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Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of Diabetic Nephropathy

Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of... ORIGINAL ARTICLE Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of Diabetic Nephropathy 1,2,3,4 2,5 6 7,8,9 Winfred W. Williams, Rany M. Salem, Amy Jayne McKnight, Niina Sandholm, 7,8 1,2,10 2 1,2,10 6 Carol Forsblom, Andrew Taylor, Candace Guiducci, Jarred B. McAteer, Gareth J. McKay, 11 12,13 12,13,14 2,5 Tamara Isakova, Eoin P. Brennan, Denise M. Sadlier, Cameron Palmer, 7,8 7,8 7,8,15 7,8 7,8 Jenny Söderlund, Emma Fagerholm, Valma Harjutsalo, Raija Lithovius, Daniel Gordin, 7,16 7,16 7,8 7,8 7,8 Kustaa Hietala, Janne Kytö, Maija Parkkonen, Milla Rosengård-Bärlund, Lena Thorn, 7,8 7,8 7,8 7,8 17 Anna Syreeni, Nina Tolonen, Markku Saraheimo, Johan Wadén, Janne Pitkäniemi, 17 15,17,18 19 19 19 Cinzia Sarti, Jaakko Tuomilehto, Karl Tryggvason, Anne-May Österholm, Bing He, 20 12,21 12,13 2,5 6 Steve Bain, Finian Martin, Catherine Godson, Joel N. Hirschhorn, Alexander P. Maxwell, 7,8 1,2,4,10 Per-Henrik Groop, and Jose C. Florez, for the GENIE Consortium P= 0.60). However, a fixed-effects meta-analysis that included the We formed the GEnetics of Nephropathy–an International Effort previously reported cohorts retained a genome-wide significant (GENIE) consortium to examine previously reported genetic asso- association with that phenotype (OR 1.31, P= 2 3 10 ). An ciations with diabetic nephropathy (DN) in type 1 diabetes. GENIE expanded investigation of the ELMO1 locus and genetic regions consists of 6,366 similarly ascertained participants of European reported to be associated with DN in the U.S. GoKinD yielded only ancestry with type 1 diabetes, with and without DN, from the nominal statistical significance for these loci. Finally, top candi- All Ireland-Warren 3-Genetics of Kidneys in Diabetes U.K. and Re- dates identified in a recent meta-analysis failed to reach genome- public of Ireland (U.K.-R.O.I.) collection and the Finnish Diabetic wide significance. In conclusion, we were unable to replicate most Nephropathy Study (FinnDiane), combined with reanalyzed data of the previously reported genetic associations for DN, and signif- from the Genetics of Kidneys in Diabetes U.S. Study (U.S. icance for the EPO promoter association was attenuated. GoKinD). We found little evidence for the association of the Diabetes 61:2187–2194, 2012 EPO promoter polymorphism, rs161740, with the combined phe- notype of proliferative retinopathy and end-stage renal disease in U.K.-R.O.I. (odds ratio [OR] 1.14, P = 0.19) or FinnDiane (OR 1.06, ype 1 diabetes has continuously increased world- From the Center for Human Genetic Research, Massachusetts General Hospi- wide, and the highest incidence is found in Finland tal, Boston, Massachusetts; the Program in Medical and Population Genetics, (1). Diabetic nephropathy (DN) is a complication Broad Institute, Cambridge, Massachusetts; the Division of Nephrology, Tthat develops in approximately 25–40% of patients Department of Medicine, Massachusetts General Hospital, Boston, Massachu- setts; the Department of Medicine, Harvard Medical School, Boston, Massa- with type 1and type2diabetes. DN is the leading cause of chusetts; the Endocrine Research Unit, Department of Endocrinology, end-stage renal disease (ESRD) in the developed world. Children’s Hospital, Boston, Massachusetts; the Nephrology Research, Currently, 44% of the new cases of ESRD in the U.S. annually Centre for Public Health, Queen’s University of Belfast, Belfast, U.K.; the Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum are attributable to DN (2). A better understanding of the Helsinki, Helsinki, Finland; the Division of Nephrology, Department causal factors of DN and its pathogenesis may lead to new of Medicine, Helsinki University Central Hospital, Helsinki, Finland; the 9 strategies to decrease its incidence, preemptively treat the Department of Biomedical Engineering and Computational Science, Aalto University, Helsinki, Finland; the Diabetes Research Center (Diabetes Unit), disorder, attenuate morbidity and mortality, and would be Department of Medicine, Massachusetts General Hospital, Boston, Massachu- a valuable contribution to global public health. setts; the Division of Nephrology, University of Miami, Miller School of Several observations suggest that DN, one of the major Medicine, Miami, Florida; the UCD Diabetes Research Centre, Conway In- complications of type 1 and type 2 diabetes, has an in- stitute, University College Dublin, Belfield, Dublin, Ireland; the School of Medicine, University College Dublin, Belfield, Dublin, Ireland; the Mater herent genetic susceptibility. Familial clustering of DN is University Hospital, Dublin, Ireland; the Department of Chronic Disease evident for both type 1 and type 2 diabetes (3–6), and ge- Prevention, Welfare and Health Promotion Division, National Institute for netic risk factors are being sought in multiple populations Health and Welfare, Helsinki, Finland; the Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland; the Department of (7–9). Unfortunately, robust replication of many initial Public Health, University of Helsinki, Helsinki, Finland; the South Ostro- associations has not been forthcoming (10). bothnia Central Hospital, Seinäjoki, Finland; the Division of Matrix Biology, This study recruited a large collection of individuals with Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; the Institute of Life Sciences, Swansea University, type 1 diabetes as part of the GEnetics of Nephropathy–an Swansea, U.K.; and the School of Biomolecular and Biomedical Sciences, International Effort (GENIE) consortium and examined se- University College Dublin, Belfield, Dublin, Ireland. lected candidate loci associated with DN from genome-wide Corresponding author: Jose C. Florez, jcflorez@partners.org. Received 1 June 2011 and accepted 5 April 2012. case-control studies or other association studies that DOI: 10.2337/db11-0751 reported high levels of statistical significance. The variants This article contains Supplementary Data online at http://diabetes examined and the rationale for their inclusion are as follows: .diabetesjournals.org/lookup/suppl/doi:10.2337/db11-0751/-/DC1 W.W.W., R.M.S., A.J.M., and N.S. contributed equally to this work. 2012 by the American Diabetes Association. Readers may use this article as 1) A single nucleotide polymorphism (SNP) (rs1617640) long as the work is properly cited, the use is educational and not for profit, within the promoter region of the EPO gene (encoding and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. erythropoietin) was identified as having a genome-wide significant (P , 5 3 10 ) association with ESRD and See accompanying commentary, p. 1923. diabetes.diabetesjournals.org DIABETES, VOL. 61, AUGUST 2012 2187 PRIOR GENETIC ASSOCIATIONS WITH DN proliferative diabetic retinopathy (PDR) (11). Interest- top-reported SNP (or proxy) for each gene in that report ingly, erythropoietin levels were elevated sevenfold for an association with DN. in the human vitreous fluid of nondiabetic individuals with the risk genotype TT compared with those with In this study, we have assembled the largest reported the wild-type GG genotype. In addition, EPO expres- case-control sample of DN in type 1 diabetes to evaluate sion levels were significantly elevated above control in the previously reported genetic associations in newly geno- the tissues and vitreous fluid of animal models of DN typed samples from the U.K., Republic of Ireland (R.O.I.), (DN in db/db mice) and in proliferative retinopathy and Finland, plus pre-existing data from the U.S. GoKinD. (murine oxygen-induced retinopathy model), respec- tively (11). RESEARCH DESIGN AND METHODS 2) The engulfment and cell motility 1 gene (ELMO1) has Cohorts been reported to be associated with DN in Japanese U.K.-R.O.I. collection. Recruited individuals were part of the All Ireland- patients with type 2 diabetes (12). Recently, Pezzolesi Warren 3-Genetics of Kidneys in Diabetes U.K. collection (U.K.-R.O.I.). All were et al. (13), using the Genetics of Kidneys in Diabetes self-reported as white, with grandparents born in the U.K. or Ireland, and type 1 U.S. Study (U.S. GoKinD) cohorts, also examined diabetes diagnosed before the age of 31 years requiring uninterrupted insulin ELMO1 for association with DN and presented evi- treatment. Case subjects (n = 903) with DN had persistent proteinuria (.0.5 dence of association of variants within this gene for g/24 h), hypertension (.135/85 mmHg and/or treatment with antihypertensive medication), and diabetic retinopathy. ESRD (28%) was defined as requiring the development of DN. However, the risk alleles for renal replacement therapy or having received a kidney transplant. Individuals ELMO1 identified in their study differed from those in the control group (n = 1,001) had had type 1 diabetes for at least 15 years, reported in the original Japanese investigation. In the had no evidence of microalbuminuria on repeated testing, and were not re- context of a genome-wide association study (GWAS), ceiving antihypertensive medication (Table 1). 118 SNPs were assessed in 1,705 individuals of Euro- Finnish Diabetic Nephropathy Study (FinnDiane). The FinnDiane study pean ancestry with type 1 diabetes (885 control subjects is a nationwide multicenter study of .4,800 adult participants with type 1 diabetes (16). This study comprises genotype data for 2,914 patients with type and 820 DN case subjects). The strongest associations 1 diabetes diagnosed before age 35 years and insulin treatment started within in ELMO1 in the U.S. study occurred at rs11769038 23 1 year of diagnosis. The disease status was defined by urine albumin excretion (odds ratio [OR] 1.24; P = 1.7 3 10 ) and rs1882080 rate (AER) or urine albumin-to-creatinine ratio (ACR) in at least two of three (OR 1.23; P = 3.2 3 10 ), located in intron 16. Two consecutive urine collections at local centers. Macroalbuminuria (n = 686) additional SNPs, located in introns 18 and 20, were was defined as AER .200 mg/min or .300 mg/24 h or an ACR .25 mg/mmol also nominally associated with DN. In total, eight for men and .35 mg/mmol for women in overnight, 24-h, or spot urine col- lections, respectively. Similarly, the limit for normal AER (n = 1,601) was ,20 ELMO1 SNPs were reported to confer risk for DN, al- mg/min or ,30 mg/24 h or ACR ,2.5 mg/mmol for men and ,3.5 mg/mmol for though none reached genome-wide significance (13). women. Control patients with normal AER were required to have type 1 Supportive evidence was also found in African Ameri- diabetes duration of at least 15 years. ESRD (n = 627) was defined as ongoing cans with type 2 diabetes and ESRD (14). dialysis treatment or receipt of a kidney transplant. From the total, 505 par- 3) The U.S. GoKinD GWAS analyzed 359,193 SNPs in 820 ticipants were included from an independent Finnish cohort collected by the case subjects (284 with proteinuria and 536 with ESRD) National Institute for Health and Welfare (17). These participants met the FinnDiane diagnosis and selection criteria and were analyzed together with and 885 control subjects with type 1 diabetes but no the FinnDiane cohort (Table 1). evidence of DN. Although no risk variant achieved U.S. GoKinD. The U.S. GoKinD study consists of a DN case-control cohort of genome-wide significance, the primary association anal- individuals diagnosed with type 1 diabetes before age 31 years, who were ysis identified 11 SNPs representing four distinct chromo- between 18 and 59 years of age at enrollment, and who began insulin treatment somal regions (P , 1 3 10 ). The strongest association within 1 year after diagnosis (18). The 905 case subjects were defined as people with DN reported in this study was on chromosome 9q aged 18–54, with type 1 diabetes for at least 10 years, and DN. The 898 control subjects were aged 18–59, had type 1 diabetes for at least 15 years, but did not with rs10868025 (OR 1.45, P =5.0 3 10 )(15). have DN. The DN definition includes individuals with ESRD (on dialysis or 4) Finally, in an effort to systematically explore and compre- having received a kidney transplant) or persistent macroalbuminuria (at least hensively capture common genetic variations that might two of three tests positive for albuminuria by dipstick $1+ or ACR .300 mg be associated with DN, we reviewed the largest meta- albumin/mg of urine creatinine). The U.K. GoKinD inclusion criteria were used analysis published to date studying genetic associations to recruit individuals to the control group. Individuals were recruited at two with the DN phenotype (7). In GENIE, we examined the study centers, George Washington University (GWU) and the Joslin Diabetes TABLE 1 Phenotypic characteristics of the GENIE cohorts (U.K.-R.O.I., FinnDiane, and U.S. GoKinD) U.K.-R.O.I. FinnDiane U.S. GoKinD† Case subjects Control subjects Case subjects Control subjects Case subjects Control subjects n 903 1,001 1,289 1,577 774 821 Sex (n) Male 531 438 764 651 402 342 Female 372 563 525 926 372 479 Type 1 diabetes Duration (years) 32.9 6 9.5 27.2 6 8.7 32.8 6 9.1 27.9 6 9.5 31.4 6 7.8 25.4 6 7.7 Age at diagnosis (years) 14.6 6 7.7 14.5 6 7.8 12.8 6 7.6 15.1 6 8.3 11 6 6.6 13 6 7.3 HbA (%) 9.0 6 1.9 8.7 6 1.6 8.8 6 1.6 8.0 6 1.2 7.5 6 1.9 7.5 6 1.2 1c BMI (kg/m ) 26.2 6 4.7 26.2 6 4.2 25.5 6 4.2 25.2 6 3.4 25.7 6 5.2 26.1 6 4.3 ESRD (%) 28.0 0 48.2 0 65.6 0 Categoric data are shown as indicated; continuous data as mean 6 SD. †Reanalysis of the U.S. GoKinD dataset using new quality control filters to account for published plate effects (see RESEARCH DESIGN AND METHODS for complete details). 2188 DIABETES, VOL. 61, AUGUST 2012 diabetes.diabetesjournals.org W.W. WILLIAMS AND ASSOCIATES Center (JDC) using differing methods of ascertainment and recruitment (see as well as sex chromosome and mitochondrial SNPs were excluded from Pezzolesi et al. [15] for details). Analysis of the U.S. GoKinD cohort was lim- analyses. After applying the QC protocol, we had access to 549,530 SNPs in ited to individuals whose reported primary ethnicity was white. 3,370 FinnDiane samples, 791,687 SNPs in 1,726 U.K.-R.O.I. samples, and Phenotype definition: DN and EPO study outcomes. DN was the primary 360,899 SNPs in 1,595 U.S. GoKinD samples. outcome for all association studies of the SNPs investigated. For the EPO study, SNP imputation. MACH 1.0 software (http://www.sph.umich.edu/csg/abecasis/ we used the phenotypic definitions used in the original report (11); namely, MACH) with the HapMap phase II CEU reference panel was used to perform ESRD as defined by case subjects who were on dialysis or who had received SNP imputation for GWAS results in each cohort. Estimates of the crossover a renal transplant; concurrently, this ESRD population also had to have evi- and error rates were obtained via 50 iteration rounds in ;300 randomly se- dence of advanced PDR on physical examination and a history of laser lected samples per cohort. A greedy algorithm was used for imputation, and treatment. Control subjects with evidence of PDR were excluded. We also the maximum likelihood method was specified to yield allele dosages. A filter examined PDR as an outcome independent of ESRD status. For this analysis, was applied to exclude SNPs with low imputation quality (r , 0.6), case subjects had clinical evidence of PDR, whereas control subjects had none resulting in ;2.4 million SNPs per cohort. (irrespective of DN status). Cohorts analyzed in the original EPO study (Fig. 1) Statistical analysis. Association tests were conducted using PLINK v1.07 (20) were composed of European-American cases, and control subjects were col- (http://pngu.mgh.harvard.edu/purcell/plink), with logistic regression adjusted lected from distinct geographic areas in the U.S. These included the GoKinD for sex and age. U.K.-R.O.I. was adjusted for recruitment center, but the two U.S. GoKinD centers, GWU and JDC, were analyzed separately as reported by cohort (Boston, Pittsburgh, and Minnesota), the Utah cohort (Salt Lake City), Pezzolesi et al. (15). Data from the GWAS genotyping was adjusted addition- and the Boston cohort (Boston Joslin Center for Diabetes) (11). ally for duration of type 1 diabetes and principal components from Eigenstrat SNP selection. SNP markers with evidence for association with DN sus- analysis. The EPO locus was analyzed with the Pearson x test in the Finn- ceptibility in reference studies (11) were selected for genotyping in GENIE. Diane dataset without adjusting for any covariates. Fixed-effects meta-analyses Where more than one SNP was associated at a particular locus with DN, the were conducted with the software package Comprehensive Meta-Analysis most strongly associated variant was selected for genotyping in the DN (Version 2.2.040, Englewood, NJ) and the software package METAL (http:// case-control cohorts. Where no genotyping assay could be developed for the www.sph.umich.edu/csg/abecasis/Metal/) (21) under the additive genetic index SNP, a proxy in strong linkage disequilibrium (LD) was genotyped using the CEU HapMap population. CEU is the official three letter code for the model. To determine the appropriate statistical cutoff for correction for HapMap samples of Utah residents with ancestry from Northern and Western multiple testing, we calculated the total number of effective tests (because Europe (see http://hapmap.ncbi.nlm.nih.gov/citinghapmap.html). For the a large portion of SNPs were in LD), using SNPSpD (http://gump.qimr.edu.au/ original U.S. GoKinD reported results and ELMO1, additional SNPs within general/daleN/SNPSpD/). SNPSpD uses correlation between analyzed SNPs to 20-kb upstream and downstream of the locus (or index SNP) were selected calculate the total number of independent tests (22). The total number of SNPs using the SNP Annotation and Proxy Search (http://www.broadinstitute.org/ tested is 2,199, with 113.7 effect-independent tests. Thus the experiment-wide mpg/snap/), specifying chromosome position, CEU samples, and 1000 Genome cutoff for statistical significance was set at 4.4 3 10 (0.05/113.7). Pilot 1 data. The expanded SNP list was extracted from GWAS results for U.K.-R.O.I. and FinnDiane (N. Sandholm et al., submitted). De novo genotyping. For the U.K.-R.O.I. collection (n = 1,904 unique indi- RESULTS viduals), SNPs were genotyped using Sequenom iPLEX (Sequenom, Hamburg, Germany) or TaqMan (Applied Biosystems, Warrington, U.K.) technology. EPO promoter polymorphism. The association of the Duplicate and no-DNA-template samples were included on all plates as ex- EPO promoter polymorphism, rs1617640, with DN was perimental controls. evaluated by de novo genotyping of the SNP in GENIE. For In FinnDiane, the EPO locus was genotyped with TaqMan chemistry (Applied this analysis, case subjects were defined as having both Biosystems, Foster City, CA) in 3,363 samples, of which 251 case subjects ESRD and PDR because the initial report showed the (ESRD + laser treatment) and 987 control subjects (no DN, no retinopathy) polymorphism was robustly associated with DN when both passed the phenotype criteria. All other SNPs were genotyped at the Institute of Molecular Medicine Finland (Helsinki, Finland) on the Illumina’sBeadArray of these “extreme” phenotypes were coexpressed. Significant 610Quad array. Illumina’s BeadStudio clustering algorithm was used to call association was not observed in the U.K.-R.O.I. (P = 0.19) genotypes in FinnDiane. SNPs were filtered for those with call rates .95%, minor or FinnDiane collections (P = 0.60), although the direc- allele frequency (MAF) .1%, and test for Hardy Weinberg equilibrium (HWE; tions of effect were consistent with the original report. P . 1 3 10 ). Sample filters included individual call rates .95% and no first- Fixed-effects meta-analysis of the association of rs1617640 degree relatives and resulted in 1,289 case subjects and 1,577 control subjects. with ESRD/PDR, including the previously reported cohorts Existing genotype data for the U.S. GoKinD genotype data were downloaded from dbGAP (phs000018.v2.p1, retrieved June 2010), containing genotype data (a total of 3,162 case and 3,845 control subjects across five from the Affymetrix 500 K array (Affymetrix, Santa Clara, CA). The version 2 separate cohorts of European and European-American genotype data differed from the original U.S. GoKinD data, containing updated ancestry) retained genome-wide statistical significance and recalled genotype calls for a previously reported problematic plate (19), 29 (OR 1.31 [95% CI 1.20–1.44], P =2 3 10 ; Fig. 1). and SNP call rate (.90%), MAF (.1%), and HWE performed by the National As an additional experimental control, we examined the Heart, Lung, and Blood Institute. We applied additional quality control (QC) potential association of the EPO promoter polymorphism filters to the data, including removing samples with evidence of contamination (extreme heterozygosity, n = 16), known parents (n = 4), non–European- with the development of PDR in case subjects, irrespective reported ancestry (n = 121), reported sex–genotype mismatch (n = 1), cryptic of ESRD status. No association was observed between related subjects (n = 4), and principal component analysis admixture outliers EPO and PDR for the individual cohorts or in the meta- (n = 5). Furthermore, SNP QC filters were applied to remove known prob- analysis of the combined results for FinnDiane (OR 0.95 lematic SNPs, HWE (P . 1 3 10 ), call rate (90%), missingness, MAF (1%), [95% CI 0.85–1.04], P = 0.25) or and U.K.-R.O.I. (0.96 [0.88– and SNPs plate effects. 1.04], P = 0.29). Furthermore, no association was observed GWAS genotyping. Genotypes for U.K.-R.O.I. (n = 1,830) and FinnDiane (n = 3,651) were supplemented with SNPs retrieved from GWAS results for after inclusion of the restricted phenotype, PDR, in U.S. each study (Sandholm et al., submitted). The FinnDiane study samples were GoKinD case and control subjects, separately, or in the genotyped using the Illumina’s BeadArray 610-Quad (Illumina, San Diego, CA) meta-analysis of all cohorts combined (results not shown). array at the Institute of Molecular Medicine Finland (FIMM, Helsinki, Finland). ELMO1. Neither single-center nor meta-analysis of de novo The U.K.-R.O.I. study samples were genotyped at the Broad Institute using the genotyping in U.K.-R.O.I., nor GWAS data for FinnDiane, Illumina Omni1-Quad array. Samples with insufficient DNA quality, quantity, revealed a significant association in subjects with type 1 or poor genotype concordance with previous available genotypes at a finger- printing stage were excluded. For all three discovery datasets (FinnDiane, diabetes between rs741301, the previously reported risk U.K.-R.O.I., U.S. GoKinD), a standardized and detailed genotype QC proce- variant within ELMO1, and DN (OR 1.04 [95% CI 0.95–1.13], dures were applied using PLINK supplemented with perl and R scripts. These P= 0.46; Supplementary Fig. 1). Pezzolesi et al. (13) also included selecting SNPs with a call rate .90%, MAF .1%, and concordance tested rs741301 but did not replicate the reported associa- with HWE (P . 10 ). We discarded samples with a call rate ,95%, extreme tion. They went on to test other SNPs in the region and heterozygosity, cryptic relatedness, or ethnic outliers by principal components reported nominal associations (P =0.002–0.05) with eight analysis, and tested for SNP missingness by haplotype (P , 10 ), by phenotype 28 27 (P . 10 ), and by plate effects (P , 10 ). Probes for copy number variation other SNPs . We examined LD between rs741301 and the diabetes.diabetesjournals.org DIABETES, VOL. 61, AUGUST 2012 2189 PRIOR GENETIC ASSOCIATIONS WITH DN FIG. 1. Previously published and new results from this study provide an estimate of the effect of the EPO promoter SNP (rs1617640) on the risk of the combined phenotype of PDR and ESRD in type 1 diabetes in five cohorts (3,162 case subjects and 3,845 control subjects) in a fixed-effects meta-analysis. SNPs reported to be associated with DN by these inves- 1.12, P = 0.08) and in meta-analysis of the two replication tigators. The r statistic between rs741301 and the other cohorts (U.K.-R.O.I. and FinnDiane; OR 1.06, P = 0.06; SNPs revealed only low to moderate LD, ranging from 0.38 Table 2). In expanded locus-region analyses (plus or minus to 0.65 (Supplementary Table 1). 20 kb of the locus of interest), no SNP reached significance As an additional and more extensive test of variants in after adjustment for multiple testing (one-tailed P = 0.03, this region, we performed an expanded analysis capturing experiment-wide threshold P = 4.3 3 10 ) for the two all available SNPs 20 kb upstream and downstream of the cohorts separately or via meta-analysis. The combined ELMO1 locus to account for LD differences presumably meta-analysis including U.S. GoKinD revealed two SNPs due to ancestry. Results of the expanded analysis did not downstream of FRMD3, rs1888747 (P = 1.5 3 10 ) and reveal any significant SNPs for either cohort individually rs13288659 (P = 9.7 3 10 ), which showed significance or for the two cohorts meta-analyzed after correcting for after adjusting for experiment-wide multiple testing (P , 24 24 multiple testing (P , 4.3 3 10 ). Furthermore, no SNP 4.3 3 10 ). However, neither SNP achieved genome-wide achieved significance with inclusion of the U.S. GoKinD significance (P , 5 3 10 ; Supplementary Table 3). results in the meta-analysis (Supplementary Table 2). Pooled meta-analyses examining variants associated Risk variants reported from U.S. GoKinD for DN in with DN in type 1 and type 2 diabetes. In the most type 1 diabetes. Eleven DN susceptibility SNPs that were comprehensive literature search for DN associated genetic first reported by the U.S. GoKinD investigators as highly variants to date, Mooyaart et al. (7) identified 24 loci. In associated with the risk of developing DN in type 1 di- GENIE, we examined all the available top-reported SNPs abetes were parsed into eight candidate loci. We selected (or their proxies) for each gene in that report. Three SNPs one representative SNP for each region of strong LD in were nominally associated (P , 0.05) with DN: rs13293564 which multiple SNPs represented the same association at UNC13B (P = 0.01) and rs179975 at the ACE (P= 0.03) signal. After performing additional QC checks (see RESEARCH in FinnDiane, and rs39075 at CPVL/CHN2 (P= 0.05) in the DESIGN AND METHODS), we first tested the eight U.S. GoKinD U.K.-R.O.I. samples. In a meta-analysis of the two cohorts, potential DN susceptibility SNPs by reanalyzing the U.S. the ACE polymorphism remained nominally significant GoKinD dataset downloaded from dbGAP (19). As in the (P= 0.04). Including the U.S. GoKinD results, the FRMD3 original report, none of the eight SNPs were associated signal at rs1888747 emerged as noted above; no other with DN at genome-wide statistical significance (Table 2). signals were significant after adjusting for multiple com- The two SNPs in the FERM (F [Band 4.1], E [Ezrin], R parisons (see Supplementary Table 4 for full details). [Radixin], M [Moesin]) domain 3 (FRMD3) region showed similar P values as in the original report (2.1 3 10 and DISCUSSION 1.6 3 10 , respectively; Table 2). In our analysis, the statistical significance of the P values for SNPs in the Using a large, homogeneous population sample of European CPVL/CHN2 and CARS regions was reduced from 6.5 3 ancestry subjects with type 1 diabetes in the GENIE con- 27 23 26 23 10 to 2 3 10 and 6.4 3 10 to 2.2 3 10 , respectively. sortium, we were unable to replicate most of the previously P values for the 6 SNPs in the 13q region were also reported genetic associations with DN that we examined. 26 25 changed from 1.8–7.0 3 10 to 1.4–9.5 3 10 . Our findings do not support previously reported genetic We next examined these SNPs in our newly genotyped associations with DN in type 1 diabetes in the largest GWAS samples. Case-control association analysis for these loci in published to date (15). None of those signals reached the U.K.-R.O.I. and FinnDiane samples revealed no signifi- genome-wide statistical significance with the addition of cant associations in either cohort. The strongest signal was larger, similarly ascertained datasets. Using ORs at the observed at rs39075 near CPVL/CHN2 in U.K.-R.O.I., (OR lower limit of the 95% CI from the original publication, our 2190 DIABETES, VOL. 61, AUGUST 2012 diabetes.diabetesjournals.org TABLE 2 Results in the reanalyzed U.S. GoKinD and additional GENIE cohorts for eight SNPs with strongest reported associations to DN in U.S. GoKinD Meta-analysis of U.K.-R.O.I. & Published U.S. GoKinD Updated U.S. GoKinD† U.K.-R.O.I. FinnDiane FinnDiane Risk allele SNP Nearest gene (nonrisk) OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P 27 23 rs39075 CPVL/CHN2 G (A) 1.43 (1.23–1.64) 6.5 3 10 1.29 (1.13–1.45) 2.0 3 10 1.12 (0.99–1.25) 0.08 1.01 (0.89–1.12) 0.92 1.06 (0.98–1.25) 0.06 27 27 rs1888747* FRMD3 G (C) 1.45 (1.25–1.67) 6.3 3 10 1.56 (1.40–1.73) 2.1 3 10 1.03 (0.86–1.22) 0.77 1.07 (0.95–1.19) 0.25 1.06 (0.96–1.17) 0.24 27 26 rs1086805 FRMD3 A (G) 1.45 (1.25–1.67) 5.0 3 10 1.47 (1.31–1.62) 1.6 3 10 1.05 (0.90–1.24) 0.52 1.07 (0.95–1.19) 0.25 1.06 (0.97–1.17) 0.19 26 23 rs451041 CARS A (G) 1.36 (1.19–1.56) 6.4 3 10 1.28 (1.12–1.43) 2.2 3 10 1.07 (0.94–1.29) 0.29 1.00 (0.88–1.11) 0.93 1.03 (0.95–1.12) 0.45 26 25 rs1041466‡ No gene (13q) A (G) 1.38 (1.20–1.58) 3.2 3 10 1.36 (1.20–1.51) 9.3 3 10 1.01 (0.88–1.14) 0.94 1.00 (0.89–1.11) 0.99 1.00 (0.93–1.09) 0.92 26 25 rs1411766/rs1741288 No gene (13q) A (G) 1.41 (1.23–1.63) 1.8 3 10 1.42 (1.26–1.57) 1.4 3 10 1.01 (0.88–1.14) 0.87 0.93 (0.82–1.05) 0.24 0.97 (0.89–1.06) 0.47 26 25 rs6492208/rs2391777 No gene (13q) T (C) 1.37 (1.20–1.59) 6.1 3 10 1.36 (1.21–1.52) 9.5 3 10 1.03 (0.90–1.16) 0.64 1.00 (0.89–1.12) 0.97 1.01 (0.93–1.10) 0.76 26 25 rs7989848 No gene (13q) A (G) 1.37 (1.19–1.56) 7.0 3 10 1.36 (1.21–1.52) 5.5 3 10 1.05 (0.93–1.18) 0.41 1.01 (0.90–1.12) 0.88 1.03 (0.95–1.11) 0.50 †Reanalysis of the U.S. GoKinD dataset using new quality control filters to account for published plate effects (see RESEARCH DESIGN AND METHODS for complete details). *In FinnDiane, 2 2 rs13289150 was used as proxy for rs1888747 (D’ =1, r = 0.82 in HapMapII CEU) and for rs10868025; (D’ =1, r = 1 in HapMapII CEU). ‡SNP rs1411765 was used as proxy for rs1041466 (D’ = 0.89, r = 0.62 in HapMapII CEU). W.W. WILLIAMS AND ASSOCIATES combined sample of U.K.-R.O.I. and FinnDiane cohorts in GENIE had 99.9% power (at a = 0.001) to detect the U.S. GoKinD reported effect sizes (Supplementary Table 5); thus, our negative results (even at a = 0.05) were un- expected. Further, after performing additional QC checks on the original U.S. GoKinD dataset and combining these samples with GENIE, we were unable to achieve genome- wide significant replication in the meta-analysis. We investigated other previously reported genetic asso- ciations with DN in subjects with type 1 diabetes, namely the EPO promoter polymorphism rs1617640 and variants within the ELMO1 gene; these also failed to replicate in GENIE. The EPO promoter polymorphism retained genome- wide significance after meta-analysis of the prior data combined with ours, for the combined phenotype of ESRD and PDR. However, the overall P value attained was at- 211 29 tenuated from 2.8 3 10 to 2 3 10 . We also did not observe evidence for replication of the association of rs741301 in the ELMO1 gene and DN in GENIE. Shimazaki et al. (12) first reported an association for this genetic variant with DN in Japanese subjects with type 2 diabetes. Subsequently, Pezzolesi et al. (13) failed to replicate the association of the same SNP in type 1 diabetes but reported that eight SNPs within the gene locus had nominal associations with DN in the European-derived subjects in U.S. GoKinD. Lack of replication may be due to different genomic patterns in populations of diverse ances- tries or the distinct genetic architecture of DN in type 1 versus type 2 diabetes (14). To address the first concern, we expanded our analysis by interrogating all available SNPs plus or minus 20 kb of ELMO1. Because in the U.S. GoKinD analysis of ELMO1 the risk variants were only in weak to modest LD with the index SNP (r between rs741301 and the U.S. GoKinD SNPs ranged from 0.38 to 0.65), we reasoned that this expanded strategy would ac- count for most of the differences in LD presumably due to ancestry. The results of this expanded analysis, however, did not reveal any significant association for the additional 670 SNPs interrogated in ELMO1 in the U.K.-R.O.I. or FinnDiane cohorts individually, for these two cohorts in meta-analysis, or when combined with previously reported risk variants reported from U.S. GoKinD. As a final step to ensure a systematic and more com- prehensive approach to candidate loci associated with DN, we reviewed the top SNPs from the 24 loci cited by the largest meta-analysis published to date that has examined candidate genetic variants for association with DN (7). From the results of the pooled meta-analyses, the strongest signal emerged at rs1888747 in FRMD3 (P =1.5 3 10 ). Variants within this gene have been reported to be associated with DN in European cohorts (7,15,23), and Freedman et al. (24) recently reported evidence for a role for gene–gene interactions between myosin heavy-chain 9 (MYH9)– apolipoprotein L1 (APOL1) haplotypes and FRMD3 in African American subjects. It should be noted, however, that risk variants within this gene have achieved only nominal but not genome-wide statistical significance in all previous reports. From the foregoing observations, we conclude that there is a high likelihood that many of the previously reported positive associations with DN represent potential false-positive findings (type I error). We emphasize that the combined sample of U.K.-R.O.I. and FinnDiane represents a substantially larger collection of case and control subjects than U.S. GoKinD and is well powered to detect the origi- nally reported effect sizes, thereby decreasing the likelihood diabetes.diabetesjournals.org DIABETES, VOL. 61, AUGUST 2012 2191 PRIOR GENETIC ASSOCIATIONS WITH DN of a false-negative finding (type II error), even accounting shared genetic susceptibility for nephropathy between for the likely overestimation of effect sizes due to the type 1 and type 2 diabetes, remain unresolved. winner’s curse phenomenon (25). We also harmonized the ascertainment criteria for case-control definitions across ACKNOWLEDGMENTS all the study populations (including U.S. GoKinD), making it unlikely that phenotypic heterogeneity across study Funding for this study was provided by National Institutes populations explains the lack of replication. of Health National Institute of Diabetes and Digestive and A crucial issue that bears on the interpretation of Kidney Diseases Grant R01-DK-081923 to J.N.H., P.-H.G., case-control studies of the genetics of DN concerns the and J.C.F., a Science Foundation Ireland U.S.-Ireland R&D adequacy of phenotype definition. In this and most studies partnership, and The Health Research Board Ireland. R.M.S. cited to date, there is the presumption that long-duration was supported by a Juvenile Diabetes Research Founda- diabetes exposure and the presence of frank protein in the tion postdoctoral fellowship (#3-2011-70). The FinnDiane urine—macroalbuminuria—defines DN and that pheno- Study was supported by grants from the Folkhälsan Research typic heterogeneity has been well controlled through this Foundation, the Wilhelm and Else Stockmann Foundation, Liv classification. These definitions are derived in large mea- och Hälsa Foundation, Helsinki University Central Hospital sure from the classic studies of Parving et al. (26), Viberti Research Funds (EVO), the Sigrid Juselius Foundation, the et al. (27), and Mogensen and Christensen (28), who docu- Signe and Arne Gyllenberg Foundation, Finska Läkaresällska- mented 30 years ago a virtually inexorable progression to pet, the European Commission, and the European Union’s ESRD in patients who developed microalbuminuria after Seventh Framework Program (FP7/2007-2013) for the In- approximately 2 decades of exposure to the diabetic met- novative Medicine Initiative under Grant Agreement No. abolic milieu. However, these longitudinal findings were IMI/115006 (the Surrogate Markers for Micro- and Macro- based on small numbers of patients. The plasticity of DN vascular Hard Endpoints for Innovative Diabetes Tools phenotypes is reflected in more recent and much larger [SUMMIT] consortium). longitudinal studies showing that most patients with type 1 J.C.F. has received consulting honoraria from Novartis, diabetes, categorized initially as having microalbuminuria, Eli Lilly, and Pfizer. No other potential conflicts of interest undergo regression to normoalbuminuria with preservation relevant to this article were reported. of renal function (29). It is not entirely clear that micro- W.W.W. contributed to study design and management, albuminuria versus macroalbuminuria, stage of chronic data acquisition, statistical and data analysis, and manu- kidney disease and attendant renal function, the rate of script preparation and review. R.M.S. contributed to data renal decline, or the occurrence of extreme phenotypes, acquisition, genotyping, statistical and data analysis, and such as ESRD/PDR, represent one disease process along manuscript review. A.J.M. contributed to data acquisition, a continuum or many distinct disease states, each of which statistical and data analysis, and manuscript preparation. may be under distinct genetic control. As pointed out re- N.S. contributed to data acquisition, statistical and data cently (24), genetic variants, such as those in MYH9 and analysis, and manuscript preparation and review. C.F. APOL1 that are common in certain ethnic groups, may contributed to data acquisition, statistical and data anal- mask the effects at other loci unless methods such as ysis, and manuscript review. A.T., C.Gu., and M.P. con- multilocus modeling and interaction analyses are used to tributed to genotyping and manuscript review. J.B.M., E.F., control for these effects. V.H., R.L., D.G., K.H., J.K., M.R.-B., N.T., M.S., J.W., and B.H. In addition, phenotypic variation may be a function of contributed to data acquisition and manuscript review. ethnicity and disease-specific gene expression. For exam- G.J.M., T.I., E.P.B., D.M.S., C.P., S.B., F.M., C.Go., and ple, Pima Indians with type 2 diabetes have very early- A.P.M. contributed to manuscript review. J.S. contributed onset DN, characterized by an accelerated loss of renal to genotyping and statistical and data analysis. L.T., J.P., function and progression to ESRD despite lower blood C.S., J.T., and A.-M.Ö. contributed to data acquisition. A.S. pressures and lipid levels, factors thought to be protective contributed to data acquisition, genotyping, and manu- (30). This has been postulated to be due to structural dif- script review. K.T. contributed to statistical and data anal- ferences in the nephron–podocyte number and density per ysis and manuscript review. J.N.H. contributed to study glomerulus (“podocyte insufficiency”), a decrease in net design and manuscript review. P.-H.G. contributed to study nephron mass (glomerulopenia) resulting in glomerulomegaly, design, statistical and data analysis, and to manuscript increased intraglomerular capillary pressure, and ultimately, review. J.C.F. contributed to study design and manage- hyperfiltration injury (30). Whether these structural and ment, statistical and data analysis, and manuscript prep- intrarenal hydraulic changes could be genetically regulated aration and review. J.C.F. is the guarantor of this work is ultimately a testable hypothesis; they warrant further in- and, as such, had full access to all of the data in the study vestigation to continue the inquiry why certain populations and takes responsibility for the integrity of the data and have an apparent disproportional susceptibility to ESRD theaccuracy of thedataanalysis. and, particularly, DN. The authors acknowledge the physicians, nurses, and In summary, we have presented evidence that several researchers at each center participating in the collection previously reported genetic associations with DN in type 1 of patients: in the FinnDiane study group and at the Hel- diabetes could not be replicated in a large, homogeneous sinki University Central Hospital, Department of Medicine, sample of subjects with type 1 diabetes. Our failure to Division of Nephrology: C. Forsblom, A. Ahola, J. Fagerudd, replicate these associations underscores the need to apply M. Feodoroff, D. Gordin, V. Harjutsalo, O. Heikkilä, K. Hietala, stringent statistical thresholds of significance, maximize J. Kytö, M. Lehto, S. Lindh, M. Parkkonen, K. Pettersson- power through meta-analysis of all available data, and seek Fernholm, M. Rosengård-Bärlund, M. Rönnback, A. Sandelin, replication in independent samples, as has been proposed A.-R. Salonen, L. Salovaara, M. Saraheimo, T. Soppela, by a number of different authors (31,32). Finally, the ap- A. Soro-Paavonen, P. Summanen, L. Thorn, N. Tolonen, plicability and generalizability of DN risk loci from type 1 J. Tuomikangas, T. Vesisenaho, and J. Wadén. Anjalankoski diabetes to type 2 diabetes, and the related question of Health Centre: S. Koivula and T. Uggeldahl. Central Finland 2192 DIABETES, VOL. 61, AUGUST 2012 diabetes.diabetesjournals.org W.W. WILLIAMS AND ASSOCIATES Central Hospital, Jyväskylä: T. Forslund, A. Halonen, Hospital: V. Javtsenko and M. Tamminen. Pietarsaari Hospi- A. Koistinen, P. Koskiaho, M. Laukkanen, J. Saltevo, and tal: M.-L. Holmbäck, B. Isomaa, and L. Sarelin. Pori City M. Tiihonen. Central Hospital of Åland Islands, Mariehamn: Hospital: P. Ahonen, P. Merensalo, and K. Sävelä. Porvoo M. Forsen, H. Granlund, A.-C. Jonsson, and B. Nyroos. Hospital: M. Kallio, B. Rask, and S. Rämö. Raahe Hospital: Central Hospital of Kanta-Häme, Hämeenlinna: P. Kinnunen, A. Holma, M. Honkala, A. Tuomivaara, and R. Vainionpää. A. Orvola, T. Salonen, and A. Vähänen. Central Hospital Rauma Hospital: K. Laine, K. Saarinen, and T. Salminen. of Länsi-Pohja, Kemi: H. Laukkanen, P. Nyländen, and Riihimäki Hospital: P. Aalto, E. Immonen, and L. Juurinen. A. Sademies. Central Ostrabothnian Hospital District, Kokkola: Salo Hospital: A. Alanko, J. Lapinleimu, P. Rautio, and S. Anderson, B. Asplund, U. Byskata, P. Liedes, M. Kuusela, M. Virtanen. Satakunta Central Hospital, Pori: M. Asola, and T. Virkkala. City of Espoo Health Centre Espoonlahti: M. Juhola, P. Kunelius, M.-L. Lahdenmäki, P. Pääkkönen, A. Nikkola and E. Ritola; Tapiola: M. Niska and H. Saarinen; and M. Rautavirta. Savonlinna Central Hospital: E. Korpi- Samaria: E. Oukko-Ruponen and T. Virtanen; and Viherlaakso: Hyövälti, T. Latvala, and E. Leijala. South Karelia Central A. Lyytinen. City of Helsinki Health Centre Puistola: Hospital, Lappeenranta: T. Ensala, E. Hussi, R. Härkönen, H. Kari and T. Simonen; Suutarila: A. Kaprio, J. Kärkkäinen, U. Nyholm, and J. Toivanen. Tampere Health Centre: and B. Rantaeskola; and Töölö: P. Kääriäinen, J. Haaga, A. Vaden, P. Alarotu, E. Kujansuu, H. Kirkkopelto-Jokinen, and A.-L. Pietiläinen. City of Hyvinkää Health Centre: M. Helin, S. Gummerus, L. Calonius, T. Niskanen, T. Kaitala, S. Klemetti, T. Nyandoto, E. Rontu, and S. Satuli-Autere. and T. Vatanen. Tampere University Hospital: I. Ala-Houhala, City of Vantaa Health Centre Korso: R. Toivonen and T. Kuningas, P. Lampinen, M. Määttä, H. Oksala, T. Oksanen, H. Virtanen; Länsimäki: R. Ahonen, M. Ivaska-Suomela, K. Salonen, H. Tauriainen, and S. Tulokas. Tiirismaa Health and A. Jauhiainen; Martinlaakso: M. Laine, T. Pellonpää, and Centre, Hollola: T. Kivelä, L. Petlin, and L. Savolainen. R. Puranen; Myyrmäki: A. Airas, J. Laakso, and K. Rautavaara; Turku Health Centre: I. Hämäläinen, H. Virtamo, and Rekola: M. Erola and E. Jatkola; and Tikkurila: R. Lönnblad, M. Vähätalo. Turku University Central Hospital: K. 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Pubmed Central
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© 2012 by the American Diabetes Association.
ISSN
0012-1797
eISSN
1939-327X
DOI
10.2337/db11-0751
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ORIGINAL ARTICLE Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of Diabetic Nephropathy 1,2,3,4 2,5 6 7,8,9 Winfred W. Williams, Rany M. Salem, Amy Jayne McKnight, Niina Sandholm, 7,8 1,2,10 2 1,2,10 6 Carol Forsblom, Andrew Taylor, Candace Guiducci, Jarred B. McAteer, Gareth J. McKay, 11 12,13 12,13,14 2,5 Tamara Isakova, Eoin P. Brennan, Denise M. Sadlier, Cameron Palmer, 7,8 7,8 7,8,15 7,8 7,8 Jenny Söderlund, Emma Fagerholm, Valma Harjutsalo, Raija Lithovius, Daniel Gordin, 7,16 7,16 7,8 7,8 7,8 Kustaa Hietala, Janne Kytö, Maija Parkkonen, Milla Rosengård-Bärlund, Lena Thorn, 7,8 7,8 7,8 7,8 17 Anna Syreeni, Nina Tolonen, Markku Saraheimo, Johan Wadén, Janne Pitkäniemi, 17 15,17,18 19 19 19 Cinzia Sarti, Jaakko Tuomilehto, Karl Tryggvason, Anne-May Österholm, Bing He, 20 12,21 12,13 2,5 6 Steve Bain, Finian Martin, Catherine Godson, Joel N. Hirschhorn, Alexander P. Maxwell, 7,8 1,2,4,10 Per-Henrik Groop, and Jose C. Florez, for the GENIE Consortium P= 0.60). However, a fixed-effects meta-analysis that included the We formed the GEnetics of Nephropathy–an International Effort previously reported cohorts retained a genome-wide significant (GENIE) consortium to examine previously reported genetic asso- association with that phenotype (OR 1.31, P= 2 3 10 ). An ciations with diabetic nephropathy (DN) in type 1 diabetes. GENIE expanded investigation of the ELMO1 locus and genetic regions consists of 6,366 similarly ascertained participants of European reported to be associated with DN in the U.S. GoKinD yielded only ancestry with type 1 diabetes, with and without DN, from the nominal statistical significance for these loci. Finally, top candi- All Ireland-Warren 3-Genetics of Kidneys in Diabetes U.K. and Re- dates identified in a recent meta-analysis failed to reach genome- public of Ireland (U.K.-R.O.I.) collection and the Finnish Diabetic wide significance. In conclusion, we were unable to replicate most Nephropathy Study (FinnDiane), combined with reanalyzed data of the previously reported genetic associations for DN, and signif- from the Genetics of Kidneys in Diabetes U.S. Study (U.S. icance for the EPO promoter association was attenuated. GoKinD). We found little evidence for the association of the Diabetes 61:2187–2194, 2012 EPO promoter polymorphism, rs161740, with the combined phe- notype of proliferative retinopathy and end-stage renal disease in U.K.-R.O.I. (odds ratio [OR] 1.14, P = 0.19) or FinnDiane (OR 1.06, ype 1 diabetes has continuously increased world- From the Center for Human Genetic Research, Massachusetts General Hospi- wide, and the highest incidence is found in Finland tal, Boston, Massachusetts; the Program in Medical and Population Genetics, (1). Diabetic nephropathy (DN) is a complication Broad Institute, Cambridge, Massachusetts; the Division of Nephrology, Tthat develops in approximately 25–40% of patients Department of Medicine, Massachusetts General Hospital, Boston, Massachu- setts; the Department of Medicine, Harvard Medical School, Boston, Massa- with type 1and type2diabetes. DN is the leading cause of chusetts; the Endocrine Research Unit, Department of Endocrinology, end-stage renal disease (ESRD) in the developed world. Children’s Hospital, Boston, Massachusetts; the Nephrology Research, Currently, 44% of the new cases of ESRD in the U.S. annually Centre for Public Health, Queen’s University of Belfast, Belfast, U.K.; the Folkhälsan Institute of Genetics, Folkhälsan Research Center, Biomedicum are attributable to DN (2). A better understanding of the Helsinki, Helsinki, Finland; the Division of Nephrology, Department causal factors of DN and its pathogenesis may lead to new of Medicine, Helsinki University Central Hospital, Helsinki, Finland; the 9 strategies to decrease its incidence, preemptively treat the Department of Biomedical Engineering and Computational Science, Aalto University, Helsinki, Finland; the Diabetes Research Center (Diabetes Unit), disorder, attenuate morbidity and mortality, and would be Department of Medicine, Massachusetts General Hospital, Boston, Massachu- a valuable contribution to global public health. setts; the Division of Nephrology, University of Miami, Miller School of Several observations suggest that DN, one of the major Medicine, Miami, Florida; the UCD Diabetes Research Centre, Conway In- complications of type 1 and type 2 diabetes, has an in- stitute, University College Dublin, Belfield, Dublin, Ireland; the School of Medicine, University College Dublin, Belfield, Dublin, Ireland; the Mater herent genetic susceptibility. Familial clustering of DN is University Hospital, Dublin, Ireland; the Department of Chronic Disease evident for both type 1 and type 2 diabetes (3–6), and ge- Prevention, Welfare and Health Promotion Division, National Institute for netic risk factors are being sought in multiple populations Health and Welfare, Helsinki, Finland; the Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland; the Department of (7–9). Unfortunately, robust replication of many initial Public Health, University of Helsinki, Helsinki, Finland; the South Ostro- associations has not been forthcoming (10). bothnia Central Hospital, Seinäjoki, Finland; the Division of Matrix Biology, This study recruited a large collection of individuals with Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; the Institute of Life Sciences, Swansea University, type 1 diabetes as part of the GEnetics of Nephropathy–an Swansea, U.K.; and the School of Biomolecular and Biomedical Sciences, International Effort (GENIE) consortium and examined se- University College Dublin, Belfield, Dublin, Ireland. lected candidate loci associated with DN from genome-wide Corresponding author: Jose C. Florez, jcflorez@partners.org. Received 1 June 2011 and accepted 5 April 2012. case-control studies or other association studies that DOI: 10.2337/db11-0751 reported high levels of statistical significance. The variants This article contains Supplementary Data online at http://diabetes examined and the rationale for their inclusion are as follows: .diabetesjournals.org/lookup/suppl/doi:10.2337/db11-0751/-/DC1 W.W.W., R.M.S., A.J.M., and N.S. contributed equally to this work. 2012 by the American Diabetes Association. Readers may use this article as 1) A single nucleotide polymorphism (SNP) (rs1617640) long as the work is properly cited, the use is educational and not for profit, within the promoter region of the EPO gene (encoding and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. erythropoietin) was identified as having a genome-wide significant (P , 5 3 10 ) association with ESRD and See accompanying commentary, p. 1923. diabetes.diabetesjournals.org DIABETES, VOL. 61, AUGUST 2012 2187 PRIOR GENETIC ASSOCIATIONS WITH DN proliferative diabetic retinopathy (PDR) (11). Interest- top-reported SNP (or proxy) for each gene in that report ingly, erythropoietin levels were elevated sevenfold for an association with DN. in the human vitreous fluid of nondiabetic individuals with the risk genotype TT compared with those with In this study, we have assembled the largest reported the wild-type GG genotype. In addition, EPO expres- case-control sample of DN in type 1 diabetes to evaluate sion levels were significantly elevated above control in the previously reported genetic associations in newly geno- the tissues and vitreous fluid of animal models of DN typed samples from the U.K., Republic of Ireland (R.O.I.), (DN in db/db mice) and in proliferative retinopathy and Finland, plus pre-existing data from the U.S. GoKinD. (murine oxygen-induced retinopathy model), respec- tively (11). RESEARCH DESIGN AND METHODS 2) The engulfment and cell motility 1 gene (ELMO1) has Cohorts been reported to be associated with DN in Japanese U.K.-R.O.I. collection. Recruited individuals were part of the All Ireland- patients with type 2 diabetes (12). Recently, Pezzolesi Warren 3-Genetics of Kidneys in Diabetes U.K. collection (U.K.-R.O.I.). All were et al. (13), using the Genetics of Kidneys in Diabetes self-reported as white, with grandparents born in the U.K. or Ireland, and type 1 U.S. Study (U.S. GoKinD) cohorts, also examined diabetes diagnosed before the age of 31 years requiring uninterrupted insulin ELMO1 for association with DN and presented evi- treatment. Case subjects (n = 903) with DN had persistent proteinuria (.0.5 dence of association of variants within this gene for g/24 h), hypertension (.135/85 mmHg and/or treatment with antihypertensive medication), and diabetic retinopathy. ESRD (28%) was defined as requiring the development of DN. However, the risk alleles for renal replacement therapy or having received a kidney transplant. Individuals ELMO1 identified in their study differed from those in the control group (n = 1,001) had had type 1 diabetes for at least 15 years, reported in the original Japanese investigation. In the had no evidence of microalbuminuria on repeated testing, and were not re- context of a genome-wide association study (GWAS), ceiving antihypertensive medication (Table 1). 118 SNPs were assessed in 1,705 individuals of Euro- Finnish Diabetic Nephropathy Study (FinnDiane). The FinnDiane study pean ancestry with type 1 diabetes (885 control subjects is a nationwide multicenter study of .4,800 adult participants with type 1 diabetes (16). This study comprises genotype data for 2,914 patients with type and 820 DN case subjects). The strongest associations 1 diabetes diagnosed before age 35 years and insulin treatment started within in ELMO1 in the U.S. study occurred at rs11769038 23 1 year of diagnosis. The disease status was defined by urine albumin excretion (odds ratio [OR] 1.24; P = 1.7 3 10 ) and rs1882080 rate (AER) or urine albumin-to-creatinine ratio (ACR) in at least two of three (OR 1.23; P = 3.2 3 10 ), located in intron 16. Two consecutive urine collections at local centers. Macroalbuminuria (n = 686) additional SNPs, located in introns 18 and 20, were was defined as AER .200 mg/min or .300 mg/24 h or an ACR .25 mg/mmol also nominally associated with DN. In total, eight for men and .35 mg/mmol for women in overnight, 24-h, or spot urine col- lections, respectively. Similarly, the limit for normal AER (n = 1,601) was ,20 ELMO1 SNPs were reported to confer risk for DN, al- mg/min or ,30 mg/24 h or ACR ,2.5 mg/mmol for men and ,3.5 mg/mmol for though none reached genome-wide significance (13). women. Control patients with normal AER were required to have type 1 Supportive evidence was also found in African Ameri- diabetes duration of at least 15 years. ESRD (n = 627) was defined as ongoing cans with type 2 diabetes and ESRD (14). dialysis treatment or receipt of a kidney transplant. From the total, 505 par- 3) The U.S. GoKinD GWAS analyzed 359,193 SNPs in 820 ticipants were included from an independent Finnish cohort collected by the case subjects (284 with proteinuria and 536 with ESRD) National Institute for Health and Welfare (17). These participants met the FinnDiane diagnosis and selection criteria and were analyzed together with and 885 control subjects with type 1 diabetes but no the FinnDiane cohort (Table 1). evidence of DN. Although no risk variant achieved U.S. GoKinD. The U.S. GoKinD study consists of a DN case-control cohort of genome-wide significance, the primary association anal- individuals diagnosed with type 1 diabetes before age 31 years, who were ysis identified 11 SNPs representing four distinct chromo- between 18 and 59 years of age at enrollment, and who began insulin treatment somal regions (P , 1 3 10 ). The strongest association within 1 year after diagnosis (18). The 905 case subjects were defined as people with DN reported in this study was on chromosome 9q aged 18–54, with type 1 diabetes for at least 10 years, and DN. The 898 control subjects were aged 18–59, had type 1 diabetes for at least 15 years, but did not with rs10868025 (OR 1.45, P =5.0 3 10 )(15). have DN. The DN definition includes individuals with ESRD (on dialysis or 4) Finally, in an effort to systematically explore and compre- having received a kidney transplant) or persistent macroalbuminuria (at least hensively capture common genetic variations that might two of three tests positive for albuminuria by dipstick $1+ or ACR .300 mg be associated with DN, we reviewed the largest meta- albumin/mg of urine creatinine). The U.K. GoKinD inclusion criteria were used analysis published to date studying genetic associations to recruit individuals to the control group. Individuals were recruited at two with the DN phenotype (7). In GENIE, we examined the study centers, George Washington University (GWU) and the Joslin Diabetes TABLE 1 Phenotypic characteristics of the GENIE cohorts (U.K.-R.O.I., FinnDiane, and U.S. GoKinD) U.K.-R.O.I. FinnDiane U.S. GoKinD† Case subjects Control subjects Case subjects Control subjects Case subjects Control subjects n 903 1,001 1,289 1,577 774 821 Sex (n) Male 531 438 764 651 402 342 Female 372 563 525 926 372 479 Type 1 diabetes Duration (years) 32.9 6 9.5 27.2 6 8.7 32.8 6 9.1 27.9 6 9.5 31.4 6 7.8 25.4 6 7.7 Age at diagnosis (years) 14.6 6 7.7 14.5 6 7.8 12.8 6 7.6 15.1 6 8.3 11 6 6.6 13 6 7.3 HbA (%) 9.0 6 1.9 8.7 6 1.6 8.8 6 1.6 8.0 6 1.2 7.5 6 1.9 7.5 6 1.2 1c BMI (kg/m ) 26.2 6 4.7 26.2 6 4.2 25.5 6 4.2 25.2 6 3.4 25.7 6 5.2 26.1 6 4.3 ESRD (%) 28.0 0 48.2 0 65.6 0 Categoric data are shown as indicated; continuous data as mean 6 SD. †Reanalysis of the U.S. GoKinD dataset using new quality control filters to account for published plate effects (see RESEARCH DESIGN AND METHODS for complete details). 2188 DIABETES, VOL. 61, AUGUST 2012 diabetes.diabetesjournals.org W.W. WILLIAMS AND ASSOCIATES Center (JDC) using differing methods of ascertainment and recruitment (see as well as sex chromosome and mitochondrial SNPs were excluded from Pezzolesi et al. [15] for details). Analysis of the U.S. GoKinD cohort was lim- analyses. After applying the QC protocol, we had access to 549,530 SNPs in ited to individuals whose reported primary ethnicity was white. 3,370 FinnDiane samples, 791,687 SNPs in 1,726 U.K.-R.O.I. samples, and Phenotype definition: DN and EPO study outcomes. DN was the primary 360,899 SNPs in 1,595 U.S. GoKinD samples. outcome for all association studies of the SNPs investigated. For the EPO study, SNP imputation. MACH 1.0 software (http://www.sph.umich.edu/csg/abecasis/ we used the phenotypic definitions used in the original report (11); namely, MACH) with the HapMap phase II CEU reference panel was used to perform ESRD as defined by case subjects who were on dialysis or who had received SNP imputation for GWAS results in each cohort. Estimates of the crossover a renal transplant; concurrently, this ESRD population also had to have evi- and error rates were obtained via 50 iteration rounds in ;300 randomly se- dence of advanced PDR on physical examination and a history of laser lected samples per cohort. A greedy algorithm was used for imputation, and treatment. Control subjects with evidence of PDR were excluded. We also the maximum likelihood method was specified to yield allele dosages. A filter examined PDR as an outcome independent of ESRD status. For this analysis, was applied to exclude SNPs with low imputation quality (r , 0.6), case subjects had clinical evidence of PDR, whereas control subjects had none resulting in ;2.4 million SNPs per cohort. (irrespective of DN status). Cohorts analyzed in the original EPO study (Fig. 1) Statistical analysis. Association tests were conducted using PLINK v1.07 (20) were composed of European-American cases, and control subjects were col- (http://pngu.mgh.harvard.edu/purcell/plink), with logistic regression adjusted lected from distinct geographic areas in the U.S. These included the GoKinD for sex and age. U.K.-R.O.I. was adjusted for recruitment center, but the two U.S. GoKinD centers, GWU and JDC, were analyzed separately as reported by cohort (Boston, Pittsburgh, and Minnesota), the Utah cohort (Salt Lake City), Pezzolesi et al. (15). Data from the GWAS genotyping was adjusted addition- and the Boston cohort (Boston Joslin Center for Diabetes) (11). ally for duration of type 1 diabetes and principal components from Eigenstrat SNP selection. SNP markers with evidence for association with DN sus- analysis. The EPO locus was analyzed with the Pearson x test in the Finn- ceptibility in reference studies (11) were selected for genotyping in GENIE. Diane dataset without adjusting for any covariates. Fixed-effects meta-analyses Where more than one SNP was associated at a particular locus with DN, the were conducted with the software package Comprehensive Meta-Analysis most strongly associated variant was selected for genotyping in the DN (Version 2.2.040, Englewood, NJ) and the software package METAL (http:// case-control cohorts. Where no genotyping assay could be developed for the www.sph.umich.edu/csg/abecasis/Metal/) (21) under the additive genetic index SNP, a proxy in strong linkage disequilibrium (LD) was genotyped using the CEU HapMap population. CEU is the official three letter code for the model. To determine the appropriate statistical cutoff for correction for HapMap samples of Utah residents with ancestry from Northern and Western multiple testing, we calculated the total number of effective tests (because Europe (see http://hapmap.ncbi.nlm.nih.gov/citinghapmap.html). For the a large portion of SNPs were in LD), using SNPSpD (http://gump.qimr.edu.au/ original U.S. GoKinD reported results and ELMO1, additional SNPs within general/daleN/SNPSpD/). SNPSpD uses correlation between analyzed SNPs to 20-kb upstream and downstream of the locus (or index SNP) were selected calculate the total number of independent tests (22). The total number of SNPs using the SNP Annotation and Proxy Search (http://www.broadinstitute.org/ tested is 2,199, with 113.7 effect-independent tests. Thus the experiment-wide mpg/snap/), specifying chromosome position, CEU samples, and 1000 Genome cutoff for statistical significance was set at 4.4 3 10 (0.05/113.7). Pilot 1 data. The expanded SNP list was extracted from GWAS results for U.K.-R.O.I. and FinnDiane (N. Sandholm et al., submitted). De novo genotyping. For the U.K.-R.O.I. collection (n = 1,904 unique indi- RESULTS viduals), SNPs were genotyped using Sequenom iPLEX (Sequenom, Hamburg, Germany) or TaqMan (Applied Biosystems, Warrington, U.K.) technology. EPO promoter polymorphism. The association of the Duplicate and no-DNA-template samples were included on all plates as ex- EPO promoter polymorphism, rs1617640, with DN was perimental controls. evaluated by de novo genotyping of the SNP in GENIE. For In FinnDiane, the EPO locus was genotyped with TaqMan chemistry (Applied this analysis, case subjects were defined as having both Biosystems, Foster City, CA) in 3,363 samples, of which 251 case subjects ESRD and PDR because the initial report showed the (ESRD + laser treatment) and 987 control subjects (no DN, no retinopathy) polymorphism was robustly associated with DN when both passed the phenotype criteria. All other SNPs were genotyped at the Institute of Molecular Medicine Finland (Helsinki, Finland) on the Illumina’sBeadArray of these “extreme” phenotypes were coexpressed. Significant 610Quad array. Illumina’s BeadStudio clustering algorithm was used to call association was not observed in the U.K.-R.O.I. (P = 0.19) genotypes in FinnDiane. SNPs were filtered for those with call rates .95%, minor or FinnDiane collections (P = 0.60), although the direc- allele frequency (MAF) .1%, and test for Hardy Weinberg equilibrium (HWE; tions of effect were consistent with the original report. P . 1 3 10 ). Sample filters included individual call rates .95% and no first- Fixed-effects meta-analysis of the association of rs1617640 degree relatives and resulted in 1,289 case subjects and 1,577 control subjects. with ESRD/PDR, including the previously reported cohorts Existing genotype data for the U.S. GoKinD genotype data were downloaded from dbGAP (phs000018.v2.p1, retrieved June 2010), containing genotype data (a total of 3,162 case and 3,845 control subjects across five from the Affymetrix 500 K array (Affymetrix, Santa Clara, CA). The version 2 separate cohorts of European and European-American genotype data differed from the original U.S. GoKinD data, containing updated ancestry) retained genome-wide statistical significance and recalled genotype calls for a previously reported problematic plate (19), 29 (OR 1.31 [95% CI 1.20–1.44], P =2 3 10 ; Fig. 1). and SNP call rate (.90%), MAF (.1%), and HWE performed by the National As an additional experimental control, we examined the Heart, Lung, and Blood Institute. We applied additional quality control (QC) potential association of the EPO promoter polymorphism filters to the data, including removing samples with evidence of contamination (extreme heterozygosity, n = 16), known parents (n = 4), non–European- with the development of PDR in case subjects, irrespective reported ancestry (n = 121), reported sex–genotype mismatch (n = 1), cryptic of ESRD status. No association was observed between related subjects (n = 4), and principal component analysis admixture outliers EPO and PDR for the individual cohorts or in the meta- (n = 5). Furthermore, SNP QC filters were applied to remove known prob- analysis of the combined results for FinnDiane (OR 0.95 lematic SNPs, HWE (P . 1 3 10 ), call rate (90%), missingness, MAF (1%), [95% CI 0.85–1.04], P = 0.25) or and U.K.-R.O.I. (0.96 [0.88– and SNPs plate effects. 1.04], P = 0.29). Furthermore, no association was observed GWAS genotyping. Genotypes for U.K.-R.O.I. (n = 1,830) and FinnDiane (n = 3,651) were supplemented with SNPs retrieved from GWAS results for after inclusion of the restricted phenotype, PDR, in U.S. each study (Sandholm et al., submitted). The FinnDiane study samples were GoKinD case and control subjects, separately, or in the genotyped using the Illumina’s BeadArray 610-Quad (Illumina, San Diego, CA) meta-analysis of all cohorts combined (results not shown). array at the Institute of Molecular Medicine Finland (FIMM, Helsinki, Finland). ELMO1. Neither single-center nor meta-analysis of de novo The U.K.-R.O.I. study samples were genotyped at the Broad Institute using the genotyping in U.K.-R.O.I., nor GWAS data for FinnDiane, Illumina Omni1-Quad array. Samples with insufficient DNA quality, quantity, revealed a significant association in subjects with type 1 or poor genotype concordance with previous available genotypes at a finger- printing stage were excluded. For all three discovery datasets (FinnDiane, diabetes between rs741301, the previously reported risk U.K.-R.O.I., U.S. GoKinD), a standardized and detailed genotype QC proce- variant within ELMO1, and DN (OR 1.04 [95% CI 0.95–1.13], dures were applied using PLINK supplemented with perl and R scripts. These P= 0.46; Supplementary Fig. 1). Pezzolesi et al. (13) also included selecting SNPs with a call rate .90%, MAF .1%, and concordance tested rs741301 but did not replicate the reported associa- with HWE (P . 10 ). We discarded samples with a call rate ,95%, extreme tion. They went on to test other SNPs in the region and heterozygosity, cryptic relatedness, or ethnic outliers by principal components reported nominal associations (P =0.002–0.05) with eight analysis, and tested for SNP missingness by haplotype (P , 10 ), by phenotype 28 27 (P . 10 ), and by plate effects (P , 10 ). Probes for copy number variation other SNPs . We examined LD between rs741301 and the diabetes.diabetesjournals.org DIABETES, VOL. 61, AUGUST 2012 2189 PRIOR GENETIC ASSOCIATIONS WITH DN FIG. 1. Previously published and new results from this study provide an estimate of the effect of the EPO promoter SNP (rs1617640) on the risk of the combined phenotype of PDR and ESRD in type 1 diabetes in five cohorts (3,162 case subjects and 3,845 control subjects) in a fixed-effects meta-analysis. SNPs reported to be associated with DN by these inves- 1.12, P = 0.08) and in meta-analysis of the two replication tigators. The r statistic between rs741301 and the other cohorts (U.K.-R.O.I. and FinnDiane; OR 1.06, P = 0.06; SNPs revealed only low to moderate LD, ranging from 0.38 Table 2). In expanded locus-region analyses (plus or minus to 0.65 (Supplementary Table 1). 20 kb of the locus of interest), no SNP reached significance As an additional and more extensive test of variants in after adjustment for multiple testing (one-tailed P = 0.03, this region, we performed an expanded analysis capturing experiment-wide threshold P = 4.3 3 10 ) for the two all available SNPs 20 kb upstream and downstream of the cohorts separately or via meta-analysis. The combined ELMO1 locus to account for LD differences presumably meta-analysis including U.S. GoKinD revealed two SNPs due to ancestry. Results of the expanded analysis did not downstream of FRMD3, rs1888747 (P = 1.5 3 10 ) and reveal any significant SNPs for either cohort individually rs13288659 (P = 9.7 3 10 ), which showed significance or for the two cohorts meta-analyzed after correcting for after adjusting for experiment-wide multiple testing (P , 24 24 multiple testing (P , 4.3 3 10 ). Furthermore, no SNP 4.3 3 10 ). However, neither SNP achieved genome-wide achieved significance with inclusion of the U.S. GoKinD significance (P , 5 3 10 ; Supplementary Table 3). results in the meta-analysis (Supplementary Table 2). Pooled meta-analyses examining variants associated Risk variants reported from U.S. GoKinD for DN in with DN in type 1 and type 2 diabetes. In the most type 1 diabetes. Eleven DN susceptibility SNPs that were comprehensive literature search for DN associated genetic first reported by the U.S. GoKinD investigators as highly variants to date, Mooyaart et al. (7) identified 24 loci. In associated with the risk of developing DN in type 1 di- GENIE, we examined all the available top-reported SNPs abetes were parsed into eight candidate loci. We selected (or their proxies) for each gene in that report. Three SNPs one representative SNP for each region of strong LD in were nominally associated (P , 0.05) with DN: rs13293564 which multiple SNPs represented the same association at UNC13B (P = 0.01) and rs179975 at the ACE (P= 0.03) signal. After performing additional QC checks (see RESEARCH in FinnDiane, and rs39075 at CPVL/CHN2 (P= 0.05) in the DESIGN AND METHODS), we first tested the eight U.S. GoKinD U.K.-R.O.I. samples. In a meta-analysis of the two cohorts, potential DN susceptibility SNPs by reanalyzing the U.S. the ACE polymorphism remained nominally significant GoKinD dataset downloaded from dbGAP (19). As in the (P= 0.04). Including the U.S. GoKinD results, the FRMD3 original report, none of the eight SNPs were associated signal at rs1888747 emerged as noted above; no other with DN at genome-wide statistical significance (Table 2). signals were significant after adjusting for multiple com- The two SNPs in the FERM (F [Band 4.1], E [Ezrin], R parisons (see Supplementary Table 4 for full details). [Radixin], M [Moesin]) domain 3 (FRMD3) region showed similar P values as in the original report (2.1 3 10 and DISCUSSION 1.6 3 10 , respectively; Table 2). In our analysis, the statistical significance of the P values for SNPs in the Using a large, homogeneous population sample of European CPVL/CHN2 and CARS regions was reduced from 6.5 3 ancestry subjects with type 1 diabetes in the GENIE con- 27 23 26 23 10 to 2 3 10 and 6.4 3 10 to 2.2 3 10 , respectively. sortium, we were unable to replicate most of the previously P values for the 6 SNPs in the 13q region were also reported genetic associations with DN that we examined. 26 25 changed from 1.8–7.0 3 10 to 1.4–9.5 3 10 . Our findings do not support previously reported genetic We next examined these SNPs in our newly genotyped associations with DN in type 1 diabetes in the largest GWAS samples. Case-control association analysis for these loci in published to date (15). None of those signals reached the U.K.-R.O.I. and FinnDiane samples revealed no signifi- genome-wide statistical significance with the addition of cant associations in either cohort. The strongest signal was larger, similarly ascertained datasets. Using ORs at the observed at rs39075 near CPVL/CHN2 in U.K.-R.O.I., (OR lower limit of the 95% CI from the original publication, our 2190 DIABETES, VOL. 61, AUGUST 2012 diabetes.diabetesjournals.org TABLE 2 Results in the reanalyzed U.S. GoKinD and additional GENIE cohorts for eight SNPs with strongest reported associations to DN in U.S. GoKinD Meta-analysis of U.K.-R.O.I. & Published U.S. GoKinD Updated U.S. GoKinD† U.K.-R.O.I. FinnDiane FinnDiane Risk allele SNP Nearest gene (nonrisk) OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P OR (95% CI) P 27 23 rs39075 CPVL/CHN2 G (A) 1.43 (1.23–1.64) 6.5 3 10 1.29 (1.13–1.45) 2.0 3 10 1.12 (0.99–1.25) 0.08 1.01 (0.89–1.12) 0.92 1.06 (0.98–1.25) 0.06 27 27 rs1888747* FRMD3 G (C) 1.45 (1.25–1.67) 6.3 3 10 1.56 (1.40–1.73) 2.1 3 10 1.03 (0.86–1.22) 0.77 1.07 (0.95–1.19) 0.25 1.06 (0.96–1.17) 0.24 27 26 rs1086805 FRMD3 A (G) 1.45 (1.25–1.67) 5.0 3 10 1.47 (1.31–1.62) 1.6 3 10 1.05 (0.90–1.24) 0.52 1.07 (0.95–1.19) 0.25 1.06 (0.97–1.17) 0.19 26 23 rs451041 CARS A (G) 1.36 (1.19–1.56) 6.4 3 10 1.28 (1.12–1.43) 2.2 3 10 1.07 (0.94–1.29) 0.29 1.00 (0.88–1.11) 0.93 1.03 (0.95–1.12) 0.45 26 25 rs1041466‡ No gene (13q) A (G) 1.38 (1.20–1.58) 3.2 3 10 1.36 (1.20–1.51) 9.3 3 10 1.01 (0.88–1.14) 0.94 1.00 (0.89–1.11) 0.99 1.00 (0.93–1.09) 0.92 26 25 rs1411766/rs1741288 No gene (13q) A (G) 1.41 (1.23–1.63) 1.8 3 10 1.42 (1.26–1.57) 1.4 3 10 1.01 (0.88–1.14) 0.87 0.93 (0.82–1.05) 0.24 0.97 (0.89–1.06) 0.47 26 25 rs6492208/rs2391777 No gene (13q) T (C) 1.37 (1.20–1.59) 6.1 3 10 1.36 (1.21–1.52) 9.5 3 10 1.03 (0.90–1.16) 0.64 1.00 (0.89–1.12) 0.97 1.01 (0.93–1.10) 0.76 26 25 rs7989848 No gene (13q) A (G) 1.37 (1.19–1.56) 7.0 3 10 1.36 (1.21–1.52) 5.5 3 10 1.05 (0.93–1.18) 0.41 1.01 (0.90–1.12) 0.88 1.03 (0.95–1.11) 0.50 †Reanalysis of the U.S. GoKinD dataset using new quality control filters to account for published plate effects (see RESEARCH DESIGN AND METHODS for complete details). *In FinnDiane, 2 2 rs13289150 was used as proxy for rs1888747 (D’ =1, r = 0.82 in HapMapII CEU) and for rs10868025; (D’ =1, r = 1 in HapMapII CEU). ‡SNP rs1411765 was used as proxy for rs1041466 (D’ = 0.89, r = 0.62 in HapMapII CEU). W.W. WILLIAMS AND ASSOCIATES combined sample of U.K.-R.O.I. and FinnDiane cohorts in GENIE had 99.9% power (at a = 0.001) to detect the U.S. GoKinD reported effect sizes (Supplementary Table 5); thus, our negative results (even at a = 0.05) were un- expected. Further, after performing additional QC checks on the original U.S. GoKinD dataset and combining these samples with GENIE, we were unable to achieve genome- wide significant replication in the meta-analysis. We investigated other previously reported genetic asso- ciations with DN in subjects with type 1 diabetes, namely the EPO promoter polymorphism rs1617640 and variants within the ELMO1 gene; these also failed to replicate in GENIE. The EPO promoter polymorphism retained genome- wide significance after meta-analysis of the prior data combined with ours, for the combined phenotype of ESRD and PDR. However, the overall P value attained was at- 211 29 tenuated from 2.8 3 10 to 2 3 10 . We also did not observe evidence for replication of the association of rs741301 in the ELMO1 gene and DN in GENIE. Shimazaki et al. (12) first reported an association for this genetic variant with DN in Japanese subjects with type 2 diabetes. Subsequently, Pezzolesi et al. (13) failed to replicate the association of the same SNP in type 1 diabetes but reported that eight SNPs within the gene locus had nominal associations with DN in the European-derived subjects in U.S. GoKinD. Lack of replication may be due to different genomic patterns in populations of diverse ances- tries or the distinct genetic architecture of DN in type 1 versus type 2 diabetes (14). To address the first concern, we expanded our analysis by interrogating all available SNPs plus or minus 20 kb of ELMO1. Because in the U.S. GoKinD analysis of ELMO1 the risk variants were only in weak to modest LD with the index SNP (r between rs741301 and the U.S. GoKinD SNPs ranged from 0.38 to 0.65), we reasoned that this expanded strategy would ac- count for most of the differences in LD presumably due to ancestry. The results of this expanded analysis, however, did not reveal any significant association for the additional 670 SNPs interrogated in ELMO1 in the U.K.-R.O.I. or FinnDiane cohorts individually, for these two cohorts in meta-analysis, or when combined with previously reported risk variants reported from U.S. GoKinD. As a final step to ensure a systematic and more com- prehensive approach to candidate loci associated with DN, we reviewed the top SNPs from the 24 loci cited by the largest meta-analysis published to date that has examined candidate genetic variants for association with DN (7). From the results of the pooled meta-analyses, the strongest signal emerged at rs1888747 in FRMD3 (P =1.5 3 10 ). Variants within this gene have been reported to be associated with DN in European cohorts (7,15,23), and Freedman et al. (24) recently reported evidence for a role for gene–gene interactions between myosin heavy-chain 9 (MYH9)– apolipoprotein L1 (APOL1) haplotypes and FRMD3 in African American subjects. It should be noted, however, that risk variants within this gene have achieved only nominal but not genome-wide statistical significance in all previous reports. From the foregoing observations, we conclude that there is a high likelihood that many of the previously reported positive associations with DN represent potential false-positive findings (type I error). We emphasize that the combined sample of U.K.-R.O.I. and FinnDiane represents a substantially larger collection of case and control subjects than U.S. GoKinD and is well powered to detect the origi- nally reported effect sizes, thereby decreasing the likelihood diabetes.diabetesjournals.org DIABETES, VOL. 61, AUGUST 2012 2191 PRIOR GENETIC ASSOCIATIONS WITH DN of a false-negative finding (type II error), even accounting shared genetic susceptibility for nephropathy between for the likely overestimation of effect sizes due to the type 1 and type 2 diabetes, remain unresolved. winner’s curse phenomenon (25). We also harmonized the ascertainment criteria for case-control definitions across ACKNOWLEDGMENTS all the study populations (including U.S. GoKinD), making it unlikely that phenotypic heterogeneity across study Funding for this study was provided by National Institutes populations explains the lack of replication. of Health National Institute of Diabetes and Digestive and A crucial issue that bears on the interpretation of Kidney Diseases Grant R01-DK-081923 to J.N.H., P.-H.G., case-control studies of the genetics of DN concerns the and J.C.F., a Science Foundation Ireland U.S.-Ireland R&D adequacy of phenotype definition. In this and most studies partnership, and The Health Research Board Ireland. R.M.S. cited to date, there is the presumption that long-duration was supported by a Juvenile Diabetes Research Founda- diabetes exposure and the presence of frank protein in the tion postdoctoral fellowship (#3-2011-70). The FinnDiane urine—macroalbuminuria—defines DN and that pheno- Study was supported by grants from the Folkhälsan Research typic heterogeneity has been well controlled through this Foundation, the Wilhelm and Else Stockmann Foundation, Liv classification. These definitions are derived in large mea- och Hälsa Foundation, Helsinki University Central Hospital sure from the classic studies of Parving et al. (26), Viberti Research Funds (EVO), the Sigrid Juselius Foundation, the et al. (27), and Mogensen and Christensen (28), who docu- Signe and Arne Gyllenberg Foundation, Finska Läkaresällska- mented 30 years ago a virtually inexorable progression to pet, the European Commission, and the European Union’s ESRD in patients who developed microalbuminuria after Seventh Framework Program (FP7/2007-2013) for the In- approximately 2 decades of exposure to the diabetic met- novative Medicine Initiative under Grant Agreement No. abolic milieu. However, these longitudinal findings were IMI/115006 (the Surrogate Markers for Micro- and Macro- based on small numbers of patients. The plasticity of DN vascular Hard Endpoints for Innovative Diabetes Tools phenotypes is reflected in more recent and much larger [SUMMIT] consortium). longitudinal studies showing that most patients with type 1 J.C.F. has received consulting honoraria from Novartis, diabetes, categorized initially as having microalbuminuria, Eli Lilly, and Pfizer. No other potential conflicts of interest undergo regression to normoalbuminuria with preservation relevant to this article were reported. of renal function (29). It is not entirely clear that micro- W.W.W. contributed to study design and management, albuminuria versus macroalbuminuria, stage of chronic data acquisition, statistical and data analysis, and manu- kidney disease and attendant renal function, the rate of script preparation and review. R.M.S. contributed to data renal decline, or the occurrence of extreme phenotypes, acquisition, genotyping, statistical and data analysis, and such as ESRD/PDR, represent one disease process along manuscript review. A.J.M. contributed to data acquisition, a continuum or many distinct disease states, each of which statistical and data analysis, and manuscript preparation. may be under distinct genetic control. As pointed out re- N.S. contributed to data acquisition, statistical and data cently (24), genetic variants, such as those in MYH9 and analysis, and manuscript preparation and review. C.F. APOL1 that are common in certain ethnic groups, may contributed to data acquisition, statistical and data anal- mask the effects at other loci unless methods such as ysis, and manuscript review. A.T., C.Gu., and M.P. con- multilocus modeling and interaction analyses are used to tributed to genotyping and manuscript review. J.B.M., E.F., control for these effects. V.H., R.L., D.G., K.H., J.K., M.R.-B., N.T., M.S., J.W., and B.H. In addition, phenotypic variation may be a function of contributed to data acquisition and manuscript review. ethnicity and disease-specific gene expression. For exam- G.J.M., T.I., E.P.B., D.M.S., C.P., S.B., F.M., C.Go., and ple, Pima Indians with type 2 diabetes have very early- A.P.M. contributed to manuscript review. J.S. contributed onset DN, characterized by an accelerated loss of renal to genotyping and statistical and data analysis. L.T., J.P., function and progression to ESRD despite lower blood C.S., J.T., and A.-M.Ö. contributed to data acquisition. A.S. pressures and lipid levels, factors thought to be protective contributed to data acquisition, genotyping, and manu- (30). This has been postulated to be due to structural dif- script review. K.T. contributed to statistical and data anal- ferences in the nephron–podocyte number and density per ysis and manuscript review. J.N.H. contributed to study glomerulus (“podocyte insufficiency”), a decrease in net design and manuscript review. P.-H.G. contributed to study nephron mass (glomerulopenia) resulting in glomerulomegaly, design, statistical and data analysis, and to manuscript increased intraglomerular capillary pressure, and ultimately, review. J.C.F. contributed to study design and manage- hyperfiltration injury (30). Whether these structural and ment, statistical and data analysis, and manuscript prep- intrarenal hydraulic changes could be genetically regulated aration and review. J.C.F. is the guarantor of this work is ultimately a testable hypothesis; they warrant further in- and, as such, had full access to all of the data in the study vestigation to continue the inquiry why certain populations and takes responsibility for the integrity of the data and have an apparent disproportional susceptibility to ESRD theaccuracy of thedataanalysis. and, particularly, DN. The authors acknowledge the physicians, nurses, and In summary, we have presented evidence that several researchers at each center participating in the collection previously reported genetic associations with DN in type 1 of patients: in the FinnDiane study group and at the Hel- diabetes could not be replicated in a large, homogeneous sinki University Central Hospital, Department of Medicine, sample of subjects with type 1 diabetes. Our failure to Division of Nephrology: C. Forsblom, A. Ahola, J. Fagerudd, replicate these associations underscores the need to apply M. Feodoroff, D. Gordin, V. Harjutsalo, O. Heikkilä, K. Hietala, stringent statistical thresholds of significance, maximize J. Kytö, M. Lehto, S. Lindh, M. Parkkonen, K. Pettersson- power through meta-analysis of all available data, and seek Fernholm, M. Rosengård-Bärlund, M. Rönnback, A. Sandelin, replication in independent samples, as has been proposed A.-R. Salonen, L. Salovaara, M. Saraheimo, T. Soppela, by a number of different authors (31,32). Finally, the ap- A. Soro-Paavonen, P. Summanen, L. Thorn, N. Tolonen, plicability and generalizability of DN risk loci from type 1 J. Tuomikangas, T. Vesisenaho, and J. Wadén. Anjalankoski diabetes to type 2 diabetes, and the related question of Health Centre: S. Koivula and T. Uggeldahl. Central Finland 2192 DIABETES, VOL. 61, AUGUST 2012 diabetes.diabetesjournals.org W.W. WILLIAMS AND ASSOCIATES Central Hospital, Jyväskylä: T. Forslund, A. Halonen, Hospital: V. Javtsenko and M. Tamminen. Pietarsaari Hospi- A. Koistinen, P. Koskiaho, M. Laukkanen, J. Saltevo, and tal: M.-L. Holmbäck, B. Isomaa, and L. Sarelin. Pori City M. Tiihonen. Central Hospital of Åland Islands, Mariehamn: Hospital: P. Ahonen, P. Merensalo, and K. Sävelä. Porvoo M. Forsen, H. Granlund, A.-C. Jonsson, and B. Nyroos. Hospital: M. Kallio, B. Rask, and S. Rämö. Raahe Hospital: Central Hospital of Kanta-Häme, Hämeenlinna: P. Kinnunen, A. Holma, M. Honkala, A. Tuomivaara, and R. Vainionpää. A. Orvola, T. Salonen, and A. Vähänen. Central Hospital Rauma Hospital: K. Laine, K. Saarinen, and T. Salminen. of Länsi-Pohja, Kemi: H. Laukkanen, P. Nyländen, and Riihimäki Hospital: P. Aalto, E. Immonen, and L. Juurinen. A. Sademies. Central Ostrabothnian Hospital District, Kokkola: Salo Hospital: A. Alanko, J. Lapinleimu, P. Rautio, and S. Anderson, B. Asplund, U. Byskata, P. Liedes, M. Kuusela, M. Virtanen. Satakunta Central Hospital, Pori: M. Asola, and T. Virkkala. City of Espoo Health Centre Espoonlahti: M. Juhola, P. Kunelius, M.-L. Lahdenmäki, P. Pääkkönen, A. Nikkola and E. Ritola; Tapiola: M. Niska and H. Saarinen; and M. Rautavirta. Savonlinna Central Hospital: E. Korpi- Samaria: E. Oukko-Ruponen and T. Virtanen; and Viherlaakso: Hyövälti, T. Latvala, and E. Leijala. South Karelia Central A. Lyytinen. City of Helsinki Health Centre Puistola: Hospital, Lappeenranta: T. Ensala, E. Hussi, R. Härkönen, H. Kari and T. Simonen; Suutarila: A. Kaprio, J. Kärkkäinen, U. Nyholm, and J. Toivanen. Tampere Health Centre: and B. Rantaeskola; and Töölö: P. Kääriäinen, J. Haaga, A. Vaden, P. Alarotu, E. Kujansuu, H. Kirkkopelto-Jokinen, and A.-L. Pietiläinen. City of Hyvinkää Health Centre: M. Helin, S. Gummerus, L. Calonius, T. Niskanen, T. Kaitala, S. Klemetti, T. Nyandoto, E. Rontu, and S. Satuli-Autere. and T. Vatanen. Tampere University Hospital: I. Ala-Houhala, City of Vantaa Health Centre Korso: R. Toivonen and T. Kuningas, P. Lampinen, M. Määttä, H. Oksala, T. Oksanen, H. Virtanen; Länsimäki: R. Ahonen, M. Ivaska-Suomela, K. Salonen, H. Tauriainen, and S. Tulokas. Tiirismaa Health and A. Jauhiainen; Martinlaakso: M. Laine, T. Pellonpää, and Centre, Hollola: T. Kivelä, L. Petlin, and L. Savolainen. R. Puranen; Myyrmäki: A. Airas, J. Laakso, and K. Rautavaara; Turku Health Centre: I. Hämäläinen, H. Virtamo, and Rekola: M. Erola and E. Jatkola; and Tikkurila: R. Lönnblad, M. Vähätalo. Turku University Central Hospital: K. 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DiabetesPubmed Central

Published: Jul 17, 2012

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