gene ( eps8l3 ) is located 47 kb further upstream but is separated from the associated snps by two recombination hot spots ( fig4 ), making it a less likely candidate . stepwise regression analysis that adjusted for population clusters and accounted for the genotypic additive effect of the three snps showed strong evidence for independent association with rs484959 ( p = 4 . 7 × 10 − 10 and rs10494112 ( p = 9 . 28 × 10 − 3 ) as compared to rs499345 ( p = 0 . 80 ; table 7 ). this is not surprising , as rs499345 is correlated with rs10494112 ( r 2 = 0 . 61 ) but rs484959 and rs10494112 are not ( r 2 = 0 . 21 ; fig5 ). an analysis of haplotypes formed by these three snps did not show a stronger association than an analysis of the individual snps ( table 8 ). csf1 encodes macrophage colony - stimulating factor ( m - csf ), which is a strong functional candidate for pdb susceptibility because it has a critical role in osteoclast formation and survival 13 , 14 . furthermore , loss - of - function mutations in rodent csf1 cause osteopetrosis due to failure of osteoclast differentiation 15 , 16 , whereas clinical studies have shown that individuals with pdb have increased serum levels of m - csf 17 . a second locus showing significant association with pdb was situated on chromosome 10p13 . three snps ( rs1561570 , rs825411 and rs2095388 ), all located within a 30 - kb region , were analyzed in both stages of the study and the strongest signal was observed for rs1561570 ( combined p = 6 . 09 × 10 − 13 ; table 1 and table 4 ). these three snps are weakly correlated with other genotyped snps ( highest r 2 & lt ; 0 . 37 ). rs825411 is not in ld with rs1561570 ( r 2 = 0 . 04 ) ( fig5 ) but did attain borderline genome - wide significance ( p = 7 . 82 × 10 − 8 ) and appeared to have an independent association with pdb as revealed by regression analysis accounting for the genotypic additive effect of rs1561570 ( p = 2 . 23 × 10 − 9 ) and rs825411 ( p = 5 . 15 × 10 − 6 ; table 7 ). haplotype analysis showed a stronger association signal for alleles formed by both rs1561570 and rs825411 combined as compared to a single - snp analysis , with the risk haplotype ‘ tc ’ showing the strongest association ( p = 2 . 50 × 10 − 17 , or = 1 . 67 ; table 8 ). the third snp ( rs2095388 ) showed no significant association with pdb in the combined analysis ( p = 3 . 08 × 10 − 6 ; table 4 ) and had no independent effect after accounting for rs1561570 and rs825411 in the analysis ( p = 0 . 14 ; table 7 ). the 10p13 locus is marked by two recombination hot spots and contains only one known gene , optn ( fig4 ). it is interesting to note that this region of chromosome 10p13 has been previously linked to familial pdb but the causal gene within this region has not been identified 6 . it is therefore possible that the risk haplotype could be tagging rare allele ( s ) within optn that markedly increase susceptibility to pdb . in this regard , there have been other reports in which genome - wide association studies have identified common variants that are associated with diseases in which the associated variants lie within regions previously mapped by linkage analysis . examples include variants associated with amyotrophic lateral sclerosis 18 and crohn &# 39 ; s disease 19 . alternatively , the risk haplotype could be tagging another common susceptibility variant within the gene . further studies will be required to investigate these possibilities . optn , which encodes optineurin , is a new candidate gene for pdb . mutations in optn have been linked to glaucoma 20 , but until now , optn has not been implicated in regulating bone metabolism . optineurin is a ubiquitously expressed cytoplasmic protein 21 that contains a ubiquitin - binding domain , similar to that present in the protein nemo . optineurin negatively regulates tnf - α - induced nf - κb activation by interacting with ubiquitylated rip proteins 22 . furthermore , a putative nf - κb binding site has been identified in the optn promoter 23 , and studies have shown that optineurin interacts with myosin vi , suggesting it plays a role in vesicular trafficking between the golgi apparatus and plasma membrane 24 . this is of interest because mutations affecting the vcp protein , which is also involved in vesicular trafficking , cause inclusion body myopathy with early - onset paget &# 39 ; s disease and frontotemporal dementia ( ibmpfd ) syndrome 25 . taken together , these data indicate that optineurin may have a hitherto unrecognized role in regulating bone metabolism through its effects on nf - κb signaling and / or vesicular trafficking . the third region showing a significant association with pdb was located on chromosome 18q21 . 33 near tnfrsf11a , which encodes the receptor activator of nf - κb ( rank ). four snps within a 300 - kb region reached genome - wide significance in the combined analysis ( rs663354 , rs2980996 , rs2957128 and rs3018362 ). regression analysis accounting for the genotypic - additive effect of the four snps showed that only rs2957128 ( p = 0 . 047 ) and rs3018362 ( p = 0 . 022 ) had independent effects ( table 7 ). analysis of haplotypes formed by alleles of rs2957128 and rs3018362 showed that a risk haplotype ‘ aa ’ was consistently over - represented in pdb cases compared with controls in the combined sample of cases and controls ( p = 8 . 71 × 10 − 14 , or = 1 . 55 ; table 8 ). these two snps are moderately correlated ( r 2 = 0 . 55 ) and are located in adjacent ld blocks about 5 kb downstream of tnfrsf11a ( fig4 c ). the tnfrsf11a gene product rank plays a critical role in osteoclast differentiation and function . mice with targeted disruption of tnftsf11a exhibit severe osteopetrosis due to complete absence of osteoclasts 26 , and loss - of - function mutations in tnfrsf11a cause osteoclast - poor osteopetrosis in humans 27 . mutations affecting the signal peptide region of rank cause the pdb - like syndromes of familial expansile osteolysis , early - onset familial pdb and expansile skeletal hyperphosphatasia 28 - 30 . mutations of tnfrsf11a have not so far been identified in individuals with classical pdb 28 , 31 , although this region of chromosome 18q22 has been linked to pdb in some families 32 . it is also interesting to note that rs3018362 and rs884205 , located downstream of tnfrsf11a , have recently been associated with bone mineral density and fracture risk 33 - 35 . the allele of rs3018362 that was associated with pbd was also associated with reduced bone mineral density , raising the possibility that this allele may be associated with increased bone turnover . rs884205 was not directly genotyped in our study , but it is moderately correlated with both rs3018362 ( r 2 = 0 . 53 ) and rs2957128 ( r 2 = 0 . 52 ). imputation analysis showed evidence for association of rs884205 with pdb ( imputed p value = 5 . 93 × 10 − 11 ), confirming the importance of tnfrsf11a in the genetic regulation of bone metabolism . the three loci on chromosomes 1p13 , 10p13 and 18q21 identified in this study appear to have independent roles , as we found no evidence to suggest that the associated snps within these loci interacted with each other to influence susceptibility to pdb ( p & gt ; 0 . 33 for all interlocus pairwise interactions ; table 9 ). these data are consistent with a multiplicative model for association with pdb . the cumulative population attributable risk for the snps showing independent association with pdb was 70 %. additionally , the risk of pdb increased with an increasing number of risk allele scores ( or per - risk allele = 1 . 34 , 95 % ci 1 . 29 - 1 . 40 , p = 5 . 81 × 10 − 45 ), with individuals carrying ten or more risk alleles having a sixfold increase in pdb risk compared to those with the median number of risk alleles ( table 10 ). it is likely that other genomic regions also contribute to pdb because the present study was powered only to detect variants with a moderate effect size ( risk allele or & gt ; 1 . 6 ). a quantile - quantile plot showing the distribution of p values after removal of all genome - wide significant snps and correlated markers showed an excess in the number of snps with low p values compared to what is expected by chance ( fig2 ). for example , we observed 19 snps with p & lt ; 1 × 10 − 5 compared to the expected 3 , suggesting that other risk variants with a modest effect remain to be identified . these may include variants located on chromosomes 3p24 , 8q22 , 10q24 and 14q32 , which did not reach genome - wide significance but could be considered suggestively associated with pdb ( combined p & lt ; 1 × 10 − 5 ; table 4 ). for example , results from analysis of the 8q22 locus in an extended cohort of 1401 cases and 3199 controls confirmed the association of variants in tm7sf4 gene with pdb risk ( p = 9 . 16 × 10 − 12 , table 1 ). tm7sf4 which encodes a dendritic cell - specific transmembrane protein ( dc - stamp ) plays an essential role in osteoclast differentiation , as reflected by the fact that osteoclast fusion was completely absent in cells cultured from mice with targeted inactivation of dc - stamp ( yagi et al 2005 ). also of particular interest is the 14q32 locus containing rin3 , encoding ras interaction / interference protein 3 , which is involved in vesicular trafficking36 and could be important in osteoclast function . in summary , we have demonstrated that common genetic variants at loci close to csf1 , optn , tm7sf4 and tnfrsf11a are independently associated with pdb . further studies are now warranted to explore the mechanisms responsible for these associations . 1 . cooper , c . et al . the epidemiology of paget &# 39 ; s disease in britain : is the prevalence decreasing ? j . bone miner . res . 14 , 192 - 197 ( 1999 ). 2 . siris , e . s . paget &# 39 ; s disease of bone . j . bone miner . res . 13 , 1061 - 1065 ( 1998 ). 3 . morales - piga , a . a ., rey - rey , j . s ., corres - gonzalez , j ., garcia - sagredo , j . m . & amp ; lopez - abente , g . frequency and characteristics of familial aggregation of paget &# 39 ; s disease of bone . j . bone miner . res . 10 , 663 - 670 ( 1995 ). 4 . laurin , n ., brown , j . p ., morissette , j . & amp ; raymond , v . recurrent mutation of the gene encoding sequestosome 1 ( sqstm 1 / p62 ) in paget disease of bone . am . j . hum . genet . 70 , 1582 - 1588 ( 2002 ). 5 . hocking , l . j . et al . domain - specific mutations in sequestosome 1 ( sqstm1 ) cause familial and sporadic paget &# 39 ; s disease . hum . mol . genet . 11 , 2735 - 2739 ( 2002 ). 6 . lucas , g . j . et al . identification of a major locus for paget &# 39 ; s disease on chromosome 10p13 in families of british descent . j . bone miner . res . 23 , 58 - 63 ( 2008 ). 7 . hocking , l . j . et al . genomewide search in familial paget disease of bone shows evidence of genetic heterogeneity with candidate loci on chromosomes 2q36 , 10p13 , and 5q35 . am . j . hum . genet . 69 , 1055 - 1061 ( 2001 ). 8 . laurin , n . et al . paget disease of bone : mapping of two loci at 5q35 - qter and 5q31 . am . j . hum . genet . 69 , 528 - 543 ( 2001 ). 9 . tenesa , a . et al . genome - wide association scan identifies a colorectal cancer susceptibility locus on 11q23 and replicates risk loci at 8q24 and 18q21 . nat . genet . 40 , 631 - 637 ( 2008 ). 10 . clayton , d . g . et al . population structure , differential bias and genomic control in a large - scale , case - control association study . nat . genet . 37 , 1243 - 1246 ( 2005 ). 11 . wellcome trust case control consortium . genome - wide association study of 14 , 000 cases of seven common diseases and 3 , 000 shared controls . nature 447 , 661 - 678 ( 2007 ). 12 . novembre , j . et al . genes mirror geography within europe . nature 456 , 98 - 101 ( 2008 ). 13 . tsurukai , t ., udagawa , n ., matsuzaki , k ., takahashi , n . & amp ; suda , t . roles of macrophage - colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis . j . bone miner . metab . 18 , 177 - 184 ( 2000 ). 14 . bouyer , p . et al . colony - stimulating factor - 1 increases osteoclast intracellular ph and promotes survival via the electroneutral na / hco3 cotransporter nbcn1 . endocrinology 148 , 831 - 840 ( 2007 ). 15 . van wesenbeeck , l . et al . the osteopetrotic mutation toothless ( tl ) is a loss - of - function frameshift mutation in the rat csf1 gene : evidence of a crucial role for csf - 1 in osteoclastogenesis and endochondral ossification . proc . natl . acad . sci . usa 99 , 14303 - 14308 ( 2002 ). 16 . wiktor - jedrzejczak , w et al . total absence of colony - stimulating factor 1 in the macrophage - deficient osteopetrotic ( op / op ) mouse . proc . natl . acad . sci . usa 87 , 4828 - 4832 ( 1990 ). 17 . neale , s . d ., schulze , e ., smith , r . & amp ; athanasou , n . a . the influence of serum cytokines and growth factors on osteoclast formation in paget &# 39 ; s disease . qjm 95 , 233 - 240 ( 2002 ). 18 . van es , m . a . et al . genome - wide association study identifies 19p13 . 3 ( unc13a ) and 9p21 . 2 as susceptibility loci for sporadic amyotrophic lateral sclerosis . nat . genet . 41 , 1083 - 1087 ( 2009 ). 19 . rioux , j . d . et al . genome - wide association study identifies new susceptibility loci for crohn disease and implicates autophagy in disease pathogenesis . nat . genet . 39 , 596 - 604 ( 2007 ). 20 . rezaie , t . et al . adult - onset primary open - angle glaucoma caused by mutations in optineurin . science 295 , 1077 - 1079 ( 2002 ). 21 . rezaie , t ., waitzman , d . m ., seeman , j . l ., kaufman , p . l . & amp ; sarfarazi , m . molecular cloning and expression profiling of optineurin in the rhesus monkey . invest . ophthalmol . vis . sci . 46 , 2404 - 2410 ( 2005 ). 22 . zhu , g ., wu , c . j ., zhao , y . & amp ; ashwell , j . d . optineurin negatively regulates tnfα - induced nf - κb activation by competing with nemo for ubiquitinated rip . curr . biol . 17 , 1438 - 1443 ( 2007 ). 23 . sudhakar , c ., nagabhushana , a ., jain , n . & amp ; swarup , g . nf - κb mediates tumor necrosis factor α - induced expression of optineurin , a negative regulator of nf - κb . plos one 4 , e5114 ( 2009 ). 24 . sahlender , d . a . et al . optineurin links myosin vi to the golgi complex and is involved in golgi organization and exocytosis . j . cell biol . 169 , 285 - 295 ( 2005 ). 25 . watts , g . d . et al . inclusion body myopathy associated with paget disease of bone and frontotemporal dementia is caused by mutant valosin - containing protein . nat . 26 . li , j . et al . rank is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism . proc . natl . acad . sci . usa 97 , 1566 - 1571 ( 2000 ). 27 . villa , a ., guerrini , m . m ., cassani , b ., pangrazio , a . & amp ; sobacchi , c . infantile malignant , autosomal recessive osteopetrosis : the rich and the poor . calcif . tissue int . 84 , 1 - 12 ( 2009 ). 28 . hughes , a . e . et al . mutations in tnfrsf11a , affecting the signal peptide of rank , cause familial expansile osteolysis . nat . genet . 24 , 45 - 48 ( 2000 ). 29 . nakatsuka , k ., nishizawa , y . & amp ; ralston , s . h . phenotypic characterization of early onset paget &# 39 ; s disease of bone caused by a 27 - bp duplication in the tnfrsf11a gene . j . bone miner . res . 18 , 1381 - 1385 ( 2003 ). 30 . whyte , m . p . & amp ; hughes , a . e . expansile skeletal hyperphosphatasia is caused by a 15 - base pair tandem duplication in tnfrsf11a encoding rank and is allelic to familial expansile osteolysis . j . bone miner . res . 17 , 26 - 29 ( 2002 ). 31 . wuyts , w . et al . evaluation of the role of rank and opg genes in paget &# 39 ; s disease of bone . bone 28 , 104 - 107 ( 2001 ). 32 . hocking , l . et al . familial paget &# 39 ; s disease of bone : patterns of inheritance and frequency of linkage to chromosome 18q . bone 26 , 577 - 580 ( 2000 ). 33 . styrkarsdottir , u . et al . multiple genetic loci for bone mineral density and fractures . n . engl . j . med . 358 , 2355 - 2365 ( 2008 ). 34 . rivadeneira , f . et al . twenty bone - mineral - density loci identified by large - scale meta - analysis of genome - wide association studies . nat . genet . 41 , 1199 - 1206 ( 2009 ). 35 . styrkarsdottir , u . et al . new sequence variants associated with bone mineral density . nat . genet . 41 , 15 - 17 ( 2009 ). 36 . saito , k . et al . a novel binding protein composed of homophilic tetramer exhibits unique properties for the small gtpase rab5 . j . biol . chem . 277 , 3412 - 3418 ( 2002 ). 37 . barrett , j . c ., fry , b ., maller , j . & amp ; daly , m . j . haploview : analysis and visualization of ld and haplotype maps . bioinformatics 21 , 263 - 265 ( 2005 ). 38 . langston , a . l . et al . randomized trial of intensive bisphosphonate treatment versus symptomatic management in paget &# 39 ; s disease of bone . j . bone miner . res . 25 , 20 - 31 ( 2010 ). 39 . purcell , s . et al . plink : a tool set for whole - genome association and population - based linkage analyses . am . j . hum . genet . 81 , 559 - 575 ( 2007 ). 40 . price , a . l . et al . long - range ld can confound genome scans in admixed populations . am . j . hum . genet . 83 , 132 - 135 ( 2008 ). 41 . li , y . & amp ; mach abecasis , g . r . 1 . 0 : rapid haplotype reconstruction and missing genotype inference . am . j . hum . genet . s 79 , 2290 ( 2006 ). 42 . li , y ., willer , c ., sanna , s . & amp ; abecasis , g . genotype imputation . annu . rev . genomics hum . genet . 10 , 387 - 406 ( 2009 ). 43 . pe &# 39 ; er , i ., yelensky , r ., altshuler , d . & amp ; daly , m . j . estimation of the multiple testing burden for genomewide association studies of nearly all common variants . genet . epidemiol . 32 , 381 - 385 ( 2008 ). raf is risk allele frequency , pdb is paget &# 39 ; s disease cases , or is odds ratio for the risk allele par defined as the expected reduction in disease load after removal of a risk allele . a results from extended cohort of 1401 pdb cases and 3199 controls snps reaching genome wide significance from each locus on chromosome 1p13 , 10p13 and 18q21 were tested for their independent effect using logistic regression analysis . results shown here are from the combined dataset . the allelic dosage of conditioned snps was entered as a covariate in the regression model along with population clusters . a risk allele score of the six snps showing independent association with pdb in the combined dataset ( chr . 1 : rs10494112 , rs484959 ; chr . 10 : rs1561570 and rs825411 ; chr . 18 : rs2957128 and rs3018362 ). allele scores were normally distributed in cases and controls . individuals carrying low - frequency scores ( allele scores labeled 0 and 1 and 11 and 12 ) were combined together . b ors are relative to the median number of risk alleles in the controls ( five risk alleles ). this study describes an extension to our previously reported gwas of pdb in which we used genotype data from 692 pdb cases from our previously described study 1 , and extended the case group by genotyping an additional 57 pdb cases . the additional cases were selected from recently recruited subjects in the prism study 23 ; a randomised trial of two different treatment strategies for pdb patients from the uk . we also increased the size of the control group by using genotype data from 2 , 930 subjects from the british 1958 birth cohort genotyped by the wellcome trust case - control consortium 7 . this control group represents a better match to our pdb cases than the previous controls which were recruited from scotland 1 since , like the prism participants , they were recruited from all over the uk . the extended samples size used in this study provided 90 % power to detect disease associated allele with maf = 0 . 2 and genotype relative risk of 1 . 4 assuming a multiplicative model and a disease with population prevalence of 2 %. this represents a substantial increase in power compared to our previous study 1 where we had 20 % power to detect alleles with genotyped relative risk of 1 . 4 . gwas stage genotyping and quality control . genotyping and quality control for the 692 pdb cases were performed using illumina humanhap300 - duo arrays as described previously 1 . the additional 57 pdb cases were genotyped using illumina human660w quad version 1 arrays and quality control measures were applied as previously described 1 . briefly ; snps with call rate & lt ; 95 % were excluded and samples with call rate & lt ; 90 % ( n = 1 ); excess heterozygosity ( n = 1 ); and non - european ancestry ( n = 6 ; supplementary fig4 ) were removed before analysis . the genotyping of the british 1958 birth cohort was previously performed by the wellcome trust case - control consortium using the illumina human 1 . 2m duo custom array ( www . wtccc . org . uk .) 7 . for the control group , snps with call rate & lt ; 95 % were excluded and we removed 231 samples because they failed at least one of the following quality control criteria : low call rate , non - european ancestry , gender mismatch , or cryptic relatedness . population ancestry was determined using multidimensional scaling analysis of identity - by - state ( ibs ) distances matrix as previously described 1 . after quality control , we analysed 741 pdb cases and 2 , 699 controls with genotype data for 290 , 115 snps which were common to the three different genotyping arrays . to ensure consistent genotyping between different platforms , a subset of samples were genotyped using at least two different platforms and cross - platform genotype concordance rate was & gt ; 99 . 7 % ( table 5 ( example 2 )). additionally , the genotype cluster plots for all snps showing association with pdb at p & lt ; 1 . 0 × 10 − 4 were visually inspected in cases and controls and only high quality genotype data were included in the analysis . furthermore , genotype call rate for the top associated snps was consistent between cases and controls ( table 6 ( example 2 )). the replication study groups were derived from clinic - based pdb patients and gender - matched controls selected from the same region . patients with sqstm1 mutations were excluded and all study participants provided informed consent . the first replication cohort comprised 175 pdb patients from the uk ; 8 pdb cases from sydney australia and 215 pdb cases from western australia . these patients were of british descent and were matched with 485 unaffected british controls . the second replication cohort ( italian replication cohort 1 ) comprised 354 pdb cases and 390 unaffected controls enrolled from various referral centres in italy who took part in the genpage project 24 . the third replication cohort ( italian replication cohort 2 ) comprised 205 italian pdb cases and 238 unaffected controls enrolled from referral centres in northern , central and southern italy as previously described 25 . the fourth replication cohort comprised 246 sporadic pdb patients recruited from various referrals centres in belgium and these were matched with 263 controls with no clinical evidence of pdb as previously described 8 . the fifth replication cohort comprised 85 pdb patients and 93 controls recruited from various centres in the netherlands as described 8 , 26 . the sixth replication comprised 186 sporadic pdb cases recruited from the salamanca region in the castilla - leon region of spain and 202 unaffected controls from the same region . genotyping of replication samples was performed by sequenom ( hamburg , germany ) using the massarray iplex platform . to minimize genotyping bias due to variations between runs ; dna from cases and controls from the six different replication cohorts were distributed into 384 well plates so that each plate had the same number of cases and controls . we included 4000 known genotypes as a quality control measure and the concordance rate between the genotype calls was & gt ; 99 . 8 %. we removed 64 samples due to low call rate (& lt ; 90 %) and the call rate for all genotyped snps was & gt ; 95 %. genome - wide genotype imputation for autosomal snps was performed using mach 27 and the hapmap european ( ceu ) phased haplotype data from release 22 were used as a reference . we excluded snps with poor imputation quality based on the estimated correlation between imputed and true genotypes ( r 2 & lt ; 0 . 3 ). additionally , a subset ( 2 %) of known genotypes were masked during imputation and then imputed genotypes were compared with true genotypes and the average per allele imputation error rate was 2 . 9 %. imputed snps were tested for association using porbabel software 28 implementing a logistic regression model in which the allelic dosage of imputed snp was used to adjust for uncertainty in imputed genotypes . statistical analyses were performed using plink ( version 1 . 07 ) 29 and r ( v2 . 11 . 1 ). in gwas stage , genotyped snps were tested for association with pdb using standard allelic ( 1 . d . f ) χ 2 statistic . we also performed association testing using regression models in which we adjusted for gender , population clusters ( as determined by multidimensional scaling analysis ) but results were essentially identical to those obtained from the standard allelic test reported here ( data not shown ). the genomic inflation factor λ was calculated based on the 90 % least significant snps as described previously 30 . the observed test statistic values were corrected using the genomic control method ( λ = 1 . 05 ; supplementary fig3 ). logistic regression was used to test for independent effects of snps where the allelic dosage of the conditioning snp was entered as a covariate in the regression model . the cut off point for genome wide significance was set as p & lt ; 5 × 10 − 8 as recently proposed 31 . association testing of replication data was performed in each replication cohort using standard ( 1 . d . f ) χ 2 statistic . to assess combined genetic effects , we performed meta - analysis of all studies using the inverse - variance method assuming fixed - effect model . we also tested random - effects model using dersimonian - laird method 32 and between - study heterogeneity was assessed using the cochran &# 39 ; s q and i 2 metrics . heterogeneity was considered significant if p het & lt ; 0 . 05 . the population attributable risk ( par ) for markers showing association with pdb was calculated according to the following formula : where p is the frequency of the risk allele in controls and or is the risk allele odds ratio . the cumulative par was calculated as follows : where n is the number of variants and par i is the individual par for the ith snp . the proportion of familial risk attributable to the identified loci was calculated as previously described 33 assuming a multiplicative model of association and a sibling relative risk λ s = 7 . 0 as estimated from previous epidemiological studies 20 . regional association plots were generated using the locuszoom tool 34 . snps showing genome wide significant association with pdb ( or those in strong ld ; d ′≧ 0 . 8 ) were tested for association with cis - allelic expression of gene transcripts located in the associated regions using publicly available eqtl data 22 , 35 - 38 . only cis - acting allelic associations located within 250 kb of either 5 ′ or 3 ′ end of the associated gene with expression p - value & lt ; 1 × 10 − 5 were considered . to avoid false detection , we excluded expression data if the gene probe contained a polymorphic snp or was located in a highly repetitive sequence . in example 1 we describe the identification of susceptibility alleles for pdb at the csf1 , optn , and tnfrsf11a loci by a genome wide association study involving 692 pdb cases and 1 , 001 controls with replication cohort of 481 cases and 520 controls 1 . in order to identify additional susceptibility loci for the disease , we performed an extended gwas involving a total of 749 pdb cases of british descent in whom sqstm1 mutations had been excluded and 2 , 930 british controls derived from the 1958 birth cohort 7 with replication in a further 1 , 474 cases and 1 , 671 controls from six independent populations . after applying quality control measures and excluding samples of non - european ancestry , the extended cohort ( henceforth referred to as the gwas stage ) comprised 741 cases and 2 , 699 controls with genotype information for 290 , 115 snps , providing a 4 - fold increase in power to detect loci of moderate effect size ( odds ratio ≧ 1 . 4 ) compared with our previous study 1 . we then genotyped the highest ranking snps identified from the gwas stage in six independent replication cohorts of sqstm1 - negative pdb cases and matched controls from the uk , australia , italy , spain , the netherlands , and belgium . details of the study groups are provided in the online methods section and table 2 ( example 2 ). to increase snp coverage , we performed genome wide snp imputation for the gwas stage samples using phased haplotype data from the hapmap project as a reference . the results of association testing of genotyped and imputed snps ( total 2 , 487 , 078 snps ) from the gwas stage are shown in fig6 . a locus on chromosomes 8q22 . 3 showed genome - wide evidence of association with pdb ( p & lt ; 5 . 0 × 10 − 8 ) in addition to the previously identified genome wide significant loci on 1p13 . 3 , 10p13 and 18q21 . 33 1 . the 8q22 . 3 locus was suggestively associated with pdb risk in our previous study ( rs2458413 ; p = 4 . 14 × 10 − 7 ) 1 but the same snp reached genome wide significance in the present study ( rs2458413 ; p = 7 . 85 × 10 − 11 ; or = 1 . 51 ). in the second stage of this study we analysed the highest ranking snps observed in the gwas stage ( p values of 5 × 10 − 5 or less ) for replication after excluding those in linkage disequilibrium ( ld ; r 2 & gt ; 0 . 8 or d ′& gt ; 0 . 95 ) with the highest ranking snp from each region . a total of 27 snps were genotyped in the replication cohorts which consisted of 1 , 474 pdb cases from six different geographic regions and 1 , 671 unaffected controls from the same regions that were matched with the cases by gender as described in the online methods section and table 2 ( example 2 ). a meta - analysis of data from the gwas stage and individual replication cohorts was performed under fixed and random effects models and the results are summarised in table 3 ( example 2 ). this strengthened the association with pdb for the csf1 , optn , and tnfrsf11a loci which were identified in our previous study 1 and confirmed the association with 8q22 . 3 locus which was suggestively associated with pdb in our previous gwas and was confirmed to be associated with pdb in a small study of belgian and dutch subjects 8 . furthermore , three additional genome wide significant loci on 7q33 , 14q32 . 12 , and 15q24 . 1 were identified in the combined data set ( p & lt ; 5 × 10 − 8 ; table 1 ( example 2 )). to assess if the reported associations were confounded by age , age of onset or recruitment centre , we performed a regression analysis using case - only data from the gwas stage to test if any of these factors were associated with the top hits using linear regression models . the results of this analysis showed no evidence to suggest that the reported association is confounded by age ( p & gt ; 0 . 11 ), age of onset ( p & gt ; 0 . 10 ) or recruitment centre ( p & gt ; 0 . 44 ). the strongest signal on 8q22 . 3 was with rs2458413 ( combined p - value = 7 . 38 × 10 − 17 ; or = 1 . 4 ). there was no significant heterogeneity between the study groups ( i 2 = 44 . 3 %; p het = 0 . 10 ; table 1 ( example 2 ), fig8 and table 3 ( example 2 )) and the direction of association was similar in all cohorts . the associated region spans ˜ 220 kb and contains three known genes ( rims2 , tm7sf4 , and dpys ) but snps with the highest association signal appear to cluster within an 18 - kb ld block spanning the entire transmembrane 7 superfamily member 4 gene ( tm7sf4 ; fig7 and supplementary fig1 ). this gene encodes dendritic cell - specific transmembrane protein ( dc - stamp ) 9 which is a strong functional candidate gene for pdb since it is required for the fusion of osteoclast precursors to form mature osteoclasts 10 . previous studies have shown that rankl induced dc - stamp expression is essential for osteoclast formation 11 and in a recent study ; nishida et al showed that the connective tissue growth factor ccn2 stimulates osteoclast fusion through interaction with dc - stamp 12 . since osteoclasts from patients with pdb are larger in size and contain more nuclei than normal osteoclasts , it seems likely that the genetic variants that predispose to pdb do so by enhancing tm7sf4 expression or by causing gain - of - function at the protein level but further studies will be required to investigate these possibilities . the first new locus for pdb susceptibility was on 7q33 tagged by rs4294134 ( combined p - value = 8 . 45 × 10 − 10 ; or = 1 . 45 ). the direction of association was similar in all study cohorts and analysis of the combined data set showed no evidence for heterogeneity between study groups ( i 2 = 0 %; p het = 0 . 83 ; table 1 ( example 2 ), fig8 and table 4 ( example 2 )). the associated region spans ˜ 350 kb and contains three known genes ( cnot4 , nup205 , and slc13a4 ) and two predicted protein coding transcripts ( pl - 5283 and fam180a ; fig7 ). the strongest signal was with rs4294134 , located within the 22 nd intron of nup205 . this gene encodes a protein called nucleoporin 205 kda which is one of the main components of the nuclear pore complex involved in the regulation of transport between the cytoplasm and nucleus 13 . all snps with p & lt ; 1 × 10 − 5 in the 350 kb associated region were in moderate to strong ld with rs4294134 ( r 2 0 . 5 ; d ′ 0 . 95 ) with the exception of two snps ( rs3110788 and rs3110794 ) which were poorly correlated with rs4294134 ( r 2 ≦ 0 . 21 ; d ′≧ 0 . 95 ; fig7 ). conditional analysis in the gwas stage indicated that the association signal appeared to be driven by rs4294134 ( p = 8 . 8 × 10 − 3 ) after adjusting for rs3110788 ( p = 0 . 31 ) and rs3110794 ( p = 0 . 10 ). none of the three genes located in this region are known to affect bone metabolism and further studies will be required to identify the functional variant ( s ) responsible for association with pdb . the second new susceptibility locus was located on 14q32 . 12 and was tagged by rs10498635 . this snp showed borderline evidence of association with pdb in our previous study ( p = 9 . 69 × 10 − 8 ) 1 but reached genome - wide significance in the present study ( combined p - value = 2 . 55 × 10 − 11 ; or = 1 . 44 ). association testing showed no evidence for heterogeneity between the study groups v ′= 0 . 0 %; p het = 0 . 62 ; table 1 ( example 2 ), fig8 and table 4 ( example 2 )). the 62 kb - associated region is bounded by two recombination hotspots and contains the gene rin3 ( fig7 ) that encodes the ras and rab interactor 3 , a protein that plays a role in vesicular trafficking through interaction with small gtpases such as ras and rab 14 , 15 . the function of rin3 in bone metabolism is currently unknown , but it could play a role in bone resorption in view of the importance that small gtpases play in vesicular trafficking and in osteoclast function 16 , 17 . it is of interest to note that mutations affecting the vcp , a protein also involved in vesicular trafficking , cause the syndrome of inclusion body myopathy with early - onset paget &# 39 ; s disease and frontotemporal dementia ( ibmpfd ) 18 . the third new susceptibility locus was located on 15q24 . 1 and the strongest association was with rs5742915 ( combined p - value = 1 . 60 × 10 − 14 ; or = 1 . 34 ; i 2 = 0 . 0 %; p het = 0 . 56 ; table 1 ( example 2 ), fig8 and table 4 ( example 2 )). the associated region is bounded by two recombination hot spots and spans ˜ 200 kb but a gap spanning ˜ 40 kb was observed in this region with no snp coverage in the illumina arrays or the hapmap ceu population . the associated snps were clustered within the promyelocytic leukaemia gene ( pml ; fig7 ) and the strongest signal was observed for rs5742915 , which results in a phenylalanine to leucine amino acid change at codon 645 ( f645l ) of the pml protein . the function of pml in bone metabolism is unclear but it is known to be involved in tgf - β signalling 19 . accordingly lin et al showed that cells from pml knock out mice were resistant to tgf - β - dependent growth arrest and apoptosis , had impaired induction of tgf - β target genes , and exhibited abnormal nuclear translocation of the tgf - β signalling proteins smad2 and smad3 19 . since tgf - β is known to play a role in the regulation of bone remodelling , it is possible that the association between pdb and pml could be mediated by an effect on tgf - β signalling , but further research will be required to investigate this possibility . the golga6a gene is also located in the associated region and encodes a protein that belongs to golgin , a family of coiled - coil proteins associated with the golgi apparatus and play a role in membrane fusion and as structural supports for the golgi cisternae . this gene is located in the 40 kb gap region that contains a large low - copy repeat sequence , but its function in bone metabolism is unknown . we were also able to replicate our previously reported association between variants at the csf1 , optn , and tnfrsf11a loci and pdb in the present study . the results of meta - analysis of the combined data set for these loci are shown in table 1 ( example 2 ) and supplementary fig2 which provide conclusive evidence for association of variants at csf1 ( rs10494112 ; p - value = 7 . 06 × 10 − 35 ; risk - allele or = 1 . 72 ; i 2 = 0 . 0 %; p het = 0 . 97 ), optn ( rs1561570 ; p - value = 4 . 37 × 10 − 38 ; risk - allele or = 1 . 67 ; i 2 = 65 . 7 %; p het = 0 . 01 ), and tnfrsf11a ( rs3018362 ; p - value = 7 . 98 × 10 − 21 ; risk - allele or = 1 . 45 ; i 2 = 0 . 0 %; p het = 0 . 46 ) with pdb . evidence of heterogeneity between study groups was observed for rs1561570 at optn but this was due to differences in effect size rather than the direction of effect and the association remained genome wide significant after accounting for heterogeneity using the random effects model ( p = 4 . 34 × 10 − 12 ; or = 1 . 68 ). the heterogeneity was caused by larger effect size observed in the dutch cohort ( supplementary fig2 ) possibly due to the small sample size of this cohort . these observations provide highly robust evidence for association between these loci and pdb and extend those recently reported 8 in the dutch and belgian populations which were also included in the present study . we next wanted to determine if the identified loci on 15q24 . 1 , 7q33 and 14q32 . 12 interacted with each other or with the previously identified loci on 1p13 . 3 , 8q22 . 3 , 10p13 and 18q21 . 33 to affect the risk of pdb . pair - wise interaction analysis showed weak evidence for interaction between 7q33 ( rs4294134 ) with 8q22 . 3 ( rs2458413 ; p = 0 . 03 ) and 10p13 ( rs1561570 ; p = 0 . 02 ). however , these interactions were not significant after adjusting for multiple testing and none of the other loci showed evidence for interaction ( p & gt ; 0 . 05 ) suggesting a multiplicative model of association with pdb risk . in order to estimate the effect size of the identified loci on the development of pdb , we calculated the proportion of familial risk explained by the genome wide significant loci in the replication sample assuming a sibling relative risk for pdb of 7 . 0 20 . this showed that the proportion of familial risk explained was ˜ 13 % which is much greater than observed for other bone diseases like osteoporosis 21 . we also estimated the cumulative population attributable risk of these loci in the replication cohort and found it to be 86 % and we found that the risk of pdb increased with increasing number of risk allele scores defined by the seven loci ( or per - riskallele = 1 . 44 , 95 % ci = 1 . 38 - 1 . 51 , p = 5 . 4 × 10 − 57 ). when allele scores were weighted according to their estimated effect size we found that subjects in the top 10 % of the allele score distribution ( d10 ; n = 315 ) had 10 . 1 fold ( 95 % ci ; 7 . 0 - 14 . 6 ) increase in risk of developing pdb compared to those in the bottom 10 % of the distribution ( d1 ; n = 315 ) from the replication dataset ( fig9 ). although these data suggest that a large part of the genetic risk of pdb in patients without sqstm1 mutations is accounted for by these loci , we acknowledge that the functional variants need to be identified before we can precisely estimate the contribution that these loci make to the risk of developing pdb . to assess the functional effect of the identified snps on gene expression , we tested the association between top pdb - associated snps ( or those in ld ; d ′≧ 0 . 8 ) from each of the seven loci and cis - allelic expression of genes located in the associated regions using publicly available expression quantitative trait loci ( eqtl ) data . this showed highly significant associations for transcripts of tm7sf4 ( rs2458415 ; expression p - value = 1 . 22 × 10 − 18 ) and optn ( rs1561570 ; expression p - value = 6 . 61 × 10 − 62 ) in peripheral blood monocytes 22 suggesting that the association with pdb risk for these loci could be mediated by influencing gene expression levels . in addition to the loci mentioned above , additional variants were identified that showed suggestive evidence for association with pdb . for example a locus on chromosome xq24 showed borderline evidence for association with pdb ( rs5910578 within slc25a43 gene ; combined p = 1 . 26 × 10 − 7 ; or = 1 . 34 ; p het = 0 . 44 ; i 2 = 0 . 0 %) as did another locus on chromosome 6p22 . 3 ( rs1341239 near prl gene ; combined p = 3 . 83 × 10 − 6 ; or = 1 . 20 ; p het = 0 . 63 ; i 2 = 0 . 0 %; table 3 ( example 2 )). given that we observed 6 genotyped variants with p & lt ; 1 × 10 − 5 in the gwas stage after removal of confirmed snps and associated variants when we only expect 3 by chance ( supplementary fig3 ), it is likely that some of the associations observed are true but our study was not sufficiently powered to detect them at a genome wide significance level ( p & lt ; 5 × 10 − 8 ). this study has been successful in identifying seven loci that contribute substantially to the risk of developing pdb . the identified loci have relatively large effect sizes compared with other common diseases such as osteoporosis and rheumatoid arthritis . this indicates that susceptibility to pdb is most probably mediated by inheritance of a relatively small number of genes with large effect sizes as opposed to a large number of genes with small effect sizes as seen in other complex diseases . many of the susceptibility variants lie within or close to genes that are known to play important roles in regulating osteoclast differentiation and function whereas other variants lie within genes not previously implicated in the regulation of bone metabolism . whilst further work will be required to identify the functional variants , the present study has provided new insights into the genetic architecture of pdb and has identified several genes that previously were not suspected to play a role in bone metabolism . finally , the large effect size of the variants identified means that it may be possible in the future to identify people at risk of developing pdb by genetic profiling . 1 . cooper , c . et al . the epidemiology of paget &# 39 ; 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ralston , s . h . phenotypic characterization of early onset paget &# 39 ; s disease of bone caused by a 27 - bp duplication in the tnfrsf11a gene . j . bone miner . res . 18 , 1381 - 1385 ( 2003 ). 30 . whyte , m . p . & amp ; hughes , a . e . expansile skeletal hyperphosphatasia is caused by a 15 - base pair tandem duplication in tnfrsf11a encoding rank and is allelic to familial expansile osteolysis . j . bone miner . res . 17 , 26 - 29 ( 2002 ). 31 . wuyts , w . et al . evaluation of the role of rank and opg genes in paget &# 39 ; s disease of bone . bone 28 , 104 - 107 ( 2001 ). 32 . hocking , l . et al . familial paget &# 39 ; s disease of bone : patterns of inheritance and frequency of linkage to chromosome 18q . bone 26 , 577 - 580 ( 2000 ). 33 . styrkarsdottir , u . et al . multiple genetic loci for bone mineral density and fractures . n engl . j . med . 358 , 2355 - 2365 ( 2008 ). 34 . rivadeneira , f . et al . twenty bone - mineral - density loci identified by large - scale meta - analysis of genome - wide association studies . nat . genet . 41 , 1199 - 1206 ( 2009 ). 35 . styrkarsdottir , u . et al . new sequence variants associated with bone mineral density . nat . genet . 41 , 15 - 17 ( 2009 ). 36 . saito , k . et al . a novel binding protein composed of homophilic tetramer exhibits unique properties for the small gtpase rab5 . j . biol . chem . 277 , 3412 - 3418 ( 2002 ). 37 . barrett , j . c ., fry , b ., mailer , j . & amp ; daly , m . j . haploview : analysis and visualization of ld and haplotype maps . bioinformatics 21 , 263 - 265 ( 2005 ). 38 . langston , a . l . et al . randomized trial of intensive bisphosphonate treatment versus symptomatic management in paget &# 39 ; s disease of bone . j . bone miner . res . 25 , 20 - 31 ( 2010 ). 39 . purcell , s . et al . plink : a tool set for whole - genome association and population - based linkage analyses . am . j . hum . genet . 81 , 559 - 575 ( 2007 ). 40 . price , a . l . et al . long - range ld can confound genome scans in admixed populations . am . j . hum . genet . 83 , 132 - 135 ( 2008 ). 41 . li , y . & amp ; mach abecasis , g . r . 1 . 0 : rapid haplotype reconstruction and missing genotype inference . am . j . hum . genet . s 79 , 2290 ( 2006 ). 42 . li , y ., willer , c ., sanna , s . & amp ; abecasis , g . genotype imputation . annu . rev . genomics hum . genet . 10 , 387 - 406 ( 2009 ). 43 . pe &# 39 ; er , i ., yelensky , r ., altshuler , d . & amp ; daly , m . j . estimation of the multiple testing burden for genomewide association studies of nearly all common variants . genet . epidemiol . 32 , 381 - 385 ( 2008 ). details of the number , age , and gender of cases and controls from each of the replication cohorts is shown . the distribution of males and females was not statistically different between cases and controls in any of the cohorts studied ( p & gt ; 0 . 18 ). 1 as a quality control measure , a subset of pdb samples used in the gwas stage were genotyped on two different platforms and genotype concordance rate was calculated . for control samples which were genotyped by wtccc using illumina human 1 . 2m - duo chip , the same samples were also genotyped using affymetrix v6 . 0 and genotype concordance rate was calculated for the 88 , 146 overlapping snps ( out of 290 , 115 snps used in gwas analysis ). only genotypes from illumina human 1 . 2m chip were used for gwas analysis .