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1.  Improved imputation quality of low-frequency and rare variants in European samples using the ‘Genome of The Netherlands' 
European Journal of Human Genetics  2014;22(11):1321-1326.
Although genome-wide association studies (GWAS) have identified many common variants associated with complex traits, low-frequency and rare variants have not been interrogated in a comprehensive manner. Imputation from dense reference panels, such as the 1000 Genomes Project (1000G), enables testing of ungenotyped variants for association. Here we present the results of imputation using a large, new population-specific panel: the Genome of The Netherlands (GoNL). We benchmarked the performance of the 1000G and GoNL reference sets by comparing imputation genotypes with ‘true' genotypes typed on ImmunoChip in three European populations (Dutch, British, and Italian). GoNL showed significant improvement in the imputation quality for rare variants (MAF 0.05–0.5%) compared with 1000G. In Dutch samples, the mean observed Pearson correlation, r2, increased from 0.61 to 0.71. We also saw improved imputation accuracy for other European populations (in the British samples, r2 improved from 0.58 to 0.65, and in the Italians from 0.43 to 0.47). A combined reference set comprising 1000G and GoNL improved the imputation of rare variants even further. The Italian samples benefitted the most from this combined reference (the mean r2 increased from 0.47 to 0.50). We conclude that the creation of a large population-specific reference is advantageous for imputing rare variants and that a combined reference panel across multiple populations yields the best imputation results.
PMCID: PMC4200431  PMID: 24896149
genotype imputation; GWAS; GoNL; rare variants; reference sets; reference panel
2.  Insight in Genome-Wide Association of Metabolite Quantitative Traits by Exome Sequence Analyses 
PLoS Genetics  2015;11(1):e1004835.
Metabolite quantitative traits carry great promise for epidemiological studies, and their genetic background has been addressed using Genome-Wide Association Studies (GWAS). Thus far, the role of less common variants has not been exhaustively studied. Here, we set out a GWAS for metabolite quantitative traits in serum, followed by exome sequence analysis to zoom in on putative causal variants in the associated genes. 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy experiments yielded successful quantification of 42 unique metabolites in 2,482 individuals from The Erasmus Rucphen Family (ERF) study. Heritability of metabolites were estimated by SOLAR. GWAS was performed by linear mixed models, using HapMap imputations. Based on physical vicinity and pathway analyses, candidate genes were screened for coding region variation using exome sequence data. Heritability estimates for metabolites ranged between 10% and 52%. GWAS replicated three known loci in the metabolome wide significance: CPS1 with glycine (P-value  = 1.27×10−32), PRODH with proline (P-value  = 1.11×10−19), SLC16A9 with carnitine level (P-value  = 4.81×10−14) and uncovered a novel association between DMGDH and dimethyl-glycine (P-value  = 1.65×10−19) level. In addition, we found three novel, suggestively significant loci: TNP1 with pyruvate (P-value  = 1.26×10−8), KCNJ16 with 3-hydroxybutyrate (P-value  = 1.65×10−8) and 2p12 locus with valine (P-value  = 3.49×10−8). Exome sequence analysis identified potentially causal coding and regulatory variants located in the genes CPS1, KCNJ2 and PRODH, and revealed allelic heterogeneity for CPS1 and PRODH. Combined GWAS and exome analyses of metabolites detected by high-resolution 1H-NMR is a robust approach to uncover metabolite quantitative trait loci (mQTL), and the likely causative variants in these loci. It is anticipated that insight in the genetics of intermediate phenotypes will provide additional insight into the genetics of complex traits.
Author Summary
Human metabolic individuality is under strict control of genetic and environmental factors. In our study, we aimed to find the genetic determinants of circulating molecules in sera of large set of individuals representing the general population. First, we performed a hypothesis-free genome wide screen in this population to identify genetic regions of interest. Our study confirmed four known gene metabolite connections, but also pointed to four novel ones. Genome-wide screens enriched for common intergenic variants may miss causal genetic variations directly changing the protein sequence. To investigate this further, we zoomed into regions of interest and tested whether the association signals obtained in the first stage were direct, or whether they represent causal variations, which were not captured in the initial panel. These subsequent tests showed that protein coding and regulatory variations are involved in metabolite levels. For two genomic regions we also found that genes harbour more than one causal variant influencing metabolite levels independent of each other. We also observed strong connection between markers of cardio-metabolic health and metabolites. Taken together, our novel loci are of interest for further research to investigate the causal relation to for instance type 2 diabetes and cardiovascular disease.
PMCID: PMC4287344  PMID: 25569235
3.  The Challenges of Genome-Wide Interaction Studies: Lessons to Learn from the Analysis of HDL Blood Levels 
PLoS ONE  2014;9(10):e109290.
Genome-wide association studies (GWAS) have revealed 74 single nucleotide polymorphisms (SNPs) associated with high-density lipoprotein cholesterol (HDL) blood levels. This study is, to our knowledge, the first genome-wide interaction study (GWIS) to identify SNP×SNP interactions associated with HDL levels. We performed a GWIS in the Rotterdam Study (RS) cohort I (RS-I) using the GLIDE tool which leverages the massively parallel computing power of Graphics Processing Units (GPUs) to perform linear regression on all genome-wide pairs of SNPs. By performing a meta-analysis together with Rotterdam Study cohorts II and III (RS-II and RS-III), we were able to filter 181 interaction terms with a p-value<1 · 10−8 that replicated in the two independent cohorts. We were not able to replicate any of these interaction term in the AGES, ARIC, CHS, ERF, FHS and NFBC-66 cohorts (Ntotal = 30,011) when adjusting for multiple testing. Our GWIS resulted in the consistent finding of a possible interaction between rs774801 in ARMC8 (ENSG00000114098) and rs12442098 in SPATA8 (ENSG00000185594) being associated with HDL levels. However, p-values do not reach the preset Bonferroni correction of the p-values. Our study suggest that even for highly genetically determined traits such as HDL the sample sizes needed to detect SNP×SNP interactions are large and the 2-step filtering approaches do not yield a solution. Here we present our analysis plan and our reservations concerning GWIS.
PMCID: PMC4203717  PMID: 25329471
4.  Genome-Wide Meta-Analysis of Myopia and Hyperopia Provides Evidence for Replication of 11 Loci 
PLoS ONE  2014;9(9):e107110.
Refractive error (RE) is a complex, multifactorial disorder characterized by a mismatch between the optical power of the eye and its axial length that causes object images to be focused off the retina. The two major subtypes of RE are myopia (nearsightedness) and hyperopia (farsightedness), which represent opposite ends of the distribution of the quantitative measure of spherical refraction. We performed a fixed effects meta-analysis of genome-wide association results of myopia and hyperopia from 9 studies of European-derived populations: AREDS, KORA, FES, OGP-Talana, MESA, RSI, RSII, RSIII and ERF. One genome-wide significant region was observed for myopia, corresponding to a previously identified myopia locus on 8q12 (p = 1.25×10−8), which has been reported by Kiefer et al. as significantly associated with myopia age at onset and Verhoeven et al. as significantly associated to mean spherical-equivalent (MSE) refractive error. We observed two genome-wide significant associations with hyperopia. These regions overlapped with loci on 15q14 (minimum p value = 9.11×10−11) and 8q12 (minimum p value 1.82×10−11) previously reported for MSE and myopia age at onset. We also used an intermarker linkage- disequilibrium-based method for calculating the effective number of tests in targeted regional replication analyses. We analyzed myopia (which represents the closest phenotype in our data to the one used by Kiefer et al.) and showed replication of 10 additional loci associated with myopia previously reported by Kiefer et al. This is the first replication of these loci using myopia as the trait under analysis. “Replication-level” association was also seen between hyperopia and 12 of Kiefer et al.'s published loci. For the loci that show evidence of association to both myopia and hyperopia, the estimated effect of the risk alleles were in opposite directions for the two traits. This suggests that these loci are important contributors to variation of refractive error across the distribution.
PMCID: PMC4169415  PMID: 25233373
5.  Meta-analysis of genome-wide association studies in five cohorts reveals common variants in RBFOX1, a regulator of tissue-specific splicing, associated with refractive error 
Human Molecular Genetics  2013;22(13):2754-2764.
Visual refractive errors (REs) are complex genetic traits with a largely unknown etiology. To date, genome-wide association studies (GWASs) of moderate size have identified several novel risk markers for RE, measured here as mean spherical equivalent (MSE). We performed a GWAS using a total of 7280 samples from five cohorts: the Age-Related Eye Disease Study (AREDS); the KORA study (‘Cooperative Health Research in the Region of Augsburg’); the Framingham Eye Study (FES); the Ogliastra Genetic Park-Talana (OGP-Talana) Study and the Multiethnic Study of Atherosclerosis (MESA). Genotyping was performed on Illumina and Affymetrix platforms with additional markers imputed to the HapMap II reference panel. We identified a new genome-wide significant locus on chromosome 16 (rs10500355, P = 3.9 × 10−9) in a combined discovery and replication set (26 953 samples). This single nucleotide polymorphism (SNP) is located within the RBFOX1 gene which is a neuron-specific splicing factor regulating a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins.
PMCID: PMC3674806  PMID: 23474815
6.  Identification of seven loci affecting mean telomere length and their association with disease 
Codd, Veryan | Nelson, Christopher P. | Albrecht, Eva | Mangino, Massimo | Deelen, Joris | Buxton, Jessica L. | Jan Hottenga, Jouke | Fischer, Krista | Esko, Tõnu | Surakka, Ida | Broer, Linda | Nyholt, Dale R. | Mateo Leach, Irene | Salo, Perttu | Hägg, Sara | Matthews, Mary K. | Palmen, Jutta | Norata, Giuseppe D. | O’Reilly, Paul F. | Saleheen, Danish | Amin, Najaf | Balmforth, Anthony J. | Beekman, Marian | de Boer, Rudolf A. | Böhringer, Stefan | Braund, Peter S. | Burton, Paul R. | de Craen, Anton J. M. | Denniff, Matthew | Dong, Yanbin | Douroudis, Konstantinos | Dubinina, Elena | Eriksson, Johan G. | Garlaschelli, Katia | Guo, Dehuang | Hartikainen, Anna-Liisa | Henders, Anjali K. | Houwing-Duistermaat, Jeanine J. | Kananen, Laura | Karssen, Lennart C. | Kettunen, Johannes | Klopp, Norman | Lagou, Vasiliki | van Leeuwen, Elisabeth M. | Madden, Pamela A. | Mägi, Reedik | Magnusson, Patrik K.E. | Männistö, Satu | McCarthy, Mark I. | Medland, Sarah E. | Mihailov, Evelin | Montgomery, Grant W. | Oostra, Ben A. | Palotie, Aarno | Peters, Annette | Pollard, Helen | Pouta, Anneli | Prokopenko, Inga | Ripatti, Samuli | Salomaa, Veikko | Suchiman, H. Eka D. | Valdes, Ana M. | Verweij, Niek | Viñuela, Ana | Wang, Xiaoling | Wichmann, H.-Erich | Widen, Elisabeth | Willemsen, Gonneke | Wright, Margaret J. | Xia, Kai | Xiao, Xiangjun | van Veldhuisen, Dirk J. | Catapano, Alberico L. | Tobin, Martin D. | Hall, Alistair S. | Blakemore, Alexandra I.F. | van Gilst, Wiek H. | Zhu, Haidong | Erdmann, Jeanette | Reilly, Muredach P. | Kathiresan, Sekar | Schunkert, Heribert | Talmud, Philippa J. | Pedersen, Nancy L. | Perola, Markus | Ouwehand, Willem | Kaprio, Jaakko | Martin, Nicholas G. | van Duijn, Cornelia M. | Hovatta, Iiris | Gieger, Christian | Metspalu, Andres | Boomsma, Dorret I. | Jarvelin, Marjo-Riitta | Slagboom, P. Eline | Thompson, John R. | Spector, Tim D. | van der Harst, Pim | Samani, Nilesh J.
Nature genetics  2013;45(4):422-427e2.
Inter-individual variation in mean leukocyte telomere length (LTL) is associated with cancer and several age-associated diseases. Here, in a genome-wide meta-analysis of 37,684 individuals with replication of selected variants in a further 10,739 individuals, we identified seven loci, including five novel loci, associated with mean LTL (P<5x10−8). Five of the loci contain genes (TERC, TERT, NAF1, OBFC1, RTEL1) that are known to be involved in telomere biology. Lead SNPs at two loci (TERC and TERT) associate with several cancers and other diseases, including idiopathic pulmonary fibrosis. Moreover, a genetic risk score analysis combining lead variants at all seven loci in 22,233 coronary artery disease cases and 64,762 controls showed an association of the alleles associated with shorter LTL with increased risk of CAD (21% (95% CI: 5–35%) per standard deviation in LTL, p=0.014). Our findings support a causal role of telomere length variation in some age-related diseases.
PMCID: PMC4006270  PMID: 23535734
7.  Association Analysis of Bitter Receptor Genes in Five Isolated Populations Identifies a Significant Correlation between TAS2R43 Variants and Coffee Liking 
PLoS ONE  2014;9(3):e92065.
Coffee, one of the most popular beverages in the world, contains many different physiologically active compounds with a potential impact on people’s health. Despite the recent attention given to the genetic basis of its consumption, very little has been done in understanding genes influencing coffee preference among different individuals. Given its markedly bitter taste, we decided to verify if bitter receptor genes (TAS2Rs) variants affect coffee liking. In this light, 4066 people from different parts of Europe and Central Asia filled in a field questionnaire on coffee liking. They have been consequently recruited and included in the study. Eighty-eight SNPs covering the 25 TAS2R genes were selected from the available imputed ones and used to run association analysis for coffee liking. A significant association was detected with three SNP: one synonymous and two functional variants (W35S and H212R) on the TAS2R43 gene. Both variants have been shown to greatly reduce in vitro protein activity. Surprisingly the wild type allele, which corresponds to the functional form of the protein, is associated to higher liking of coffee. Since the hTAS2R43 receptor is sensible to caffeine, we verified if the detected variants produced differences in caffeine bitter perception on a subsample of people coming from the FVG cohort. We found a significant association between differences in caffeine perception and the H212R variant but not with the W35S, which suggests that the effect of the TAS2R43 gene on coffee liking is mediated by caffeine and in particular by the H212R variant.
No other significant association was found with other TAS2R genes. In conclusion, the present study opens new perspectives in the understanding of coffee liking. Further studies are needed to clarify the role of the TAS2R43 gene in coffee hedonics and to identify which other genes and pathways are involved in its genetics.
PMCID: PMC3960174  PMID: 24647340
8.  The Genome of the Netherlands: design, and project goals 
Within the Netherlands a national network of biobanks has been established (Biobanking and Biomolecular Research Infrastructure-Netherlands (BBMRI-NL)) as a national node of the European BBMRI. One of the aims of BBMRI-NL is to enrich biobanks with different types of molecular and phenotype data. Here, we describe the Genome of the Netherlands (GoNL), one of the projects within BBMRI-NL. GoNL is a whole-genome-sequencing project in a representative sample consisting of 250 trio-families from all provinces in the Netherlands, which aims to characterize DNA sequence variation in the Dutch population. The parent–offspring trios include adult individuals ranging in age from 19 to 87 years (mean=53 years; SD=16 years) from birth cohorts 1910–1994. Sequencing was done on blood-derived DNA from uncultured cells and accomplished coverage was 14–15x. The family-based design represents a unique resource to assess the frequency of regional variants, accurately reconstruct haplotypes by family-based phasing, characterize short indels and complex structural variants, and establish the rate of de novo mutational events. GoNL will also serve as a reference panel for imputation in the available genome-wide association studies in Dutch and other cohorts to refine association signals and uncover population-specific variants. GoNL will create a catalog of human genetic variation in this sample that is uniquely characterized with respect to micro-geographic location and a wide range of phenotypes. The resource will be made available to the research and medical community to guide the interpretation of sequencing projects. The present paper summarizes the global characteristics of the project.
PMCID: PMC3895638  PMID: 23714750
whole-genome sequence; trio-design; population genetics
9.  Analytical and simulation methods for estimating the potential predictive ability of genetic profiling: a comparison of methods and results 
European Journal of Human Genetics  2012;20(12):1270-1274.
Various modeling methods have been proposed to estimate the potential predictive ability of polygenic risk variants that predispose to various common diseases. However, it is unknown whether differences between them affect their conclusions on predictive ability. We reviewed input parameters, assumptions and output of the five most common methods and compared their estimates of the area under the receiver operating characteristic (ROC) curve (AUC) using hypothetical data representing effect sizes and frequencies of genetic variants, population disease risk and number of variants. To assess the accuracy of the estimated AUCs, we aimed to reproduce the AUCs of published empirical studies. All methods assumed that the combined effect of genetic variants on disease risk followed a multiplicative risk model of independent genetic effects, but they either assumed per allele, per genotype or dominant/recessive effects for the genetic variants. Modeling strategy and input parameters differed. Methods used simulation analysis or analytical formulas with effect sizes quantified by odds ratios (ORs) or relative risks. Estimated AUC values were similar for lower ORs (<1.2). When AUCs were larger (>0.7) due to variants with strong effects, differences in estimated AUCs between methods increased. The simulation methods accurately reproduced the AUC values of empirical studies, but the analytical methods did not. We conclude that despite differences in input parameters, the modeling methods estimate similar AUC for realistic values of the ORs. When one or more variants have stronger effects and AUC values are higher, the simulation methods tend to be more accurate.
PMCID: PMC3499740  PMID: 22643180
risk prediction; modeling; discriminative accuracy; AUC; complex disease
10.  Genome-wide meta-analyses of multi-ethnic cohorts identify multiple new susceptibility loci for refractive error and myopia 
Verhoeven, Virginie J.M. | Hysi, Pirro G. | Wojciechowski, Robert | Fan, Qiao | Guggenheim, Jeremy A. | Höhn, René | MacGregor, Stuart | Hewitt, Alex W. | Nag, Abhishek | Cheng, Ching-Yu | Yonova-Doing, Ekaterina | Zhou, Xin | Ikram, M. Kamran | Buitendijk, Gabriëlle H.S. | McMahon, George | Kemp, John P. | St. Pourcain, Beate | Simpson, Claire L. | Mäkelä, Kari-Matti | Lehtimäki, Terho | Kähönen, Mika | Paterson, Andrew D. | Hosseini, S. Mohsen | Wong, Hoi Suen | Xu, Liang | Jonas, Jost B. | Pärssinen, Olavi | Wedenoja, Juho | Yip, Shea Ping | Ho, Daniel W. H. | Pang, Chi Pui | Chen, Li Jia | Burdon, Kathryn P. | Craig, Jamie E. | Klein, Barbara E. K. | Klein, Ronald | Haller, Toomas | Metspalu, Andres | Khor, Chiea-Chuen | Tai, E-Shyong | Aung, Tin | Vithana, Eranga | Tay, Wan-Ting | Barathi, Veluchamy A. | Chen, Peng | Li, Ruoying | Liao, Jiemin | Zheng, Yingfeng | Ong, Rick T. | Döring, Angela | Evans, David M. | Timpson, Nicholas J. | Verkerk, Annemieke J.M.H. | Meitinger, Thomas | Raitakari, Olli | Hawthorne, Felicia | Spector, Tim D. | Karssen, Lennart C. | Pirastu, Mario | Murgia, Federico | Ang, Wei | Mishra, Aniket | Montgomery, Grant W. | Pennell, Craig E. | Cumberland, Phillippa M. | Cotlarciuc, Ioana | Mitchell, Paul | Wang, Jie Jin | Schache, Maria | Janmahasathian, Sarayut | Igo, Robert P. | Lass, Jonathan H. | Chew, Emily | Iyengar, Sudha K. | Gorgels, Theo G.M.F. | Rudan, Igor | Hayward, Caroline | Wright, Alan F. | Polasek, Ozren | Vatavuk, Zoran | Wilson, James F. | Fleck, Brian | Zeller, Tanja | Mirshahi, Alireza | Müller, Christian | Uitterlinden, Andre’ G. | Rivadeneira, Fernando | Vingerling, Johannes R. | Hofman, Albert | Oostra, Ben A. | Amin, Najaf | Bergen, Arthur A.B. | Teo, Yik-Ying | Rahi, Jugnoo S. | Vitart, Veronique | Williams, Cathy | Baird, Paul N. | Wong, Tien-Yin | Oexle, Konrad | Pfeiffer, Norbert | Mackey, David A. | Young, Terri L. | van Duijn, Cornelia M. | Saw, Seang-Mei | Wilson, Joan E. Bailey | Stambolian, Dwight | Klaver, Caroline C. | Hammond, Christopher J.
Nature genetics  2013;45(3):314-318.
Refractive error is the most common eye disorder worldwide, and a prominent cause of blindness. Myopia affects over 30% of Western populations, and up to 80% of Asians. The CREAM consortium conducted genome-wide meta-analyses including 37,382 individuals from 27 studies of European ancestry, and 8,376 from 5 Asian cohorts. We identified 16 new loci for refractive error in subjects of European ancestry, of which 8 were shared with Asians. Combined analysis revealed 8 additional loci. The new loci include genes with functions in neurotransmission (GRIA4), ion channels (KCNQ5), retinoic acid metabolism (RDH5), extracellular matrix remodeling (LAMA2, BMP2), and eye development (SIX6, PRSS56). We also confirmed previously reported associations with GJD2 and RASGRF1. Risk score analysis using associated SNPs showed a tenfold increased risk of myopia for subjects with the highest genetic load. Our results, accumulated across independent multi-ethnic studies, considerably advance understanding of mechanisms involved in refractive error and myopia.
PMCID: PMC3740568  PMID: 23396134
11.  The Role of Adiposity in Cardiometabolic Traits: A Mendelian Randomization Analysis 
Fall, Tove | Hägg, Sara | Mägi, Reedik | Ploner, Alexander | Fischer, Krista | Horikoshi, Momoko | Sarin, Antti-Pekka | Thorleifsson, Gudmar | Ladenvall, Claes | Kals, Mart | Kuningas, Maris | Draisma, Harmen H. M. | Ried, Janina S. | van Zuydam, Natalie R. | Huikari, Ville | Mangino, Massimo | Sonestedt, Emily | Benyamin, Beben | Nelson, Christopher P. | Rivera, Natalia V. | Kristiansson, Kati | Shen, Huei-yi | Havulinna, Aki S. | Dehghan, Abbas | Donnelly, Louise A. | Kaakinen, Marika | Nuotio, Marja-Liisa | Robertson, Neil | de Bruijn, Renée F. A. G. | Ikram, M. Arfan | Amin, Najaf | Balmforth, Anthony J. | Braund, Peter S. | Doney, Alexander S. F. | Döring, Angela | Elliott, Paul | Esko, Tõnu | Franco, Oscar H. | Gretarsdottir, Solveig | Hartikainen, Anna-Liisa | Heikkilä, Kauko | Herzig, Karl-Heinz | Holm, Hilma | Hottenga, Jouke Jan | Hyppönen, Elina | Illig, Thomas | Isaacs, Aaron | Isomaa, Bo | Karssen, Lennart C. | Kettunen, Johannes | Koenig, Wolfgang | Kuulasmaa, Kari | Laatikainen, Tiina | Laitinen, Jaana | Lindgren, Cecilia | Lyssenko, Valeriya | Läärä, Esa | Rayner, Nigel W. | Männistö, Satu | Pouta, Anneli | Rathmann, Wolfgang | Rivadeneira, Fernando | Ruokonen, Aimo | Savolainen, Markku J. | Sijbrands, Eric J. G. | Small, Kerrin S. | Smit, Jan H. | Steinthorsdottir, Valgerdur | Syvänen, Ann-Christine | Taanila, Anja | Tobin, Martin D. | Uitterlinden, Andre G. | Willems, Sara M. | Willemsen, Gonneke | Witteman, Jacqueline | Perola, Markus | Evans, Alun | Ferrières, Jean | Virtamo, Jarmo | Kee, Frank | Tregouet, David-Alexandre | Arveiler, Dominique | Amouyel, Philippe | Ferrario, Marco M. | Brambilla, Paolo | Hall, Alistair S. | Heath, Andrew C. | Madden, Pamela A. F. | Martin, Nicholas G. | Montgomery, Grant W. | Whitfield, John B. | Jula, Antti | Knekt, Paul | Oostra, Ben | van Duijn, Cornelia M. | Penninx, Brenda W. J. H. | Davey Smith, George | Kaprio, Jaakko | Samani, Nilesh J. | Gieger, Christian | Peters, Annette | Wichmann, H.-Erich | Boomsma, Dorret I. | de Geus, Eco J. C. | Tuomi, TiinaMaija | Power, Chris | Hammond, Christopher J. | Spector, Tim D. | Lind, Lars | Orho-Melander, Marju | Palmer, Colin Neil Alexander | Morris, Andrew D. | Groop, Leif | Järvelin, Marjo-Riitta | Salomaa, Veikko | Vartiainen, Erkki | Hofman, Albert | Ripatti, Samuli | Metspalu, Andres | Thorsteinsdottir, Unnur | Stefansson, Kari | Pedersen, Nancy L. | McCarthy, Mark I. | Ingelsson, Erik | Prokopenko, Inga
PLoS Medicine  2013;10(6):e1001474.
In this study, Prokopenko and colleagues provide novel evidence for causal relationship between adiposity and heart failure and increased liver enzymes using a Mendelian randomization study design.
Please see later in the article for the Editors' Summary
The association between adiposity and cardiometabolic traits is well known from epidemiological studies. Whilst the causal relationship is clear for some of these traits, for others it is not. We aimed to determine whether adiposity is causally related to various cardiometabolic traits using the Mendelian randomization approach.
Methods and Findings
We used the adiposity-associated variant rs9939609 at the FTO locus as an instrumental variable (IV) for body mass index (BMI) in a Mendelian randomization design. Thirty-six population-based studies of individuals of European descent contributed to the analyses.
Age- and sex-adjusted regression models were fitted to test for association between (i) rs9939609 and BMI (n = 198,502), (ii) rs9939609 and 24 traits, and (iii) BMI and 24 traits. The causal effect of BMI on the outcome measures was quantified by IV estimators. The estimators were compared to the BMI–trait associations derived from the same individuals. In the IV analysis, we demonstrated novel evidence for a causal relationship between adiposity and incident heart failure (hazard ratio, 1.19 per BMI-unit increase; 95% CI, 1.03–1.39) and replicated earlier reports of a causal association with type 2 diabetes, metabolic syndrome, dyslipidemia, and hypertension (odds ratio for IV estimator, 1.1–1.4; all p<0.05). For quantitative traits, our results provide novel evidence for a causal effect of adiposity on the liver enzymes alanine aminotransferase and gamma-glutamyl transferase and confirm previous reports of a causal effect of adiposity on systolic and diastolic blood pressure, fasting insulin, 2-h post-load glucose from the oral glucose tolerance test, C-reactive protein, triglycerides, and high-density lipoprotein cholesterol levels (all p<0.05). The estimated causal effects were in agreement with traditional observational measures in all instances except for type 2 diabetes, where the causal estimate was larger than the observational estimate (p = 0.001).
We provide novel evidence for a causal relationship between adiposity and heart failure as well as between adiposity and increased liver enzymes.
Please see later in the article for the Editors' Summary
Editors' Summary
Cardiovascular disease (CVD)—disease that affects the heart and/or the blood vessels—is a major cause of illness and death worldwide. In the US, for example, coronary heart disease—a CVD in which narrowing of the heart's blood vessels by fatty deposits slows the blood supply to the heart and may eventually cause a heart attack—is the leading cause of death, and stroke—a CVD in which the brain's blood supply is interrupted—is the fourth leading cause of death. Globally, both the incidence of CVD (the number of new cases in a population every year) and its prevalence (the proportion of the population with CVD) are increasing, particularly in low- and middle-income countries. This increasing burden of CVD is occurring in parallel with a global increase in the incidence and prevalence of obesity—having an unhealthy amount of body fat (adiposity)—and of metabolic diseases—conditions such as diabetes in which metabolism (the processes that the body uses to make energy from food) is disrupted, with resulting high blood sugar and damage to the blood vessels.
Why Was This Study Done?
Epidemiological studies—investigations that record the patterns and causes of disease in populations—have reported an association between adiposity (indicated by an increased body mass index [BMI], which is calculated by dividing body weight in kilograms by height in meters squared) and cardiometabolic traits such as coronary heart disease, stroke, heart failure (a condition in which the heart is incapable of pumping sufficient amounts of blood around the body), diabetes, high blood pressure (hypertension), and high blood cholesterol (dyslipidemia). However, observational studies cannot prove that adiposity causes any particular cardiometabolic trait because overweight individuals may share other characteristics (confounding factors) that are the real causes of both obesity and the cardiometabolic disease. Moreover, it is possible that having CVD or a metabolic disease causes obesity (reverse causation). For example, individuals with heart failure cannot do much exercise, so heart failure may cause obesity rather than vice versa. Here, the researchers use “Mendelian randomization” to examine whether adiposity is causally related to various cardiometabolic traits. Because gene variants are inherited randomly, they are not prone to confounding and are free from reverse causation. It is known that a genetic variant (rs9939609) within the genome region that encodes the fat-mass- and obesity-associated gene (FTO) is associated with increased BMI. Thus, an investigation of the associations between rs9939609 and cardiometabolic traits can indicate whether obesity is causally related to these traits.
What Did the Researchers Do and Find?
The researchers analyzed the association between rs9939609 (the “instrumental variable,” or IV) and BMI, between rs9939609 and 24 cardiometabolic traits, and between BMI and the same traits using genetic and health data collected in 36 population-based studies of nearly 200,000 individuals of European descent. They then quantified the strength of the causal association between BMI and the cardiometabolic traits by calculating “IV estimators.” Higher BMI showed a causal relationship with heart failure, metabolic syndrome (a combination of medical disorders that increases the risk of developing CVD), type 2 diabetes, dyslipidemia, hypertension, increased blood levels of liver enzymes (an indicator of liver damage; some metabolic disorders involve liver damage), and several other cardiometabolic traits. All the IV estimators were similar to the BMI–cardiovascular trait associations (observational estimates) derived from the same individuals, with the exception of diabetes, where the causal estimate was higher than the observational estimate, probably because the observational estimate is based on a single BMI measurement, whereas the causal estimate considers lifetime changes in BMI.
What Do These Findings Mean?
Like all Mendelian randomization studies, the reliability of the causal associations reported here depends on several assumptions made by the researchers. Nevertheless, these findings provide support for many previously suspected and biologically plausible causal relationships, such as that between adiposity and hypertension. They also provide new insights into the causal effect of obesity on liver enzyme levels and on heart failure. In the latter case, these findings suggest that a one-unit increase in BMI might increase the incidence of heart failure by 17%. In the US, this corresponds to 113,000 additional cases of heart failure for every unit increase in BMI at the population level. Although additional studies are needed to confirm and extend these findings, these results suggest that global efforts to reduce the burden of obesity will likely also reduce the occurrence of CVD and metabolic disorders.
Additional Information
Please access these websites via the online version of this summary at
The American Heart Association provides information on all aspects of cardiovascular disease and tips on keeping the heart healthy, including weight management (in several languages); its website includes personal stories about stroke and heart attacks
The US Centers for Disease Control and Prevention has information on heart disease, stroke, and all aspects of overweight and obesity (in English and Spanish)
The UK National Health Service Choices website provides information about cardiovascular disease and obesity, including a personal story about losing weight
The World Health Organization provides information on obesity (in several languages)
The International Obesity Taskforce provides information about the global obesity epidemic
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
MedlinePlus provides links to other sources of information on heart disease, on vascular disease, on obesity, and on metabolic disorders (in English and Spanish)
The International Association for the Study of Obesity provides maps and information about obesity worldwide
The International Diabetes Federation has a web page that describes types, complications, and risk factors of diabetes
PMCID: PMC3692470  PMID: 23824655
12.  Assessment of the 9p21.3 locus in severity of coronary artery disease in the presence and absence of type 2 diabetes 
BMC Medical Genetics  2013;14:11.
The 9p21.3 locus is strongly associated with the risk of coronary artery disease (CAD) and with type 2 diabetes (T2D). We investigated the association of 9p21.3 variants with severity of CAD (defined by the number of vessel diseased [VD]) in the presence and absence of T2D.
We tested 11 9p21.3-variants for association in a white Italian study (N = 2,908), and carried out replication in 2 independent white populations, a German study (N = 2,028) and a Canadian Study (N=950). SNP association and permutation analyses were conducted.
We identified two 9p21.3-variants, rs4977574 (P < 4×10-4) and rs2383207 (P < 1.5×10-3) that were associated with severity of CAD in subjects without T2D. Association of rs4977574 with severity of CAD was confirmed in the Canadian Study. Results from subgroup analysis among patients with T2D showed an interaction between rs10738610 and T2D with P = 4.82×10-2. Further investigation showed that rs10738610 (P < 1.99×10-2) was found to be significantly associated with severity of CAD in subjects with T2D.
The 9p21.3 locus is significantly associated with severity of CAD. The number of associations of 9p21.3 variants with severity of CAD is variable to the presence and absence of T2D. In a CAD-susceptible region of 115 kb, there is only one variant associated with the severity of coronary vessel disease in the presence of type 2 diabetes.
PMCID: PMC3556499  PMID: 23343465
Severity of CAD; Coronary artery disease; Diabetes mellitus; T2D; 9p21.3; Genetics; Single nucleotide polymorphism
13.  Large scale international replication and meta-analysis study confirms association of the 15q14 locus with myopia. The CREAM consortium 
Verhoeven, Virginie J. M. | Hysi, Pirro G. | Saw, Seang-Mei | Vitart, Veronique | Mirshahi, Alireza | Guggenheim, Jeremy A. | Cotch, Mary Frances | Yamashiro, Kenji | Baird, Paul N. | Mackey, David A. | Wojciechowski, Robert |  Ikram, M. Kamran | Hewitt, Alex W. | Duggal, Priya | Janmahasatian, Sarayut | Khor, Chiea-Chuen | Fan, Qiao | Zhou, Xin | Young, Terri L. | Tai, E-Shyong | Goh, Liang-Kee | Li, Yi-Ju | Aung, Tin | Vithana, Eranga | Teo, Yik-Ying | Tay, Wanting | Sim, Xueling | Rudan, Igor | Hayward, Caroline | Wright, Alan F. | Polasek, Ozren | Campbell, Harry | Wilson, James F. | Fleck, Brian W. | Nakata, Isao | Yoshimura, Nagahisa | Yamada, Ryo | Matsuda, Fumihiko | Ohno-Matsui, Kyoko | Nag, Abhishek | McMahon, George | Pourcain, Beate St. | Lu, Yi | Rahi, Jugnoo S. | Cumberland, Phillippa M. | Bhattacharya, Shomi | Simpson, Claire L. | Atwood, Larry D. | Li, Xiaohui | Raffel, Leslie J. | Murgia, Federico | Portas, Laura | Despriet, Dominiek D. G. | van Koolwijk, Leonieke M. E. | Wolfram, Christian | Lackner, Karl J. | Tönjes, Anke | Mägi, Reedik | Lehtimäki, Terho | Kähönen, Mika | Esko, Tõnu | Metspalu, Andres | Rantanen, Taina | Pärssinen, Olavi | Klein, Barbara E. | Meitinger, Thomas | Spector, Timothy D. | Oostra, Ben A. | Smith, Albert V. | de Jong, Paulus T. V. M. | Hofman, Albert | Amin, Najaf | Karssen, Lennart C. | Rivadeneira, Fernando | Vingerling, Johannes R. | Eiríksdóttir, Guðný | Gudnason, Vilmundur | Döring, Angela | Bettecken, Thomas | Uitterlinden, André G. | Williams, Cathy | Zeller, Tanja | Castagné, Raphaële | Oexle, Konrad | van Duijn, Cornelia M. | Iyengar, Sudha K. | Mitchell, Paul | Wang, Jie Jin | Höhn, René | Pfeiffer, Norbert | Bailey-Wilson, Joan E. | Stambolian, Dwight | Wong, Tien-Yin | Hammond, Christopher J. | Klaver, Caroline C. W.
Human Genetics  2012;131(9):1467-1480.
Myopia is a complex genetic disorder and a common cause of visual impairment among working age adults. Genome-wide association studies have identified susceptibility loci on chromosomes 15q14 and 15q25 in Caucasian populations of European ancestry. Here, we present a confirmation and meta-analysis study in which we assessed whether these two loci are also associated with myopia in other populations. The study population comprised 31 cohorts from the Consortium of Refractive Error and Myopia (CREAM) representing 4 different continents with 55,177 individuals; 42,845 Caucasians and 12,332 Asians. We performed a meta-analysis of 14 single nucleotide polymorphisms (SNPs) on 15q14 and 5 SNPs on 15q25 using linear regression analysis with spherical equivalent as a quantitative outcome, adjusted for age and sex. We calculated the odds ratio (OR) of myopia versus hyperopia for carriers of the top-SNP alleles using a fixed effects meta-analysis. At locus 15q14, all SNPs were significantly replicated, with the lowest P value 3.87 × 10−12 for SNP rs634990 in Caucasians, and 9.65 × 10−4 for rs8032019 in Asians. The overall meta-analysis provided P value 9.20 × 10−23 for the top SNP rs634990. The risk of myopia versus hyperopia was OR 1.88 (95 % CI 1.64, 2.16, P < 0.001) for homozygous carriers of the risk allele at the top SNP rs634990, and OR 1.33 (95 % CI 1.19, 1.49, P < 0.001) for heterozygous carriers. SNPs at locus 15q25 did not replicate significantly (P value 5.81 × 10−2 for top SNP rs939661). We conclude that common variants at chromosome 15q14 influence susceptibility for myopia in Caucasian and Asian populations world-wide.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-012-1176-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3418496  PMID: 22665138
14.  Genome-Wide Association Study Identifies Novel Loci Associated with Circulating Phospho- and Sphingolipid Concentrations 
PLoS Genetics  2012;8(2):e1002490.
Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10−204) and 10 loci for sphingolipids (smallest P-value = 3.10×10−57). After a correction for multiple comparisons (P-value<2.2×10−9), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.
Author Summary
Phospho- and sphingolipids are integral to membrane formation and are involved in crucial cellular functions such as signalling, membrane fluidity, membrane protein trafficking, neurotransmission, and receptor trafficking. In addition to severe monogenic diseases resulting from defective phospho- and sphingolipid function and metabolism, the evidence suggests that variations in these lipid levels at the population level are involved in the determination of cardiovascular and neurologic traits and subsequent disease. We took advantage of modern laboratory methods, including microarray-based genotyping and electrospray ionization tandem mass spectrometry, to hunt for genetic variation influencing the levels of more than 350 phospho- and sphingolipid phenotypes. We identified nine novel loci, in addition to confirming a number of previously described loci. Several other genetic regions provided substantial evidence of their involvement in these traits. All of these loci are strong candidates for further research in the field of lipid biology and are likely to yield considerable insights into the complex metabolic pathways underlying circulating phospho- and sphingolipid levels. Understanding these mechanisms might help to illuminate factors leading to the development of common cardiovascular and neurological diseases and might provide molecular targets for the development of new therapies.
PMCID: PMC3280968  PMID: 22359512
15.  A Genome-Wide Screen for Interactions Reveals a New Locus on 4p15 Modifying the Effect of Waist-to-Hip Ratio on Total Cholesterol 
Surakka, Ida | Isaacs, Aaron | Karssen, Lennart C. | Laurila, Pirkka-Pekka P. | Middelberg, Rita P. S. | Tikkanen, Emmi | Ried, Janina S. | Lamina, Claudia | Mangino, Massimo | Igl, Wilmar | Hottenga, Jouke-Jan | Lagou, Vasiliki | van der Harst, Pim | Mateo Leach, Irene | Esko, Tõnu | Kutalik, Zoltán | Wainwright, Nicholas W. | Struchalin, Maksim V. | Sarin, Antti-Pekka | Kangas, Antti J. | Viikari, Jorma S. | Perola, Markus | Rantanen, Taina | Petersen, Ann-Kristin | Soininen, Pasi | Johansson, Åsa | Soranzo, Nicole | Heath, Andrew C. | Papamarkou, Theodore | Prokopenko, Inga | Tönjes, Anke | Kronenberg, Florian | Döring, Angela | Rivadeneira, Fernando | Montgomery, Grant W. | Whitfield, John B. | Kähönen, Mika | Lehtimäki, Terho | Freimer, Nelson B. | Willemsen, Gonneke | de Geus, Eco J. C. | Palotie, Aarno | Sandhu, Manj S. | Waterworth, Dawn M. | Metspalu, Andres | Stumvoll, Michael | Uitterlinden, André G. | Jula, Antti | Navis, Gerjan | Wijmenga, Cisca | Wolffenbuttel, Bruce H. R. | Taskinen, Marja-Riitta | Ala-Korpela, Mika | Kaprio, Jaakko | Kyvik, Kirsten O. | Boomsma, Dorret I. | Pedersen, Nancy L. | Gyllensten, Ulf | Wilson, James F. | Rudan, Igor | Campbell, Harry | Pramstaller, Peter P. | Spector, Tim D. | Witteman, Jacqueline C. M. | Eriksson, Johan G. | Salomaa, Veikko | Oostra, Ben A. | Raitakari, Olli T. | Wichmann, H.-Erich | Gieger, Christian | Järvelin, Marjo-Riitta | Martin, Nicholas G. | Hofman, Albert | McCarthy, Mark I. | Peltonen, Leena | van Duijn, Cornelia M. | Aulchenko, Yurii S. | Ripatti, Samuli
PLoS Genetics  2011;7(10):e1002333.
Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene–environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA) data from 18 population-based cohorts with European ancestry (maximum N = 32,225). We collected 8 further cohorts (N = 17,102) for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR) on total cholesterol (TC) with a combined P-value of 4.79×10−9. There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd) difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.
Author Summary
Circulating serum lipids contribute greatly to the global health by affecting the risk for cardiovascular diseases. Serum lipid levels are partly inherited, and already 95 loci affecting high- and low-density lipoprotein cholesterol, total cholesterol, and triglycerides have been found. Serum lipids are also known to be affected by multiple epidemiological risk factors like body composition, lifestyle, and sex. It has been hypothesized that there are loci modifying the effects between risk factors and serum lipids, but to date only candidate gene studies for interactions have been reported. We conducted a genome-wide screen with meta-analysis approach to identify loci having interactions with epidemiological risk factors on serum lipids with over 30,000 population-based samples. When combining results from our initial datasets and 8 additional replication cohorts (maximum N = 17,102), we found a genome-wide significant locus in chromosome 4p15 with a joint P-value of 4.79×10−9 modifying the effect of waist-to-hip ratio on total cholesterol. In the area surrounding this genetic variant, there were two genes having association between the genotypes and the gene expression in adipose tissue, and we also found enrichment of association in genes belonging to lipid metabolism related functions.
PMCID: PMC3197672  PMID: 22028671
16.  Meta-analysis of genome-wide association studies identifies novel loci that influence cupping and the glaucomatous process 
Springelkamp, Henriët. | Höhn, René | Mishra, Aniket | Hysi, Pirro G. | Khor, Chiea-Chuen | Loomis, Stephanie J. | Bailey, Jessica N. Cooke | Gibson, Jane | Thorleifsson, Gudmar | Janssen, Sarah F. | Luo, Xiaoyan | Ramdas, Wishal D. | Vithana, Eranga | Nongpiur, Monisha E. | Montgomery, Grant W. | Xu, Liang | Mountain, Jenny E. | Gharahkhani, Puya | Lu, Yi | Amin, Najaf | Karssen, Lennart C. | Sim, Kar-Seng | van Leeuwen, Elisabeth M. | Iglesias, Adriana I. | Verhoeven, Virginie J. M. | Hauser, Michael A. | Loon, Seng-Chee | Despriet, Dominiek D. G. | Nag, Abhishek | Venturini, Cristina | Sanfilippo, Paul G. | Schillert, Arne | Kang, Jae H. | Landers, John | Jonasson, Fridbert | Cree, Angela J. | van Koolwijk, Leonieke M. E. | Rivadeneira, Fernando | Souzeau, Emmanuelle | Jonsson, Vesteinn | Menon, Geeta | Weinreb, Robert N. | de Jong, Paulus T. V. M. | Oostra, Ben A. | Uitterlinden, André G. | Hofman, Albert | Ennis, Sarah | Thorsteinsdottir, Unnur | Burdon, Kathryn P. | Spector, Timothy D. | Mirshahi, Alireza | Saw, Seang-Mei | Vingerling, Johannes R. | Teo, Yik-Ying | Haines, Jonathan L. | Wolfs, Roger C. W. | Lemij, Hans G. | Tai, E-Shyong | Jansonius, Nomdo M. | Jonas, Jost B. | Cheng, Ching-Yu | Aung, Tin | Viswanathan, Ananth C. | Klaver, Caroline C. W. | Craig, Jamie E. | Macgregor, Stuart | Mackey, David A. | Lotery, Andrew J. | Stefansson, Kari | Bergen, Arthur A. B. | Young, Terri L. | Wiggs, Janey L. | Pfeiffer, Norbert | Wong, Tien-Yin | Pasquale, Louis R. | Hewitt, Alex W. | van Duijn, Cornelia M. | Hammond, Christopher J.
Nature Communications  2014;5:4883.
Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.
Glaucoma is the most common cause of irreversible blindness worldwide. Here, the authors carry out a large meta-analysis of genetic data from individuals of European and Asian ancestry and identify 10 new loci associated with vertical cup-disc ratio, a key factor in the clinical assessment of patients with glaucoma.
PMCID: PMC4199103  PMID: 25241763

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