An 11-year-old, intact male German Shepherd dog (GSD) was referred to the emergency service of the Department of Clinical Sciences of Companion Animals of the Faculty of Veterinary Medicine of Utrecht University with a 2-day history of acute onset dyspnea and generalized weakness. Vaccination and deworming were performed regularly. The dog had visited Southern Europe 6 months before presentation. No drugs were administered prior to the development of the clinical signs and no environmental circumstances that could cause dyspnea (e.g. tobacco, organic solvent exposure, dust) were reported. Physical examination showed a responsive but lethargic dog with generalized weakness, severe dyspnea, cyanotic mucous membranes, prolonged capillary refill time, weak peripheral pulses, tachycardia (heart rate 180 beats/min) and a grade one out of six systolic murmur with the point of maximal intensity over the right cardiac apex. Harsh lung sounds were heard on lung auscultation. Complete blood count (CBC) showed a mild mature leukocytosis (white blood cells: 18.9 × 109/L; reference interval 4.5–14.6 × 109/L) and a haematocrit of 56% (reference interval 42–61%). Biochemistry did not show any abnormalities. Arterial blood gas analysis showed a severe hypoxemia (PaO2: 48.5 mm Hg; reference interval 85–103 mm Hg) and mild hypocapnia (PaCO2: 27.0 mm Hg; reference interval: 32–43 mm Hg). The suspected cause for the hypocapnia was hyperventilation. D-dimer and antithrombin concentrations were within the reference intervals. Dirofilaria immitis antigen snap test (SNAP® Heartworm RT Test, IDEXX Laboratories) and faecal examination (flotation and Baermann larval isolation technique) were both negative. Thoracic radiographs showed a dilation of the pulmonary artery trunk and right-sided cardiomegaly. Echocardiography was severely complicated due to the severe anxiety and panting of the dog and was therefore limited. It showed severe right ventricular dilation, mild uniform dilation of the main pulmonary artery, systolic flattening of the interventricular septum and moderate tricuspid regurgitation. Application of the modified Bernoulli equation to the velocity of the tricuspid regurgitation jet showed an estimated systolic pulmonary artery pressure of 77 mm Hg, graded as severe PH (reference < 25 mm, severe > 75 mm Hg) []. The left ventricular dimensions were markedly reduced, consistent with left-sided volume depletion. A saline contrast echocardiography was performed, which was negative, thereby excluding intra- and extra-cardiac right-to-left shunting. To address the severe hypoxemia and PH the dog was placed in an oxygen cage with an inspired concentration of oxygen between 40 and 50%. Furthermore, 1.5 mg/kg/8 h oral sildenafil (Viagra®, Pfizer, New York, USA) and 0.25 mg/kg/12 h oral pimobendan (Vetmedin®, Boehringer Ingelheim, Germany) were administered. This therapy did not significantly affect the clinical condition of the dog. However, the arterial hypoxemia mildly improved after the first day of therapy (PaO2 increased from 48.5 to 53 mm Hg, reference interval 85–103 mm Hg). The echocardiogram was repeated on the third day of therapy and the echocardiographic changes and the severity of the PH were markedly reduced. The right ventricular dilation had dramatically decreased, the interventricular septum was no longer flattened, and the pressure gradient of the tricuspid regurgitation was reduced from 77 mm Hg to 41 mm Hg (reference < 25 mm Hg). Therefore, therapy with pimobendan and sildenafil was continued during the hospitalized period (7 days). As further diagnostic steps a (pre-and post-contrast) computed tomography (CT) scan with an apnoea, followed by a surgical lung biopsy in the same anaesthetic session were performed 4 days after the initial presentation. A single slice helical CT scanner (Philips Secura, Philips NV, Eindhoven, the Netherlands) was used. Technical settings included 3 mm helical slices, 120 kV, 200 mA, 292 mm field of view, 512 × 512 matrix and a high spatial frequency algorithm. On CT images, the lung parenchyma showed subtle centrilobular ground glass nodules and an enlarged pulmonary artery. Septal lines, pleural effusion and lymphadenopathy were absent. CT findings were compatible with PH without a conclusive diagnosis. Following the CT scan, a lung biopsy from the left cranial lung lobe (3 × 2 cm) was taken for histopathological examination with a mini-thoracotomy. Directly after this procedure a therapy with dexamethasone (0.25 mg/kg q24 h I.V., Rapidexon ®, Eurovet Animal Health, Bladel, the Netherlands) was initiated as a last resort while histopathological results were pending (3 days). Histopathology of the surgical lung biopsy showed a moderate chronic interstitial histiocytic pneumonia of unknown aetiology and atelectasis by haematoxylin and eosin, periodic acid–Schiff, and Van Gieson’s stainings. Vascular changes were initially not clearly identified. Because of the poor response to the initiated treatment and the suspected poor prognosis based on the histopathologic results, the dog was euthanized. Autopsy was performed with the owner’s informed consent. Gross pathology of the lungs showed moderately collapsed firm lungs with a diffuse mottled appearance with multiple dark red foci of 1 × 1 × 3 mm, and small numbers of white foci of 1 × 1 × 1 mm, often surrounded by a dark red zone (demarcation) randomly distributed throughout all lung lobes. Routine histopathology of the lungs showed multifocal vascular remodelling. In order to differentiate between small arteries and veins, essential to diagnose PVOD, an additional stain visualising elastic fibers and collagen (Weigert’s Resorcin Fuchsin) was added to identify elastic laminae. In this dog, like in people with PVOD, all three compartments (arteries, veins and capillaries) of the pulmonary microcirculation were affected, although the changes in the pulmonary venous system were the most pronounced. Venular lesions included severe concentric intimal proliferation, partial to complete obliteration of the lumina and post-thrombotic recanalization. Capillary lesions were organized in foci, most obvious adjacent to remodelled venules and were characterized by proliferation of plumb endothelial cells (similar to PCH). Segmental congestion of alveolar capillaries was also regularly associated with the foci of PCH. Arterial lesions resembled those of PAH with concentric intimal thickening by increased extracellular matrix and medial hypertrophy, but complex plexiform lesions were absent. These findings were consistent with the findings described by Williams et al. [] and therefore the human equivalent of PVOD. The genomic DNA of the case was isolated from EDTA-blood using a semi-automated Chemagen extraction robot (PerkinElmer Chemagen Technologie GmbH) and was stored at − 20 °C. Approximately 6 years later, the genomic DNA of the case and an unrelated healthy GSD were analysed by whole genome sequencing. Integrity of DNA was checked on a Bioanalyzer (Agilent, Santa Clara, USA) and quantified using Qubit dsDNA HS (Thermo Fisher Scientific, Waltham, USA). DNA libraries were prepared using TruSeq Nano library prep kit (TruSeq Nano library prep kit, Illumina, San Diego CA, USA) using 200 ng gDNA input. Whole genome sequencing information at 30× coverage was obtained using a HiSeqX Ten instrument (HiSeqX Ten instrument, Illumina, San Diego CA, USA) and 2 × 150 base pair paired-end reads. Data was processed with our in-house developed pipeline v1.2.1 () including somatic mutation analysis (Strelka, VarScan, FreeBayes, and MuTect) and a genome analysis toolkit (GATK v. 3.2.2) [] according to best practices guidelines []. Sequence reads were mapped against the Canine Reference Genome (CanFam 3.1) using Burrows-Wheeler alignment with maximal exact matches (BWA-MEM) v0.7.5a [] followed by marked duplicates, merging of lanes, and realignment of indels. Base recalibration was not performed. In human medicine, mutations in BMPR2, ACVRL1, ENG, KCNK3, CAV-1, and SMAD9 have been suggested to be causative for an autosomal dominant form of PAH []. These genes were included in analyses to prevent missing candidate mutations due to phaenotypical misclassification. Comparing the case and control, analysis of these genes revealed 196 unique intronic variants. Comparing the sequence of EIF2AK4 in the case and the healthy GSD revealed 124 unique variants, of which 9 were located in the 3`-UTR, 112 were intronic and 3 were exonic variants. The case was homozygous mutant for c.2961T>C and c.1266G>A, a heterozygous variation was found at c.2092G>A. Variant validation was performed by Sanger sequencing on amplified polymerase chain reaction products from genomic DNA using Platinum Taq Polymerase (Invitrogen). After exonuclease I treatment DNA sequence reactions were performed using BigDye v3.1 on, sequenced on an ABI3130XL and analyzed in Lasergene (version 12.0 DNASTAR). Four of the five additional GSD's, unknown for any form of respiratory stress and aged ≥ 10 years revealed the identical genotype as the case and therefore excluded these as causal variants for PVOD.