families used for initial screening were selected from nine different sources : the four type 1 diabetes genetics consortium ( t1dgc ) networks ( asia - pacific [ n = 228 ] , europe [ n = 585 ] , north america [ n = 370 ] , and the u.k . [ n = 124 ] ) , the diabetes u.k . warren repository ( n = 429 ) , the human biological data interchange repository ( n = 424 ) , the joslin diabetes center ( n = 112 ) , the steno diabetes center ( n = 146 ) , and sardinia ( n = 78 ) . details on recruitment sites and the numbers of families contributed for each of the t1dgc networks are accessible from the public side of the t1dgc web site ( www.t1dgc.org ) . minimum entry criteria for families included the presence of at least one affected sibling pair , and availability of one or both biological parents was preferred . eligibility requirements for affected individuals included documented type 1 diabetes with onset earlier than 35 years of age , insulin use within 6 months of diagnosis , and the absence of any concomitant disease or disorder associated with diabetes . in total , 2,496 families were genotyped . both parents were available for 72.9% of families ; 18.8% had only a single parent . most families ( 91.7% ) had exactly two affected full siblings ; 4.1% had three and 0.3% had four or more affected siblings . the family replication set of samples consisted of 2,214 parent - affected child trio families derived from two sources . the genetics of kidneys in diabetes ( gokind ) study of diabetic nephropathy contributed 578 families . affected offspring in these families had type 1 diabetes diagnosed before 31 years of age , had initiated insulin therapy within 1 year of diagnosis , and had continued insulin treatment uninterrupted since initiation . the remaining 1,636 families were selected from the danish society of childhood diabetes and were all of danish caucasoid origin . probands in these families were diagnosed with type 1 diabetes before 15 years of age and had continued insulin treatment since diagnosis . the case - control replication set consisted of 17,400 subjects , including 7,721 type 1 diabetic case subjects recruited as part of the juvenile diabetes research foundation international / wellcome trust british case collection and 9,679 control subjects from the british 1958 birth cohort and the wellcome trust case control blood service ( 13,17 ) . families included in the original genome - wide linkage scan were all genotyped at the center for inherited disease research using the illumina human linkage-12 genotyping beadchip consisting of 6,090 snps . genotyping of snp rs876498 in replication samples was performed using an eclipse genotyping assay ( nanogen ) . as quality control for accuracy of this assay , a blinded resampling and genotyping of 366 families with type 1 diabetes from the initial genome - wide scan was performed . before statistical genetic analyses , genotype data were evaluated for mendelian errors using pedcheck ( 18 ) . prest ( 19 ) was used to estimate the likelihood of each specified relationship in pedigrees given the genotyping information . based on these analyses , 43 families were removed from further analysis due to the presence of a duplicate family , a duplicate sample within the family , nonresolvable family errors , or missing genotype information . allelic association with type 1 diabetes within multiplex families was assessed using the pedigree disequilibrium test ( pdt ) ( 20 ) , which provides a valid test of association in the presence of linkage in families with multiple affected individuals . the pdt examines the discrepancy between the alleles of heterozygote parents and those transmitted to affected offspring , as well as the allelic difference between affected and unaffected siblings . both pdt - sum and pdt - ave tests ( 21 ) were carried out . because these tests are asymptotically equivalent , the pdt - ave test , in which counts of trios and pairs for each pedigree are weighted , is primarily reported here . plink ( v.1.02 ) ( 22 ) was used to assess allelic association to type 1 diabetes in trio families , via the transmission / disequilibrium test ( 23 ) , and in case - control samples . a logistic regression model was used to estimate effect sizes , with familial correlations being taken into account by the generalized estimating equation method ( 24 ) . to combine results of the original genome scan and those of follow - up studies , a weighted z score , a z statistic summarizing the magnitude of the p value for association and direction of effect was generated for each study . an overall z statistic was then computed as a weighted average of the individual statistics , and a corresponding p value for that statistic was computed . the weights were proportional to the square root of the number of individuals in each study and scaled such that the squared weights summed to 1 . for the meta - analysis of the effect size , the inverse variance was used as the weight for each study . families included in the original genome - wide linkage scan were all genotyped at the center for inherited disease research using the illumina human linkage-12 genotyping beadchip consisting of 6,090 snps . genotyping of snp rs876498 in replication samples was performed using an eclipse genotyping assay ( nanogen ) . as quality control for accuracy of this assay , a blinded resampling and genotyping of 366 families with type 1 diabetes from the initial genome - wide scan was performed . before statistical genetic analyses , genotype data were evaluated for mendelian errors using pedcheck ( 18 ) . prest ( 19 ) was used to estimate the likelihood of each specified relationship in pedigrees given the genotyping information . based on these analyses , 43 families were removed from further analysis due to the presence of a duplicate family , a duplicate sample within the family , nonresolvable family errors , or missing genotype information . allelic association with type 1 diabetes within multiplex families was assessed using the pedigree disequilibrium test ( pdt ) ( 20 ) , which provides a valid test of association in the presence of linkage in families with multiple affected individuals . the pdt examines the discrepancy between the alleles of heterozygote parents and those transmitted to affected offspring , as well as the allelic difference between affected and unaffected siblings . both pdt - sum and pdt - ave tests ( 21 ) were carried out . because these tests are asymptotically equivalent , the pdt - ave test , in which counts of trios and pairs for each pedigree are weighted , is primarily reported here . plink ( v.1.02 ) ( 22 ) was used to assess allelic association to type 1 diabetes in trio families , via the transmission / disequilibrium test ( 23 ) , and in case - control samples . a logistic regression model was used to estimate effect sizes , with familial correlations being taken into account by the generalized estimating equation method ( 24 ) . to combine results of the original genome scan and those of follow - up studies , a weighted z score , a z statistic summarizing the magnitude of the p value for association and direction of effect was generated for each study . an overall z statistic was then computed as a weighted average of the individual statistics , and a corresponding p value for that statistic was computed . the weights were proportional to the square root of the number of individuals in each study and scaled such that the squared weights summed to 1 . for the meta - analysis of the effect size , the inverse variance was used as the weight for each study . after data cleaning , pdt was performed on the data from 5,943 markers genotyped in the family collection . table 1 lists all snps with p < 0.001 in this analysis . only a single marker , rs1004446 , displayed evidence of association with type 1 diabetes at a genome - wide significance level ( p = 2.5  10 ) . this snp is located in the ins - igf2 region on chromosome 11p15 and has previously been reported to be associated with type 1 diabetes ( 14 ) . indeed , among the 16 snps listed in table 1 , 5 corresponded to previously confirmed type 1 diabetes loci identified through genome - wide scanning : rs1004446 ( ins - igf2 ) , a nonsynonymous coding snp rs1990760 ( ifih1 ) , rs887864 ( intron 18 of clec16a / kiaa0350 ) , and two snps , rs169679 and rs1011094 , that flank the hla region . given that the top three most associated snps ( ranked by statistical significance in table 1 ) correspond to confirmed type 1 diabetes risk loci , we examined the fourth - ranked snp , rs876498 , for which there was also substantial evidence of association with type 1 diabetes ( p = 1.0  10 for pdt - ave test and p = 1.0  10 for pdt - sum test ) . further impetus for follow - up study of this snp came from its location within the sixth intron of the gene ubash3a , which is expressed predominantly in t - cells and is involved in the regulation of signaling from the antigen receptor ( 25 ) . the putative disease - associated allele was relatively common , with a minor allele frequency of 0.45 in unaffected founders . blinded validation genotyping was carried out in 366 of the original 2,496 t1dgc families by a second methodology to confirm the initial finding . a concordance rate of 100% was observed between the original and the validation study . to confirm and extend the initial finding , rs876498 was genotyped in additional independent populations , both family based and case control ( table 2 ) . overall , there was highly significant evidence of association between the minor allele at rs876498 and type 1 diabetes ( or 1.10 [ 95% ci 1.071.13 ] ; p = 4.4  10 ) . in the current study , we took advantage of available snp genotyping data from a linkage study of 2,496 multiplex families with type 1 diabetes the ability to use family - based testing for association allowed the combined analysis of subjects selected from multiple different geographic areas while reducing concerns regarding the potential effects of population stratification . two approaches were used as an additional check for potential heterogeneity from family collection in the nine t1dgc regions . first , a meta - analysis combining the nine individual pdt results provided a p = 2.8  10 , the same magnitude of significance as our combined data analysis . second , a q statistic based on the association effect and se was computed . the association heterogeneity test ( q 5.15 , 7 d.f . [ one group has missing estimates ] ; p = 0.64 ) indicates no statistically significant heterogeneity across the regions of the t1dgc data . the validness of our test for association can also be shown from the genomic control number 1.02 obtained when the whole - genome scan results were examined ( 26 ) . although snp coverage was extremely sparse for genome - wide association testing , an advantage was that the snp selection in the linkage panel of markers differs from the higher density panels used in previous genome - wide association studies . three of the top four markers , ranked by p value , corresponded to previously established type 1 diabetes risk loci . the fourth - ranked snp mapped to chromosome 21q22.3 ; no locus associated at genome - wide significance levels with type 1 diabetes or any other autoimmune disorder has previously been mapped to this chromosome . the region had , however , been previously linked to type 1 diabetes in scandinavian families ( 27 ) , and a region 10 mb centromeric to rs876498 has been fine - mapped in danish families ( 28 ) . furthermore , a type 1 diabetes risk locus on chromosome 21 might have been anticipated given the increased frequency of downs syndrome , caused by trisomy for chromosome 21 , among type 1 diabetic patients ( 29 ) . the findings reported here for rs876498 were validated by a blinded 15% resample using an alternative genotyping approach and then replicated in independent sets of parent - affected child trio families and case - control subjects . these studies provide compelling evidence for association of the minor allele at this snp , rs876498 , with type 1 diabetes . the snp rs876498 is located within the sixth intron of a gene variously referred to as ubiquitin associated and sh3 domain containing , a ( ubash3a ) ( 30 ) , t - cell ubiquitin ligand ( tula ) ( 31 ) , and suppressor of t - cell signaling 2 ( sts-2 ) ( 32 ) . based on the available data in hapmap ( release 23a ) , rs876498 is centrally located in a haplotype block of 14 kb contained entirely within ubash3a . flanking genes in the region are not obvious candidates , on functional grounds , for involvement in type 1 diabetes etiology , and they include the trefoil domain containing family members tff1 and tff2 ; the transmembrane serine protease tmprss3 ; rsph1 , which encodes a male meiotic metaphase chromosome associated protein ; and the glycerol 3-phosphate transporter slc37a1 . ubash3a is expressed predominantly in t - cells , where it interacts with c - cbl through its sh3 domain and binds to ubiquitin and ubiquitylated proteins via its ubiquitin - associated domain ( ( 25 ) . in t - cells , it acts in part by inhibiting c - cbl  mediated downregulation of protein tyrosine kinases such as zap70 that are activated upon t - cell receptor stimulation ( 32 ) . in this regard , ubash3a is reminiscent of another identified type 1 diabetes risk locus , ptpn22 , whose product , lyp , also interacts with c - cbl but directly downregulates some of the same protein tyrosine kinases by dephosphorylation ( 6,33 ) . ubash3a has other functions in t - cells , including participation in proapoptotic pathways ( 34 ) . the functions of ubash3a make it an appealing candidate to contribute to type 1 diabetes risk . while it may be premature to focus exclusively on this single gene based upon the present data , the significant and replicated findings of association reported here strongly suggest that a gene or genes in this region contributes to risk for type 1 diabetes .
objective the type 1 diabetes genetics consortium ( t1dgc ) has assembled and genotyped a large collection of multiplex families for the purpose of mapping genomic regions linked to type 1 diabetes . in the current study , we tested for evidence of loci associated with type 1 diabetes utilizing genome - wide linkage scan data and family - based association methods.research design and methods a total of 2,496 multiplex families with type 1 diabetes were genotyped with a panel of 6,090 single nucleotide polymorphisms ( snps ) . 
 evidence of association to disease was evaluated by the pedigree disequilibrium test . 
 significant results were followed up by genotyping and analyses in two independent sets of samples : 2,214 parent - affected child trio families and a panel of 7,721 case and 9,679 control subjects.results three of the snps most strongly associated with type 1 diabetes localized to previously identified type 1 diabetes risk loci : ins , ifih1 , and kiaa0350 . 
 a fourth strongly associated snp , rs876498 ( p = 1.0  104 ) , occurred in the sixth intron of the ubash3a locus at chromosome 21q22.3 . support for this disease association 
 was obtained in two additional independent sample sets : families with type 1 diabetes ( odds ratio [ or ] 1.06 [ 95% ci 1.001.11 ] ; p = 0.023 ) and case and control subjects ( 1.14 [ 1.091.19 ] ; p = 7.5  108).conclusions the t1dgc 6k snp scan and follow - up studies reported here confirm previously reported type 1 diabetes associations at ins , ifih1 , and kiaa0350 and identify an additional disease association on chromosome 21q22.3 in the ubash3a locus ( or 1.10 [ 95% ci 1.071.13 ] ; p = 4.4  1012 ) . 
 this gene and its flanking regions are now validated targets for further resequencing , genotyping , and functional studies in type 1 diabetes .