Cardiovascular and Metabolic Risk ORIGINAL ARTICLE

ABC Transporter and Risk of Type 2 Diabetes A study of 40,000 individuals from the general population

1,2 2,3,4,6 JESPER SCHOU, MSC BØRGE G. NORDESTGAARD, MD, DMSC secretion in mice (2). In humans, family 1,2,3,4 1,2,4 ANNE TYBJÆRG-HANSEN, MD, DMSC RUTH FRIKKE-SCHMIDT, MD, DMSC 5 studies and small case-control studies HOLGER J. MØLLER, MD, PHD have suggested that loss-of-function muta- tions in ABCA1 associate with decreased in- sulin secretion (8) or type 2 diabetes (9). OBJECTIVEd b b Alterations of pancreatic -cell cholesterol content may contribute to -cell Further, macrophages from diabetic mice dysfunction. Two important determinants of intracellular cholesterol content are the ATP- (10) and humans (11) display decreased binding cassette (ABC) transporters A1 (ABCA1) and -G1 (ABCG1). Whether genetic variation ABCG1 expression. Finally, we recently re- in ABCA1 and ABCG1 predicts risk of type 2 diabetes in the general population is unknown. ported that two functional variants, ABCA1 RESEARCH DESIGN AND METHODSdWe tested whether genetic variation in the N1800H and ABCG1 g.-376C.T, were as- promoter and coding regions of ABCA1 and ABCG1 predicted risk of type 2 diabetes in the sociated with, respectively, substantial re- general population. Twenty-seven variants, identified by previous resequencing of both genes, ductions in levels of HDL cholesterol or were genotyped in the Copenhagen City Heart Study (CCHS) (n = 10,185). Two loss-of-function reduced ABCG1 mRNA expression levels mutations (ABCA1 N1800H and ABCG1 g.-376C.T) (n = 322) and a common variant (ABCG1 . n and increased risk of ischemic heart disease g.-530A G) were further genotyped in the Copenhagen General Population Study (CGPS) ( = and myocardial infarction (12,13)drisk 30,415). factors and disease entities closely related RESULTSdOnly one of the variants examined, ABCG1 g.-530A.G, predicted a decreased risk to diabetes. Collectively, these data suggest of type 2 diabetes in the CCHS (P for trend = 0.05). Furthermore, when validated in the CGPS or that decreased ABCA1 and ABCG1 func- in the CCHS and CGPS combined (n = 40,600), neither the two loss-of-function mutations tion may influence normal b-cell action (ABCA1 N1800H, ABCG1 g.-376C.T) nor ABCG1 g.-530A.G were associated with type 2 and susceptibility to type 2 diabetes. For diabetes (P values .0.57 and .0.30, respectively). clarification of whether genetic variation in ABCA1 and ABCG1 contributes to risk CONCLUSIONSdGenetic variations in ABCA1 and ABCG1 were not associated with in- creased risk of type 2 diabetes in the general population. These data were obtained in general of type 2 diabetes in humans, large studies population samples harboring the largest number of heterozygotes for loss-of-function muta- of the general population are needed. tions in ABCA1 and ABCG1. We tested the following hypotheses: 1) genetic variation in ABCA1 and ABCG1 associates with measures of glucose me- tabolism, markers of inflammation, and b lipid and lipoprotein levels in the general he cellular mechanisms underlying insulin secretion in cells (2,3). The 2 pancreatic b-cell dysfunction in type question of whether genetic variation in population and ) genetic variation in T ABCA1 and ABCG1 is associated with 2 diabetes are incompletely under- ABCA1 and ABCG1 predicts risk of type 2 stood. Recent evidence suggests that al- diabetes in the general population re- risk of type 2 diabetes in the general pop- terations of b-cell cholesterol content mains unanswered. ulation. These hypotheses were tested in may contribute to islet dysfunction and Homozygosity for mutations in ABCA1 two studies of the general population, the loss of insulin secretion (1). Two impor- causes a rare HDL-deficiency syndrome, Copenhagen City Heart Study (CCHS) (n = 10,185) and the Copenhagen General tant determinants of intracellular choles- Tangier disease, a disorder characterized n terol content are the ATP-binding cassette by accumulation of cholesterol esters in pe- Population Study (CGPS) ( = 30,415), (ABC) transporters A1 (ABCA1) and -G1 ripheral tissues (4–7). Deficiency of ABCA1 harboring the largest known number of b heterozygotes for loss-of-function muta- (ABCG1), which have been suggested to in cells results in altered intracellular cho- ABCA1 ABCG1 n regulate cholesterol homeostasis and lesterol homeostasis and impaired insulin tions in and ( =322). ccccccccccccccccccccccccccccccccccccccccccccccccc RESEARCH DESIGN AND From the1Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark; the 2Faculty of Health METHODSdStudies were approved by Sciences, University of Copenhagen, Copenhagen, Denmark; the 3Copenhagen City Heart Study, institutional review boards and Danish Bispebjerg Hospital, Copenhagen, Denmark; the 4Copenhagen General Population Study, Herlev Hospital, 5 ethics committees (nos. KF-100.2039/91, Copenhagen, Denmark; the Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, KF-01-144/01, and H-KF-01-144/01) and Denmark; and the 6Department of Clinical Biochemistry, Herlev Hospital, Herlev, Denmark. Corresponding author: Ruth Frikke-Schmidt, [email protected]. conducted according to the Declaration of Received 13 January 2012 and accepted 18 June 2012. Helsinki. Informed consent was obtained DOI: 10.2337/dc12-0082 from all participants. All participants were This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10 white and of Danish descent. No partic- .2337/dc12-0082/-/DC1. © 2012 by the American Diabetes Association. Readers may use this article as long as the work is properly ipants appeared in more than one of the cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/ two studies, permitting independent con- licenses/by-nc-nd/3.0/ for details. firmation of the findings in each group. care.diabetesjournals.org DIABETES CARE 1 Diabetes Care Publish Ahead of Print, published online November 8, 2012 ABC transporter genes and type 2 diabetes

CCHS. This was a prospective study of used to measure HDL cholesterol and tri- Events the general population initiated in 1976– glycerides (Boehringer Mannheim, Man- Informationondiagnosesoftype2di- 1978 with follow-up examinations in nheim, Germany), and LDL cholesterol abetes (World Health Organization; 1981–1983, 1991–1994, and 2001– was calculated using the Friedewald ICD-8, 250; and ICD-10, E11, E13, 2003. Individuals were selected based equation (19), when plasma triglycerides E14) was collected from the National on the National Danish Civil Registration were ,4.00 mmol/L (,354.00 mg/dL), Danish Patient Registry (94% of end System to reflect the adult Danish popu- and otherwise was measured directly points) and the National Danish Causes lation aged 20 to $80 years. Data were (Thermo Fisher Scientific, Waltham, of Death Registry (6% of end points). All obtained from a questionnaire, a physical MA). diagnoses in these registries are given by a examination, and blood samples. Blood samples for DNA extraction were avail- able on 10,185 participants attending the 1991–1994 and/or 2001–2003 examinations. CGPS. This is a prospective study of the general population initiated in 2003 with ongoing enrollment. For the current study, we used the CGPS cross-sectionally in order to include all available events oc- curring from the establishment of the National Danish Patient Registry and Na- tional Danish Causes of Death Registry in 1976 to the end of follow-up in 2010. Participants were recruited from the gen- eral population and examined as in the CCHS. At the time of genotyping, 30,415 had been included.

Genotyping The ABI PRISM 7900HT Sequence De- tection System (Applied Biosystems, Fos- ter City, CA) was used to genotype the CCHS for all variants in the core promoter and nonsynonymous variants identified by previous resequencing of the ABCG1 and ABCA1 genes (12–14). Two loss-of- function mutations (ABCA1 N1800H and ABCG1 g.-376C.T),aswellasacommon variant associated with reduced risk of type 2 diabetes in the CCHS (ABCG1 g.-530A.G), were further genotyped in the CGPS. TaqMan-based assays were used.

Measures of glucose metabolism, markers of inflammation, and lipid and lipoprotein levels Glucose concentrations were measured using a standard hexokinase/glucose-6- phosphate-dehydrogenease assay (15), and HbA1c was measured on a Kone analyzer (Kone-Laboratory, Helsinki, Finland). Soluble CD163, a marker of low-grade inflammation and a strong predictor of type 2 diabetes (16), was measured using an in-house sandwich enzyme-linked immunosorbent assay Figure 1dMeasures of glucose metabolism, inflammatory markers, and levels of lipids and on a BEP-2000 ELISA analyzer as previ- lipoproteins as a function of 13 ABCA1 variants. Values shown are means 6 SEM. P values by ously described (16,17). High-sensitivity Kruskal-Wallis one-way ANOVA or Mann Whitney U test. Parametric ANOVA and Student t C-reactive (hsCRP) was measured tests gave similar results. All calculations were performed for 10,185 individuals in CCHS except by standard nephelometric or turbidimet- for HbA1c, soluble CD163 (sCD163), and hsCRP, for which measurements were available for ric assays (18). Colorimetric assays were 5,661, 8,191, and 9,752 individuals, respectively.

2 DIABETES CARE care.diabetesjournals.org Schou and Associates hospital-employed physician on the day of their death date. In the CGPS and in the [20]), logistic regression analysis ad- discharge or death of the patient and are CCHS and CGPS combined, logistic re- justed for age in 10-year age-groups thus based on international criteria, med- gression analysis was used to estimate and sex was used to estimate odds ratios ical records, and pharmaceutical therapy. odds ratios. Multifactorial adjustment in the CCHS. Missing values were im- Follow-up ended in August 2010 and was was for age, sex, BMI, hypertension, puted. Burden testing for rare variants 100% complete; i.e., no individual was lost smoking, and physical inactivity, which (21) was performed in two ways: 1)col- to follow-up in either study. are the most important risk factors for lapsing rare variants with minor allele fre- type 2 diabetes (20). For the end point pre- quencies ,1% and 2) collapsing rare 2 Other covariates diabetes (BMI $25 kg/m ,HbA1c $5.7%, variants that were experimentally veri- BMI was measured as weight (kilograms) physical inactivity, and hypertension fied to be functional. Fischer exact test divided by the square of measured height in meters. Use of lipid-lowering therapy was self-reported. Hypertension was de- fined as systolic blood pressure $140 mmHg or diastolic blood pressure $90 mmHg and/or use of antihypertensive drugs. Smoking was defined as current smoking. Physical inactivity was defined as ,2–4 h per week of light physical ac- tivity at leisure time.

Statistical analysis Data were analyzed using Stata/SE statis- tical software (version 12.0; StataCorp, College Station, TX). Two-sided proba- bility values ,0.05 were considered sig- nificant. Mann-Whitney U test and Kruskal-Wallis one-way ANOVA evalu- ated two- and three-group comparisons for continuous variables, and Pearson x2 or Fischer exact tests were used for cate- gorical variables. For trend tests, groups of individuals were classified by ABCA1 or ABCG1 genotypes and ranked 0, 1, and 2, with 0 (noncarriers) as the reference group. Pairwise linkage disequilibria and haplotype association analyses for all 23 common variants in ABCA1 and ABCG1 were estimated using the software Haplo- view 4.0 (available at www.broad.mit. edu/mpg/haploview/download.php). To test our first hypothesis that ge- netic variation in ABCA1 and ABCG1 as- sociates with measures of glucose metabolism, inflammatory markers, and levels of lipids and lipoproteins in the general population, we used nonparamet- ric Mann-Whitney U and trend tests across genotypes with two and three groups, respectively. To test our second hypothesis, that genetic variation in ABCA1 and ABCG1 is associated with risk of type 2 diabetes in the general pop- ulation, we used Cox proportional haz- ards regression models, with age as time scale and use of left truncation (delayed entry), to estimate the hazard ratios of Figure 2dMeasures of glucose metabolism, inflammatory markers, and levels of lipids and type 2 diabetes in the prospective lipoproteins as a function of 14 ABCG1 variants. Values shown are means 6 SEM. P values by CCHS; individuals diagnosed with an Kruskal-Wallis one-way ANOVA or Mann Whitney U test. Parametric ANOVA and Student end point before entry were excluded t tests gave similar results. All calculations were performed for 10,185 individuals in CCHS except from Cox regression analyses, and those for HbA1c, soluble CD163 (sCD163), and hsCRP, for which measurements were available for dying during follow-up were censored at 5,661, 8,191, and 9,752 individuals, respectively. care.diabetesjournals.org DIABETES CARE 3 ABC transporter genes and type 2 diabetes evaluated whether collapsed rare variant individuals) showed 100% concordance plasma levels of HDL cholesterol were re- frequencies differed between type 2 di- with Taqman results. duced by 0.45 mmol/L in heterozygotes for abetes case subjects and participants ABCA1 N1800H compared with noncarri- without type 2 diabetes. Genetic variation in ABCA1 and ers (P 5 0.0001) (Fig. 1); the correspond- ABCG1 and biochemical phenotypes ing reduction for apoAI was 27.6 mg/dL. RESULTS Measures of glucose metabolism, mark- Similar findings for HDL cholesterol and ers of inflammation, and lipid and lipo- apoAI levels were seen in the CGPS (P val- Characteristics of participants and protein levels are presented for 13 ues = 0.0001) (data not shown). resequencing of ABCA1 and ABCG1 ABCA1 variants and 14 ABCG1 variants Characteristics of subjects in the CCHS and in Figs. 1 and 2, respectively. After appli- Genetic variation in ABCA1 and in the CGPS with or without type 2 diabetes cation of a Bonferroni-corrected P value ABCG1 and risk of type 2 diabetes are shown in Supplementary Table 1. Indi- of 0.0002 (0.05/8 biochemical markers Risk of type 2 diabetes for all 27 genetic viduals with type 2 diabetes were older and multiplied by 27 genetic variants), only variants in ABCA1 and ABCG1 is presented more often men, had lower HDL choles- one association remained significant: in Fig. 3. The g.-530A.G ABCG1 variant terol and apolipoprotein (apo)A-I levels and higher HbA1c and triglyceride levels, were more obese, were more often on lipid-lowering therapy, more often had hy- pertension, and were less physically active compared with control subjects. Resequencing of the core promoter and coding regions of ABCA1 and ABCG1 in individuals from the CCHS with the 1–2% lowest (n =95–180) and 1–2% high- est (n =95–180) plasma HDL cholesterol levels identified 27 promoter and nonsy- nonymous variants with a minor allele fre- quency .0.02% (13,14) (Supplementary Table 2). Except for three very rare ABCG1 variants (g.-686G.A, g.-269G. A, and S630 L), all variants identified in the current study were also reported in the publicly available 1000 Genomes Pro- ject (Supplementary Table 2). In the 1000 Genomes Project, 31 additional very rare variants, not identified in the CCHS, were reported. (For detailed information, see the legend to Supplementary Table 2.) All 27 variants identified by resequencing ex- treme HDL cholesterol groups in the CCHS were further genotyped in the entire CCHS cohort. Four variants were rare (al- lele frequency 0.02–0.07%), and 23 were more common (minor allele frequency 0.13–34%). The linkage disequilibrium patterns for these 23 common variants are shown for each in Supplementary Figs. 1 and 2. Two loss-of-function muta- tions, as well as a common variant with effect on risk of type 2 diabetes in the CCHS, were further genotyped in the CGPS and displayed minor allele frequen- cies similar to those in the CCHS: 0.1, 0.2, and 6.7% for ABCA1 N1800H, ABCG1 g.- 376C.T, and ABCG1 g.-530A.G, respec- tively. Genotype frequencies did not differ from those predicted by Hardy-Weinberg equilibrium (P values 0.11–0.98), except for ABCA1 E1172D (P =0.03),in the CCHS. However, sequencing 299 Figure 3dRisk of type 2 diabetes as a function of ABCA1 and ABCG1 genotype in CCHS. Hazard base pairs spanning this variant in all ratios were multifactorially adjusted for age, sex, BMI, hypertension, smoking, and physical heterozygotes and homozygotes (.500 inactivity. P values from Cox regression or Cox regression trend test.

4 DIABETES CARE care.diabetesjournals.org Schou and Associates associated with decreased risk of type 2 di- in Supplementary Fig. 3. No test for trends case-control study of 244 patients with abetes in the CCHS (P for trend = 0.05). The fulfilled a Bonferroni-corrected P value of type2diabetesversus202nondiabeticcon- hazard ratios for type 2 diabetes (multifac- 0.002 (0.05/27 genetic variants) (Supple- trol subjects, ABCA1 R230C was reported to torially adjusted) did not differ significantly mentary Fig. 3). associate with type 2 diabetes in a Mexican- from 1.0 for any of the 26 remaining genetic Mestizopopulation(9).Inthesamestudy, variants in the CCHS (Fig. 3). Sixteen CONCLUSIONSdThe principal find- these results were validated in a second sam- ABCA1 and five ABCG1 haplotypes with fre- ings of the current study are that genetic ple of similar size and ethnicity. quencies .1% were estimated on the basis variants in ABCA1 and ABCG1 are not The negative findings between ge- of 13 common ABCA1 variants and 10 com- associated with increased risk of type 2 netic variation in ABCA1 and ABCG1 and mon ABCG1 variants, respectively. None of diabetes or related disease entities in the risk of type 2 diabetes in the current study the estimated haplotypes associated with general population. These findings were are in accordance with recent genome- type 2 diabetes in the CCHS (Supplemen- observed in two large studies of the gen- wide association studies (22–24). In tary Table 3) (all P values .0.27 after 1,000 eral population comprising .40,000 in- these large datasets, 22 genes were asso- permutations). Neither the two loss-of- dividuals by applying a series of ciated with b-cell dysfunction, and function mutations nor the ABCG1 g.- statistical analyses including Cox regres- ABCA1 and ABCG1 or other ABC trans- 530A.G associated with type 2 diabetes sion, logistic regression, haplotype asso- porter genes were not among them. Be- in the CGPS (ABCA1 N1800H AC vs. AA ciation testing, and burden testing of rare cause the chromosomal region spanning odds ratio 0.7 [95% CI 0.2–2.8], ABCG1 g.- variants. ABCA1 and ABCG1 (ABCA1, 376C.TCTvs.CC1.1[0.5–2.3], ABCG1 This is the largest study to date of 9: 107543283–107690527; ABCG1, chro- g.-530A.GAGvs.AA1.1[0.9–1.3], and genetic variation in ABCA1 and ABCG1 mosome 21: 43619799–43717354) is GG vs. AA 0.8 [0.3–2.2]) or in CCHS and and risk of type 2 diabetes and, in partic- well tagged on commercial arrays (170 CGPS combined (N1800H AC vs. AA 0.6 ular, is the largest study of loss-of- single nucleotide polymorphisms [SNPs] [0.2–1.8], g.-376C.TCTvs.CC1.0 function mutations, including 94 ABCA1 on the Affymetrix 500 K chip and 585 [0.5–1.8], g.-530A.GAGvs.AA1.0 N1800H heterozygotes and 228 ABCG1 SNPs on the 6.0 chip [www.Affymetrix. [0.8–1.1], and GG vs. AA 1.1 [0.5–2.0]) g.-376C.T heterozygotes. A previous com, assessed November 2011]), it is (Fig. 4). Finally, when burden testing of Dutch study suggested that ABCA1 influ- highly unlikely that common variants in rare variants was performed, neither col- ences b-cell function in humans (8). ABCA1 and ABCG1 would add to risk pre- lapsed genotypes including all rare variants Compared with family control subjects, diction of type 2 diabetes. For rare variants with minor allele frequencies ,1% (CCHS: ABCA1 heterozygotes had mild hypergly- with minor allele frequencies ,5%, the P = 0.34) nor collapsed genotypes includ- cemia but no difference in insulin re- genome-wide association study approach ing rare variants that were experimentally sponse after an oral glucose challenge does not appropriately determine risk verified to be functional (ABCA1 N1800H compared with family control subjects. (25) because capture requires that the and ABCG1 g.-376C.T) associated with With use of hyperglycemic clamps, variant is either on the array or tagged type 2 diabetes (CCHS and CGPS com- ABCA1 heterozygotes had reduced first- bycommonSNPsonthearray,whichis bined: P =0.70). phase insulin response to hyperglycemia unlikely for rare variants (25). By identi- and normal insulin sensitivity. Collec- fying low-frequency loss-of-function var- Genetic variation in ABCA1 and tively, these findings were suggested to iants by detailed resequencing and ABCG1 and risk of prediabetes point to a defect in b-cell function in subsequent large-scale genotyping (12– Risk of prediabetes for all 27 genetic ABCA1 heterozygotes but without de- 14), we suggest that rare functional var- variants in ABCA1 and ABCG1 is presented velopment of type 2 diabetes (8). In a iants in ABCA1 and ABCG1 also are not

Figure 4dRisk of type 2 diabetes as a function of ABCA1 and ABCG1 genotype in CCHS, CGPS, and CCHS and CGPS combined. Hazard ratios and odds ratios were multifactorially adjusted for age, sex, BMI, hypertension, smoking, and physical inactivity. P values from Cox and logistic regression or from Cox and logistic regression trend test. care.diabetesjournals.org DIABETES CARE 5 ABC transporter genes and type 2 diabetes associated with risk of type 2 diabetes in J.S. and A.T.-H. researched data and wrote the in a Mexican population. Diabetes 2008; the general population. manuscript. H.J.M. contributed to biochemical 57:509–513 Even though this study is performed measurements and reviewed and edited the 10. Mauldin JP, Srinivasan S, Mulya A, et al. in large, well-characterized cohorts of the manuscript. B.G.N. contributed to the discus- Reduction in ABCG1 in type 2 diabetic general population, our study has limita- sion and reviewed and edited the manuscript. mice increases macrophage foam cell for- R.F.-S. researched data and wrote the manu- mation. J Biol Chem 2006;281:21216– tions that need to be addressed. As the script. R.F.-S. is the guarantor of this work and, 21224 variants selected for genotyping were as such, had full access to all the data in the study 11. Mauldin JP, Nagelin MH, Wojcik AJ, et al. obtained after resequencing participants and takes responsibility for the integrity of the Reduced expression of ATP-binding cas- with extremes of HDL cholesterol levels, data and the accuracy of the data analysis. sette transporter G1 increases cholesterol they may not be representative of the The authors thank Mette Refstrup (De- accumulation in macrophages of patients general population and may thus be partment of Clinical Biochemistry, Rig- with type 2 diabetes mellitus. Circulation limited when they are tested against an- shospitalet), and Christina Dam (Department 2008;117:2785–2792 other conditiondin this case, type 2 of Clinical Biochemistry, Rigshospitalet) for 12. Frikke-Schmidt R, Nordestgaard BG, diabetes. A comparison between the pres- their persistent attention to the details of the Stene MC, et al. Association of loss-of- ently identified variation with that ob- large-scalegenotyping.Theauthorsarein- function mutations in the ABCA1 gene debted to the staff and participants of the tained in public databases indicates that with high-density lipoprotein cholesterol CCHS and the CGPS for their important levels and risk of ischemic heart disease. variants with minor allele frequencies – . – contributions. JAMA 2008;299:2524 2532 0.2 0.3% (21 of 27 variants in the cur- 13. Schou J, Frikke-Schmidt R, Kardassis D, rent study), are representative of the gen- et al. Genetic variation in ABCG1 and risk eral population, whereas variants below References of myocardial infarction and ischemic this frequency are more likely to be pop- 1. Brunham LR, Kruit JK, Verchere CB, heart disease. Arterioscler Thromb Vasc ulation specific. Further, because we do Hayden MR. Cholesterol in islet dys- Biol 2012;32:506–515 nothaveadirectmeasureofb-cell func- function and type 2 diabetes. J Clin Invest 14. Frikke-Schmidt R, Nordestgaard BG, tion, we cannot exclude that genetic var- 2008;118:403–408 Jensen GB, Tybjaerg-Hansen A. Genetic iation in ABCA1 and ABCG1 impacts 2. Brunham LR, Kruit JK, Pape TD, et al. variation in ABC transporter A1 contrib- fl pancreatic b-cell function as observed Beta-cell ABCA1 in uences insulin secre- utes to HDL cholesterol in the general population. J Clin Invest 2004;114: previously in a family study (8). We can, tion, glucose homeostasis and response to thiazolidinedione treatment. 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Methods for detecting as- shospitalet, Copenhagen University Hospital 8. Vergeer M, Brunham LR, Koetsveld J, et al. sociations with rare variants for common (Copenhagen, Denmark), Chief Physician Johan Carriers of loss-of-function mutations in diseases: application to analysis of se- Boserup and Lise Boserup’s Fund (Copenhagen, ABCA1 display pancreatic beta-cell dys- quence data. Am J Hum Genet 2008;83: Denmark), Ingeborg and Leo Dannin’s Grant function. Diabetes Care 2010;33:869– 311–321 (Copenhagen, Denmark), and Henry Hansen 874 22. Voight BF, Scott LJ, Steinthorsdottir V, and Wife’s Grant (Copenhagen, Denmark). The 9. Villarreal-Molina MT, Flores-Dorantes et al.; MAGIC investigators; GIANT funding sources did not direct the subject matter MT, Arellano-Campos O, et al.; Metabolic Consortium. Twelve type 2 diabetes sus- of research. Study Group. 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