© 2013 Nature America, Inc. All rights reserved. NATURE GENETICS ADVANCE ONLINE PUBLICATION 1 LETTERS Pulmonary veno-occlusive disease (PVOD) is a rare and devastating cause of pulmonary hypertension that is characterized histologically by widespread fibrous intimal proliferation of septal veins and preseptal venules and is frequently associated with pulmonary capillary dilatation and proliferation 1,2 . PVOD is categorized into a separate pulmonary arterial hypertension–related group in the current classification of pulmonary hypertension 3 . PVOD presents either sporadically or as familial cases with a seemingly recessive mode of transmission 4 . Using whole-exome sequencing, we detected recessive mutations in EIF2AK4 (also called GCN2) that cosegregated with PVOD in all 13 families studied. We also found biallelic EIF2AK4 mutations in 5 of 20 histologically confirmed sporadic cases of PVOD. All mutations, either in a homozygous or compound-heterozygous state, disrupted the function of the gene. These findings point to EIF2AK4 as the major gene that is linked to PVOD development and contribute toward an understanding of the complex genetic architecture of pulmonary hypertension. PVOD was first recognized as a specific entity of pulmonary hyperten- sion in the 1960s 5 . PVOD is characterized by a low diffusing capacity for carbon monoxide, occult alveolar hemorrhage and high-resolution computed tomography of the chest that shows patchy centrilobular ground-glass opacities, septal lines and lymph node enlargement 6 . The true incidence of PVOD remains unknown because many cases are probably misclassified as idiopathic pulmonary arterial hypertension (PAH). The proportion of idiopathic cases of PAH that in reality fulfill the criteria for PVOD is likely around 10% 1 . Mutations in BMPR2 are found in approximately 75% of familial cases of PAH and in almost 20% of apparently sporadic cases of PAH. Mutations in ACVRL1, which can complicate hereditary hemorrhagic telangiectasia, have also been described in PAH 7,8 . PAH that is due to BMPR2 mutations segregates as an autosomal-dominant trait with incomplete penetrance 9 . Familial cases of PVOD have been described in three different studies, and the disease typically occurs in the young siblings of one generation 4,10,11 . In the French referral center, we identified 13 PVOD families. In eight families (PVOD1, PVOD2, PVOD3, PVOD5, PVOD6, PVOD7, PVOD8 and PVOD12), we confirmed the PVOD diagnosis histologically after lung transplantation or lung biopsy in at least one family member (Fig. 1 and Supplementary Tables 1 and 2). In the five remaining PVOD families (PVOD4, PVOD9, PVOD10, PVOD11 and PVOD13), we considered the diagnosis to be highly likely on the basis of clinical and paraclinical data (Supplementary Tables 1 and 2). All PVOD families were characterized by the presence of at least two affected siblings and unaffected parents, suggesting that the disease segregates as a recessive trait. To identify the genetic basis of familial forms of PVOD, we first adopted a genetic linkage mapping strategy in three families (PVOD1, PVOD2 and PVOD3). We observed suggestive linkage sig- nals at six regions, with a maximum log 10 odds (LOD)-score peak above 1.5 at each locus, but we detected no genome wide–signifi- cant linkage (LOD > 3) (Supplementary Fig. 1). We then performed whole-exome sequencing on subjects from five families (PVOD1, PVOD2, PVOD3, PVOD4 and PVOD5). We selected homozygous or compound-heterozygous variants that were rare (minor allele fre- quency (MAF) <0.1%) or unknown in either the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project Exome Variant Server (EVS) or the 1000 Genomes Project and that were shared by both affected subjects and were present in a heterozygous state in the unaffected parents. We found that variants of a single gene, EIF2AK4, met these criteria in two families, PVOD1 and PVOD4. In PVOD1, the two affected siblings carried heterozygous frameshift and EIF2AK4 mutations cause pulmonary veno-occlusive disease, a recessive form of pulmonary hypertension Mélanie Eyries 1–3 , David Montani 4–6 , Barbara Girerd 4–6 , Claire Perret 3,7 , Anne Leroy 2 , Christine Lonjou 8 , Nadjim Chelghoum 8 , Florence Coulet 2,3 , Damien Bonnet 9,10 , Peter Dorfmüller 6,11 , Elie Fadel 6,12 , Olivier Sitbon 4–6 , Gérald Simonneau 4–6 , David-Alexandre Tregouët 3,7 , Marc Humbert 4–6 & Florent Soubrier 1–3 1 Unité Mixte de Recherche en Santé (UMR_S 956), Université Pierre and Marie Curie Université Paris 06 (UPMC) and Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France. 2 Genetics Department, Hôpital Pitié-Salpêtrière, Assistance Publique–Hôpitaux de Paris (AP-HP), Paris, France. 3 Institute for Cardiometabolism and Nutrition (ICAN), Paris, France. 4 Université Paris-Sud, Faculté de Médecine, Le Kremlin Bicêtre, France. 5 Département Hospitalo-Universitaire (DHU) Thorax Innovation (TORINO), Service de Pneumologie, Hôpital Bicêtre, AP-HP, Le Kremlin Bicêtre, France. 6 INSERM UMR_S 999, Laboratoire d’Excellence en Recherche sur le Médicament et l’Innovation Thérapeutique (LERMIT), Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France. 7 UMR_S 937, UPMC, INSERM, Paris, France. 8 Post-Genomic Platform (P3S), UPMC, INSERM, Paris, France. 9 Cardiac Surgery Department, Hôpital Necker-Enfants Malades, AP-HP, Paris, France. 10 UMR_S 765, INSERM and Université Paris Descartes, Paris, France. 11 Department of Pathology, Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France. 12 Thoracic Surgery Department, Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France. Correspondence should be addressed to F.S. (florent.soubrier@upmc.fr). Received 6 August; accepted 6 November; published online 1 December 2013; doi:10.1038/ng.2844