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Pulmonaryarterialhypertension(PAH)isarare,severediseaseresultingfromprogressiveobliterationofsmall-caliberpulmonaryarteriesbyproliferatingvascularcells.PAHcanoccurwithoutrecognizedetiology(idiopathicPAH),beassociatedwithasystemicdiseaseoroccurasaheritableform,withBMPR2mutatedinapproximately80%offamilialand15%ofidiopathicPAHcases1–3.Weconductedagenome-wideassociationstudy(GWAS)basedon2independentcase-controlstudiesforidiopathicandfamilialPAH(withoutBMPR2mutations),includingatotalof625casesand1,525healthyindividuals.WedetectedasignificantassociationattheCBLN2locusmappingto18q22.3,withtheriskalleleconferringanoddsratioforPAHof1.97(1.59–2.45;P=7.47×10−10).CBLN2isexpressedinthelung,anditsexpressionishigherinexplantedlungsfromindividualswithPAHandinendothelialcellsculturedfromexplantedPAHlungs.

Idiopathic PAH has an estimated incidence of approximately two cases per million people per year, with an untreated mean survival time of less than 3 years4,5, and this form represents approximately 40% of all PAH cases1,6. To identify variants predisposing to idiopathic or familial PAH, we conducted a two-stage GWAS of individuals with

idiopathic or familial PAH without detectable BMPR2 mutations. During the discovery stage of our analysis, we typed and performed quality control on 472,421 SNPs using the Illumina Human610-Quad DNA BeadChip and tested them for association with PAH in a sample of 340 idiopathic or familial PAH cases and 1,068 controls of French

Genome-wide association analysis identifies a susceptibility locus for pulmonary arterial hypertensionMarine Germain1,2, Mélanie Eyries2–4, David Montani5–7, Odette Poirier2,3, Barbara Girerd5–7, Peter Dorfmüller5–8, Florence Coulet4, Sophie Nadaud2,3, Svetlana Maugenre2,3, Christophe Guignabert5–7, Wassila Carpentier9, Anton Vonk-Noordegraaf10, Marilyne Lévy11,12, Ari Chaouat13, Jean-Charles Lambert14, Marion Bertrand15, Anne-Marie Dupuy16, Luc Letenneur17, Mark Lathrop18, Philippe Amouyel14,19, Thomy J L de Ravel20, Marion Delcroix21,22, Eric D Austin23, Ivan M Robbins24, Anna R Hemnes24, James E Loyd24, Erika Berman-Rosenzweig25, Robyn J Barst25, Wendy K Chung25, Gerald Simonneau5–7, David A Trégouët1,2, Marc Humbert5–7 & Florent Soubrier2–4

1Unité Mixte de Recherche en Santé (UMRS) 937, Université Pierre & Marie Curie (UPMC) Université Paris 6 and Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France. 2Institut Hospitalo-Universitaire (IHU) Cardiométabolisme et Nutrition (ICAN), Paris, France. 3UMRS 956, UPMC Université Paris 6 and INSERM, Paris, France. 4Department of Genetics, Hôpital Pitié-Salpêtrière, Assistance Publique–Hôpitaux de Paris (AP-HP), Paris, France. 5Université Paris–Sud, Faculté de Médecine, Le Kremlin Bicêtre, France. 6Centre National de Référence de l’Hypertension Pulmonaire Sévère, Département Hospitalo-Universitaire (DHU) Thorax Innovation, Service de Pneumologie, Hôpital de Bicêtre, AP-HP, Le Kremlin Bicêtre, France. 7UMRS 999, INSERM and Université Paris–Sud, Laboratoire d’Excellence (LabEx) en Recherche sur le Médicament et l’Innovation Thérapeutique (LERMIT), Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France. 8Department of Pathology, Centre Chirurgical Marie-Lannelongue, Le Plessis-Robinson, France. 9Post-Genomic Platform (P3S), UPMC Université Paris 6, Paris, France. 10Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands. 11Cardiac Surgery Department, Hôpital Necker–Enfants Malades, AP-HP, Paris, France. 12UMRS 765, INSERM and Université Paris Descartes, Paris, France. 13Respiratory Disease Department, Centre Hospitalier Universitaire (CHU) Brabois, Vandoeuvre-lès-Nancy, France. 14UMRS 744, Université de Lille Nord de France and INSERM, Institut Pasteur, Lille, France. 15UMRS 708, UPMC Université Paris 6 and INSERM, Paris, France. 16UMRS 888, Université Montpellier and INSERM, Montpellier, France. 17U897, Université Bordeaux and INSERM, Institut de Santé Publique d’Epidémiologie et de Développement, Bordeaux, France. 18Commissariat à l’Energie Atomique, Institut de Génomique, Centre National de Génotypage, Evry, France. 19Centre Hospitalier Régional Universitaire de Lille, Lille, France. 20Centre for Human Genetics, University Hospitals of Leuven, Leuven, Belgium. 21Department of Pneumology, Catholic University of Leuven (KU Leuven), Leuven, Belgium. 22Department of Pneumology, Gasthuisberg University Hospital, Leuven, Belgium. 23Department of Pediatrics, Division of Pulmonary, Allergy, and Immunology Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 24Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 25Department of Pediatrics, Columbia University, New York, New York, USA. Correspondence should be addressed to F.S. (florent.soubrier@upmc.fr).

Received 1 August 2012; accepted 15 February 2013; published online 17 March 2013; doi:10.1038/ng.2581

table 1 Association of CBLN2 rs2217560 with idiopathic and familial PAH in two independent case-control studies

Discovery Replication Combined

Controls Cases Controls Cases Controls Cases

rs2217560 n = 1,068 n = 340 n = 456 n = 284 n = 1,524 n = 624

AA 925 (87%) 262 (77%) 400 (88%) 211 (74%) 1,325 (87%) 473 (76%)

AG 136 (13%) 72 (21%) 52 (11%) 71 (25%) 188 (12%) 143 (23%)

GG 7 (<1%) 6 (2%) 4 (1%) 2 (1%) 11 (1%) 8 (1%)

MAF (G) 0.070 0.123 0.066 0.132 0.069 0.127

Pa 1.56 × 10−5 1.63 × 10−5 7.47 × 10−10

Allelic

ORb 1.87 (1.41–2.48) 2.16 (1.51–3.09) 1.97 (1.59–2.45)

The Cochran Q statistical test did not detect any heterogeneity between the ORs obtained for the two cohorts (P = 0.811). The Mantel-Haenszel estimate of common allelic OR derived from the meta-analysis of the results obtained in the two cohorts separately was 1.98 (1.58–2.47), P = 1.65 × 10−9.aP value of the Cochran-Armitage trend test. The P value of the association test in the discovery cohort after controlling for any underlying population stratification (EIGENSTRAT software) was 3.9 × 10−4. bAllelic OR calculated from the observed genotype counts.

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origin (Online Methods and Supplementary Table 1). Application of the EIGENSTRAT program to test for SNP association with idiopathic or familial PAH7 did not show any evidence of population stratifica-tion, with a genomic inflation coefficient of 1.02 (Supplementary Fig. 1). No SNP achieved the statistical threshold for genome-wide significance (P < 5 × 10−8) (Supplementary Fig. 2), but the 384 most significant SNPs with corresponding association P values ranging from 1.82 × 10−6 to 6.87 × 10−4 were nevertheless selected for further association testing in an independent replication sample of 285 cases and 457 controls. Of the 384 SNPs genotyped by a dedicated Illumina GoldenGate assay (Online Methods), 319 passed quality control. After Bonferroni correction for the number of tested SNPs, 2 of the 319 SNPs showed significant association with idiopathic and familial PAH (Supplementary Table 2), rs2217560 (P = 1.63 × 10−5) and rs9916909 (P = 3.50 × 10−5). These two SNPs are located 52 kb downstream of the CBLN2 gene at 18q22.3 and are in almost complete linkage disequilibrium (r2 = 0.99).

The association pattern of rs2217560 with idiopathic and familial PAH was homogeneous in both the discovery and replication cohorts (Table 1). In the discovery GWAS, the rs2217560[G] allele was more frequent in cases than in controls (0.123 versus 0.070) and was asso-ciated with a higher risk of PAH of 1.87 (1.41–2.48; P = 3.88 × 10−4). In the replication population, the rs2217560[G] allele had a higher frequency in cases than in controls (0.132 versus 0.066), and the cor-responding odds ratio (OR) was 2.16 (1.51–3.09; P = 1.63 × 10−5). In the combined sample, the rs2217560[G] allele was associated with a higher risk of PAH of 1.97 (1.59–2.45), with overall statistical sig-nificance reaching P = 7.47 × 10−10 (Table 1). No evidence for any sex-specific association was observed (Supplementary Table 3). We further examined whether the observed association could be explained by untyped SNP(s) located in the vicinity of rs2217560. Using the August 2010 release of the 1000 Genomes Project data set as a reference, we conducted imputation analysis in the GWAS discovery cohort (Online Methods). No single imputed SNP at the 18q22.3 locus showed stronger evidence of association than rs2217560 (Supplementary Fig. 3 and Supplementary Table 4). Additional

conditional regression analysis of the effect of rs2217560 did not detect any other independent association signal at the CBLN2 locus (Supplementary Table 4).

Because of the rarity of PAH, it was not possible to collect a larger sample of cases for GWAS analysis, which is the current practice for more common complex diseases8. As a consequence, the power of the single discovery sample was rather limited. For example, there was only 35% power to detect an allelic OR of 2.0 for a SNP with a minor allele frequency (MAF) of 0.10 at the genome-wide significance level of 5 × 10−8. Accordingly, we used a two-stage GWAS strategy, which provided ~75% power to detect such genetic effects. However, the two-stage strategy did not have enough power to detect moderate genetic effects, with a maximum power of ~25% to detect an OR of 1.5 or less for a SNP with a MAF of 0.20.

The rs2217560 SNP identified through our two-stage GWAS strat-egy lies 52 kb downstream of the CBLN2 gene. CBLN2 belongs to the cerebellin gene family, which encodes a group of secreted neuronal glycoproteins (CBLN1–CBLN4) and the precursor of CBLN2, a hexa-decapeptide with 94% and 44% sequence homology to the CBLN1 and CBLN3 peptides, respectively9,10. CBLN2 has previously been

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Figure 2 Immunostaining of pulmonary arteries with an antibody to CBLN2. (a) Endothelial labeling of a pulmonary artery with intimal fibrosis (black arrows). (b) Endothelial labeling of a plexiform lesion (red arrows), labeling of intravascular cellular elements (erythrocytes and leukocytes; purple arrow) and background staining in some areas (black arrow). (c) Pulmonary artery from a control individual without substantial endothelial labeling. Scattered bronchial cells are positive (black arrows). Scale bars, 100 µm.

Figure 1 CBLN2 mRNA levels are higher in lungs from individuals with PAH. CBLN2 mRNA levels were measured by RT-PCR in lungs from controls (n = 9) and individuals with PAH (n = 6) and normalized to GAPDH mRNA levels. Data are expressed as mean fold change ± s.d. ***P < 0.001, relative to controls.

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reported to mainly be expressed in different regions of the brain11, but, because CBLN2 is in close proximity to the associated SNPs, we assessed CBLN2 expression in the lung and other cell types. We found that CBLN2 mRNA was expressed in the whole lung and, at lower levels, in circulating cells such as lymphocytes. CBLN2 mRNA levels were significantly higher in lungs explanted from indi-viduals with PAH than in histologically normal lung tissue derived from subjects undergoing surgery for lung cancer (control lungs) (Fig. 1 and Supplementary Fig. 4). CBLN2 protein was detected in endothelial, epithelial and circulating cells from subjects with PAH and in control lung samples via immunohistochemistry, but the protein amounts varied widely from sample to sample (Fig. 2). In cultured pulmonary vascular cells, we found that CBLN2 mRNA was expressed in endothelial cells isolated from the pulmonary artery (PA-ECs); in contrast, expression was detected at very low levels in pulmonary artery vascular smooth muscle cells (PA-SMCs) (Fig. 3). CBLN2 mRNA levels in primary cultured endothelial cells explanted from PAH lung samples collected during lung transplantation were higher than in endothelial cells from control lung samples (Fig. 3). We hypothesized that the CBLN2 synthesized by endothelial cells might act on vascular SMC proliferation in a paracrine fashion. Therefore, we added mature CBLN2 peptide at concentrations rang-ing from 0.01 to 10 nM10 to serum-deprived PA-SMCs in primary cultures. A significant log-linear trend for proliferation inhibition was observed in cultured PA-SMCs with increasing concentra-tions of CBLN2 (Fig. 4). A generic toxic effect of CBLN2 peptide on PA-SMCs was excluded by measuring the viability of the cells to which CBLN2 was added (Supplementary Fig. 5). Preliminary data did not show any evidence for an association between the rs2217560 genotype and CBLN2 mRNA levels in monocytes (data not shown), but these results do not preclude a tissue-specific effect in lungs.

CBLN2 was previously shown to bind the S4 domain of neurexin-1β (a presynaptic cell adhesion molecule encoded by NRXN1) as well as the postsynaptic δ2 glutamate receptor (GluD2), contributing to synapse formation by bridging the two molecules12. NRXN1, which encodes both neurexin-1α and neurexin-1β, is expressed in the vas-cular wall. The neurexin-1β isoform, transcribed from an internal promoter and containing a specific N-terminal peptide absent in the neurexin-1α isoform, is present in a subset of SMCs and also in

chicken embryonic arteries13. Our results show that the neurexin-1β transcript is expressed in the lung, without there being a significant difference between control and PAH lung samples (Supplementary Fig. 6). Antibody to neurexin-1β was shown to decrease fibroblast growth factor 2 (FGF2)-induced angiogenesis in the chorioallantoic membrane chicken model and to decrease noradrenaline-induced vessel tension in isolated chicken embryonic arteries13. In recent years, several molecules have been implicated in both neurogenesis and angiogenesis14.

Altogether, these results strongly support a role for the CBLN2 locus as a new contributor to the physiopathology of idiopathic and familial PAH. The CBLN2 gene is a promising candidate because of its vascular expression and its effects in vascular cells. CBLN2, which we find to be produced by endothelial cells, was previously shown to bind an adhesion molecule present in SMCs. Further studies are needed to document the putative function of CBLN2 in pulmonary vessels.

In conclusion, we report the first GWAS of idiopathic and famil-ial PAH negative for BMPR2 mutation, which identified an allele associated with twofold greater risk of the disease. The associated locus is new with respect to PAH pathogenesis. Fine mapping and deep sequencing of the entire locus is now required to identify and characterize the functional variant(s) responsible for the observed association. These results pave the way for improved understanding of pathophysiology as well as new therapeutic approaches for PAH.

URLs. US National Institutes of Health (NIH) Clinical Trials website, http://clinicaltrials.gov/; MACH, http://www.sph.umich.edu/csg/abecasis/mach/; Minimac, http://genome.sph.umich.edu/wiki/Minimac; Mach2dat (v1.08.18) software, http://www.sph.umich.edu/csg/abecasis/MACH/download/.

MetHodSMethods and any associated references are available in the online version of the paper.

Note: Supplementary information is available in the online version of the paper.

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Figure 3 CBLN2 mRNA levels in pulmonary vascular cells from individuals with PAH. CBLN2 mRNA levels were measured by RT-PCR in cultured PA-ECs and PA-SMCs from controls (n = 10 for PA-ECs and 10 for PA-SMCs) and individuals with PAH (n = 8 for PA-ECs and 5 for PA-SMCs) and normalized to GAPDH mRNA levels. Data are expressed as mean fold change ± s.d. *P = 0.053, relative to controls (after adjustment for age and sex).

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ACKNOWLEDGMENTSWe thank E. Fadel and O. Mercier from the Centre Médico-Chirurgical Marie-Lannelongue for their help in lung sample collection. The Pulmonary Hypertension Allele-Associated Risk (PHAAR) project was financially supported by the Agence Nationale pour la Recherche (Project ANR-07-MRAR-021) and by PHRC AOM07-041, INSERM and UPMC. The 3C Study is conducted under a partnership agreement between INSERM, the Victor Segalen–Bordeaux II University and Sanofi-Synthelabo. The Fondation pour la Recherche Médicale funded the preparation and first phase of the study. The 3C Study is also supported by the Caisse Nationale Maladie des Travailleurs Salariés, the Direction Générale de la Santé, the Mutuelle Générale de l’Education Nationale, the Institut de la Longévité, the Agence Française de Sécurité Sanitaire des Produits de Santé, the regional governments of Aquitaine, Bourgogne and Languedoc-Roussillon and the Fondation de France, and the Ministry of Research–INSERM Programme Cohorts and Collection of Biological Material. The Lille Genopole received an unconditional grant from Eisai. The financial supporters had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. M.G. was funded by a grant from the Agence Nationale pour la Recherche (Project PHAAR, ANR-07-MRAR-021) and the Program Hospitalier de Recherche Clinique (PHRC2009 RENOVA-TV). Statistical analyses used the C2BIG computing centre funded by the Fondation pour la Recherche Médicale and Région Ile de France. Collection and management of samples from Vanderbilt University were supported by US NIH grants P01 HL072058 and K23 HL0987431 and Vanderbilt General Clinical Research Center (GCRC) RR000095. Collection of the samples from Columbia University was supported by US NIH grant R01 HL060056.

AUTHOR CONTRIBUTIONSF.S. initiated and supervised the study. F.S., D.A.T., G.S. and M.H. conceived and designed the experiments. D.M., B.G., G.S., M.H., A.V.-N., M.Lévy, A.C., T.J.L.d.R. and M.D. analyzed clinical data of collected individuals with PAH for the discovery cohort. J.-C.L., M.B., A.-M.D., L.L., M.Lathrop and P.A. provided genotyped data for control subjects. E.D.A., I.M.R., A.R.H., J.E.L., E.B.-R., R.J.B. and W.K.C. analyzed the clinical data of the collected individuals with PAH for the replication cohort. M.E. and F.C. managed DNA samples. W.C. performed genotyping. M.G. and D.A.T. performed statistical analysis. M.E., O.P., S.N. and S.M. performed functional analyses. C.G. isolated vascular cells. P.D. performed tissue imaging. D.A.T., M.G., M.E., O.P., S.N. and F.S. analyzed data. F.S., M.G., M.E. and D.A.T. wrote the manuscript. O.P., S.N., M.H., D.M., E.D.A., J.E.L. and W.K.C. reviewed the manuscript .

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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2. Sztrymf, B. et al. Clinical outcomes of pulmonary arterial hypertension in carriers of BMPR2 mutation. Am. J. Respir. Crit. Care Med. 177, 1377–1383 (2008).

3. Girerd, B. et al. Absence of influence of gender and BMPR2 mutation type on clinical phenotypes of pulmonary arterial hypertension. Respir. Res. 11, 73 (2010).

4. Gaine, S.P. & Rubin, L.J. Primary pulmonary hypertension. Lancet 352, 719–725 (1998).

5. McLaughlin, V.V. & McGoon, M.D. Pulmonary arterial hypertension. Circulation 114, 1417–1431 (2006).

6. Humbert, M. et al. Pulmonary arterial hypertension in France: results from a national registry. Am. J. Respir. Crit. Care Med. 173, 1023–1030 (2006).

7. Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat. Genet. 38, 904–909 (2006).

8. Schunkert, H. et al. Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat. Genet. 43, 333–338 (2011).

9. Yiangou, Y., Burnet, P., Nikou, G., Chrysanthou, B.J. & Bloom, S.R. Purification and characterisation of cerebellins from human and porcine cerebellum. J. Neurochem. 53, 886–889 (1989).

10. Nilsson, A. et al. Neuropeptidomics of mouse hypothalamus after imipramine treatment reveal somatostatin as a potential mediator of antidepressant effects. Neuropharmacology 62, 347–357 (2012).

11. Miura, E., Iijima, T., Yuzaki, M. & Watanabe, M. Distinct expression of Cbln family mRNAs in developing and adult mouse brains. Eur. J. Neurosci. 24, 750–760 (2006).

12. Matsuda, K. & Yuzaki, M. Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions. Eur. J. Neurosci. 33, 1447–1461 (2011).

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oNLINeMetHodSStudy population. Diagnosis with PAH was defined by hemodynamic measurement during right-heart catheterization for all cases included in the study (discovery stage and replication stage), including those identified by the French PAH Network between 1 January, 2003, and 1 April, 2010. For all cases, PAH was defined as a mean pulmonary arterial pressure equal to or exceeding 25 mm Hg associated with normal pulmonary capillary wedge pressure. Hemodynamic evaluation by right-heart catheterization was performed at baseline in all subjects according to previously described protocols15,16. PAH was considered to be idiopathic after clinical and biologi-cal investigations eliminated all known causes. Cases with idiopathic PAH were screened for BMPR2 mutations, and cases with a family history of PAH were screened for BMPR2 and ACVRL1 mutations. Screening for point muta-tions and large chromosomal rearrangements was performed as previously reported2,17. Familial cases carrying a mutation in either of these genes were excluded. When cases had a family history of PAH without evidence of either BMPR2 or ACVRL1 mutation, a single index case from the family was included in the GWAS analysis.

In the discovery stage, a total of 378 cases meeting these criteria were included. All clinical characteristics at PAH diagnosis and follow-up were stored in the Registry of the French PAH Network6. This registry was set up in agreement with French bioethics laws (French Commission Nationale de l’Informatique et des Libertés) and with informed consent from participants2. The control group was composed of a random sample of 1,140 subjects who were free of any chronic disease from the 3C Study18. The 3C Study is a population-based prospective cohort with a 4-year follow-up carried out in three French cities: Bordeaux (southwest France), Montpellier (southeast France) and Dijon (central eastern France). This study has served as a control population for several French GWAS projects19–22.

In the replication stage, specimens (297 PAH cases and 479 healthy controls) from participants in the Vanderbilt Prospective Pulmonary Hypertension Research Cohort study and from the Columbia University Pulmonary Hypertension Center were included. These subjects were recruited via the Vanderbilt and Columbia Pulmonary Hypertension Centers and the NIH Clinical Trials website and in collaboration with the Pulmonary Hypertension Association Conference Research Recruitment Room (2010 Conference, Anaheim, California). The Vanderbilt University Medical Center and the Columbia University Institutional Review Boards approved all study pro-tocols. All participants gave informed written consent to participate in genetic and clinical studies and underwent genetic counseling, when appro-priate, in accordance with the guidelines of the American College of Chest Physicians23.

As in the discovery stage, PAH was defined by a mean pulmonary arte-rial pressure equal to or exceeding 25 mm Hg associated with normal pul-monary capillary wedge pressure. Hemodynamic evaluation by right-heart catheterization was performed at baseline in all subjects according to previ-ously described protocols15,16. PAH was considered to be idiopathic, after clinical and biological investigations eliminated all known causes, or familial, as appropriate. Screening for mutations in BMPR2 and ACVRL1 was the same as for the discovery stage.

Genotyping. In the discovery stage, the sample of 378 idiopathic or familial PAH cases and 1,140 healthy controls were typed with the Illumina Human610-Quad BeadChip. Individuals with genotyping success rates lower than 95% were excluded from the analysis, as were individuals showing close relatedness. The latter individuals were assessed by pairwise identity clustering by state distance (IBS) and multidimensional scaling (MDS) using PLINK software24. The EIGENSTRAT program7 was further used to detect individuals of non-European ancestry. A total of 104 subjects (32 cases and 72 healthy controls) were thus excluded from the analysis because of close relatedness and/or evi-dence of non-European ancestry. SNPs showing significant (P < 1 × 10−5) deviation from Hardy-Weinberg equilibrium (HWE) in controls, MAF of <1% in controls or <5% in cases, or genotyping call rate of <99% were filtered out. This resulted in a final analysis of 462,499 autosomal and 9,922 X-linked SNPs in a sample of 340 PAH cases and 1,068 healthy controls.

In the replication stage, the 384 SNPs showing the strongest associa-tion with PAH and assigned an Illumina ScoreDesign greater than 0.4 were

selected for genotyping in an independent sample of 297 idiopathic or familial PAH cases and 479 controls using the Illumina GoldenGate assay. A total of 34 individuals (12 cases and 22 controls) were discarded owing to low geno-type calling rates (<80%). SNPs showing significant (P < 1 × 10−5) deviation from HWE in controls or call rates of <99% were removed from the analysis, resulting in the statistical analysis of 319 SNPs in a sample of 285 cases and 457 controls.

Statistical analysis. At the discovery stage, the genome-wide association analysis of SNPs was conducted using the EIGENSTRAT program that cor-rects for any uncontrolled population stratification7. The genomic control inflation factor was also computed according to the median test statistic25. At the replication stage, the association of SNPs with PAH was assessed by use of the Cochran-Armitage trend test26. Homogeneity of associations across the two stages was tested using the Mantel-Haenszel method27.

The imputation of 11,572,501 autosomal SNPs was conducted using MACH and Minimac software according to the August 2010 release of the 1000 Genomes Project CEU reference data set (Utah residents of Northern and Western European ancestry). The association of each imputed SNP with PAH was tested by logistic regression analysis in which allele dosage (0, 1 or 2 copies of the minor allele) for imputed SNPs was assessed. Analyses were adjusted for the first four principal components and were performed using mach2dat (v 1.08.18) software.

RT-PCR expression studies. To study CBLN2 expression in pulmonary vascu-lar cells, lung specimens were obtained at the time of lung transplantation from individuals with idiopathic PAH. Control lung specimens were obtained from subjects without any evidence of pulmonary vascular disease who underwent lobectomy or pneumonectomy for localized lung cancer, with normal tissue collected at a distance from the tumors. Both control and PAH lung specimens were collected under the same conditions by the same surgical department (Supplementary Table 5). This study was approved by the local ethics commit-tee (Comité de Protection des Personnes Ile-de-France, Le Kremlin-Bicêtre, France), and participants gave informed consent before the study. For RNA extraction, lung samples were washed in PBS and then immediately frozen in liquid nitrogen and stored at −80 °C. Subsequently, PA-ECs and PA-SMCs were isolated from idiopathic PAH and control lung samples (Supplementary Table 5) and cultured as previously described28,29. Cells were used from passages 3–6. RT-PCR assays were performed as previously described30 using the primers listed in Supplementary Table 6. Data are expressed as mean fold change ± s.d. from at least three independent experiments. Statistical analysis was performed using a non-parametric Mann-Whitney U test for single comparisons and ANOVA for multiple comparisons (XLSTAT software, Addinsoft). P values less than 0.05 were considered to be significant.

Immunohistochemical studies. Lung sections were stained and analyzed as described31. Immunohistochemical analysis was performed on formalin-fixed samples. Paraffin-embedded sections were stained with polyclonal rabbit pri-mary antibody against human CBLN2 (Abgent, AP11835b; 1:200 dilution). Biotinylated secondary antibody to rabbit IgG (Vector, BA-1000) was used for the detection of primary antibody, and Vector Red alkaline phosphatase substrate (Vector, Sk-5100) was the chromogen.

Proliferation assays. PA-SMCs purchased from Lonza were seeded in SmGM-2 BulletKit medium (Lonza) at a density of 5,000 cells per well in 96-well plates. The following day, cells were starved in SmGM-2 medium with 0.2% FCS for 24 h before being stimulated with the CBLN2 peptide (SGSAKVAFSATRSTNH; ProteoGenix) at concentrations of 0.01, 0.1, 1 and 10 nM. After 24 h of incuba-tion with the CBLN2 peptide, proliferation was measured by a BrdU incor-poration assay using a BrdU cell proliferation ELISA kit (Roche Diagnostics) according to the manufacturer’s instructions. Statistical analysis was performed using a log-linear regression test.

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