Hadinnapola C, Bleda M, Haimel M, Screaton N, Swift A, Dorfmüller P, Preston SD, Southwood M, Hernandez-Sanchez J, Martin J, Treacy C, Yates K, Bogaard H, Church C, Coghlan G, Condliffe R, Corris PA, Gibbs S, Girerd B, Holden S, Humbert M, Kiely DG, Lawrie A, Machado R, Ross RM, Moledina S, Montani D, Newnham M, Peacock A, Pepke-Zaba J, Rayner-Matthews P, Shamardina O, Soubrier F, Southgate L, Suntharalingam J, Toshner M, Trembath R, Vonk Noordegraaf A, Wilkins MR, Wort SJ, Wharton J, NIHR BioResource–Rare Diseases Consortium, UK National Cohort Study of Idiopathic and Heritable PAH, Gräf S and Morrell NW

Circulation 2017;136:2022-2033. DOI: 10.1161/CIRCULATIONAHA.117.028351

Article Summary:

Background: Pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) are rare, overlapping clinical and pathologic variants of World Health Organization group 1 pulmonary hypertension. PVOD/PCH has a poor prognosis due to the lack of long-term responses to medical therapy and possible occurrence of vasodilator-induced pulmonary edema. Based on these observations, early diagnosis is crucial to facilitating referral for lung transplantation in eligible patients. However, PVOD/PCH can masquerade as PAH and distinguishing between PVOD/PCH and PAH on clinical grounds is challenging due to broadly similar physical and hemodynamic findings.

Recently, an autosomal recessive form of PVOD/PCH caused by biallelic mutations of the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) was detected in both familial (100%) and sporadic (20-25%) cases of PVOD/PCH. EIF2AK4 encodes for general control non-derepressible 2 (GCN2), a serine-threonine kinase present in all eukaryotes that phosphorylates the α-subunit of eukaryotic initiation factor 2. GCN2 belongs to a family of four kinases that modulate responses to distinct cellular stressors. Detection of biallelic mutations in EIF2AK4 establishes a precise, accurate molecular diagnosis and unmasks the heritable nature of the disease. In the current study, Hadinnapola et al. report the genotypic and phenotypic characteristics of a large European cohort of PAH patients assessed for rare, pathogenic variants of EIF2AK4 and BMPR2 by whole-genome sequencing.

Methods: From January 2013 to June 2016, prevalent and incident patients with idiopathic PAH, heritable PAH, and PVOD/PCH were recruited to the National Institute of Health Research (NIHR) BRIDGE study (BioResource-Rare Diseases) from pulmonary hypertension centers in the United Kingdom, the Netherlands and France. Patients with other rare diseases and their unaffected relatives enrolled in the BRIDGE study served as controls without PAH. Next-generation sequencing with paired-end sequencing was performed on DNA libraries created from genomic DNA with Illumina HiSeq 2500 and HiSeq X. Reads were aligned against the Genome Reference Consortium human genome (build 37), and variants were called with the Isaac Aligner and Variant Caller (version 2). Variants in BMPR2 and EIF2AK4 were extracted and annotated with the Ensembl Variant Effect Predictor version 84. Likely causal variants were identified on the basis of minor allele frequency and predicted deleteriousness. Patient medical records were reviewed to capture demographic and phenotypic variables from the time of diagnosis and follow-up. Survival data for UK patients were obtained from recruiting centers through the NHS National Spine and local databases. Chest CT images, when available, were independently reviewed by two radiologists with expertise in pulmonary hypertension who were blinded to the clinical diagnoses.

Results: Whole-genome sequencing was performed on 880 patients with a clinical diagnosis of idiopathic PAH (n=808), familial PAH (n=56), or PVOD/PCH (n=16). BMPR2 mutations were detected in 41/56 (73.3%) familial PAH patients and 89/808 (11.0%) idiopathic PAH patients. While no BMPR2 mutations were detected in patients with a clinical diagnosis of PVOD/PCH, almost one-third of these patients carried biallelic EIF2AK4 mutations (31.3%; 5/16, 2 homozygotes and 3 compound heterozygotes). Twenty-five EIF2AK4 variants were also detected in 19 patients clinically diagnosed with PAH and 9 of these patients had biallelic mutations (5 homozygotes and 4 compound heterozygotes). Two patients were heterozygous for rare variants of BMPR2 and EIF2AK4. PAH patients with biallelic EIF2AK4 mutations presented at a younger age, but with similar hemodynamic profiles compared to PAH patients without mutations in PAH-associated genes (median 29 years [IQR, 23-38] versus 51 years [IQR, 37-65]; P=0.024). Despite the absence of any obstructive or restrictive defects in PAH patients with biallelic EIF2AK4 mutations, the transfer coefficient for carbon monoxide (KCO; expressed as percent predicted) was significantly lower in these patients (33% [IQR, 30-35%]) compared to PAH patients with BMPR2 mutations (81% [IQR, 73-92%]; P<0.001) and PAH patients without mutations in PAH-associated genes (71% [IQR, 51-85%]; P=0.001). The prevalence of subtle or gross centrilobular ground glass opacification was not significantly different among patients without PAH-associated mutations (38%, 8/21 CT scans available for analysis), patients with BMPR2 mutations (67%, 14/21), patients with biallelic EIF2AK4 mutations (86%, 6/7), and those diagnosed clinically with PVOD/PCH (50%, 7/14), P=0.122. In contrast, the interlobular septal thickening and mediastinal lymphadenopathy were both more frequent in patients with biallelic EIF2AK4 mutations (29% and 57%, respectively) and PVOD/PCH patients (64% and 79%) compared with patients with BMPR2 mutations (5% and 10%) or those without any mutations (5% and 0%). Pulmonary edema due to PAH-specific therapy was not detected in any of the patients with biallelic EIF2AK4 mutations and a clinical diagnosis of PAH. However, survival time was significantly shorter in these patients compared to PAH patients with BMPR2 mutations (P<0.001) and patients without any PAH-associated gene mutations (P<0.001).

Conclusions: Patients with predicted pathogenic biallelic EIF2AK4 mutations present with a spectrum of phenotypic, radiological and histological features that overlap with PAH but they tend to be younger, have a lower transfer coefficient for carbon monoxide and may have a worse prognosis. Early identification of this high-risk  group with targeted clinical genetic testing may expedite appropriate management including genetic counseling and referral for lung transplantation.

Expert Commentary:

Idiopathic and heritable PVOD and PCH are rare, difficult to diagnose, overlapping variants of pulmonary arterial hypertension (PAH) with an estimated prevalence of 1-2 cases per million (1, 2). However, this estimate does not account for PVOD associated with connective tissue disease (mainly systemic sclerosis), other associated risk factors (e.g. chemotherapy or exposure to organic solvents), or patients misclassified with Idiopathic PAH (IPAH) (1, 3). Up to 10% of patients diagnosed clinically with IPAH are ultimately found to have histologic evidence of PVOD/PCH on lung biopsy or at autopsy (4). Therefore a high index of suspicion for PVOD/PCH and an accurate diagnosis are crucial in order to avoid potential harmful effects (e.g. fatal pulmonary edema triggered by pulmonary arterial vasodilators) and delays in definitive therapy (i.e. lung transplantation) (2). While microscopic examination of lung tissue is necessary to make a definitive diagnosis of PVOD/PCH, histological confirmation requires diligence and expertise, and histologic confirmation is usually only available on post-mortem or explanted lung specimens due to the inherent risks of lung biopsy in patients with severe pulmonary hypertension. Therefore, a systematic strategy integrating a noninvasive evaluation, genetic testing, and lung biopsy in patients with low risk for surgical lung biopsy is necessary when PVOD/PCH is suspected (1).

The landmark discovery that mutations in the gene (BMPR2) that encodes the bone morphogenetic protein type II receptor  caused familial primary pulmonary hypertension provided a foundation for precision medicine in the diagnosis and management of pulmonary hypertension (5, 6). Investigators quickly recognized that many patients diagnosed with IPAH actually had an inherited disease caused by BMPR2 mutations (7). Over the next 15 years additional Mendelian causes of familial PAH were discovered, and in 2014 mutations in EIF2AK4 were found to cause Mendelian forms of PCH (8) and PVOD (9). These discoveries allowed investigators to apply molecular tests to identify heritable forms of PAH and to aid with the difficult problem of differentiating PAH from PVOD and PCH (9, 10).

Best et al provided the first report of EIF2AK4 mutations in patients diagnosed with PAH (10). Clinicians diagnosed IPAH or HPAH at 30 North American medical centers. Pathogenic mutations in BMPR2 were identified in 8 of 72 (11.1%) patients diagnosed with IPAH and 6 of 9 (66.7%) patients diagnosed with familial PAH. None of the 72 patients diagnosed with IPAH had pathogenic biallelic EIF2AK4 mutations; but 1 of  9 (11.1%) patients diagnosed with familial PAH had homozygous pathogenic EIF2AK4 mutations. Identification of biallelic EIF2AK4 mutations established the correct diagnosis and clarified the autosomal recessive inheritance pattern in the affected family.

The  study by Hadinnapola et al. (11) complements the earlier report by providing molecular and clinical data from a large European cohort of patients diagnosed with PAH. Whole genome sequencing in over 800 patients diagnosed clinically with IPAH or HPAH, identified a small subset of patients with biallelic EIF2AK4 mutations who were misclassified clinically as idiopathic (n=8/808, 1.0%) or familial (n=1/56, 1.8%) PAH (11). These observations provide important independent confirmation of the report from North America. Based upon these two studies we now know that clinical misclassification of heritable PVOD/PCH as IPAH is possible, but very unlikely. However, misclassification of heritable PVOD/PCH as familial PAH (FPAH) is more likely, especially when the inheritance pattern does not allow differentiation between an autosomal recessive disorder and inheritance of an autosomal dominant mutation with incomplete penetrance.

Hadinnapola et al (11) provide additional important data from their analysis of a large cohort. First, Hadinnapola’s observation that  patients with pathogenic EIF2AK4 mutations had a significantly lower transfer coefficient for carbon monoxide (DLCO) and were diagnosed with PAH at a younger age than PAH patients without EIF2AK4 mutations suggests that younger patients with a low DLCO may benefit from genetic tests to differentiate IPAH from PCH/PVOD caused by EIF2AK4 mutations. Such testing is also warranted for patients diagnosed with FPAH with an autosomal recessive inheritance pattern (9, 10). Identification of pathogenic EIF2AK4 mutations confirms a molecular diagnosis, obviating the costs and risks of lung biopsy. It also provides evidence of heritable disease; which has important implications for the patient and the patient’s family. Current guidelines recommend that clinicians offer genetic counselling for all patients with heritable PAH or heritable PCH/PVOD (12). Finally, detection of pathogenic EIF2AK4 mutations in patients suspected to have IPAH or HPAH informs clinicians that their patient has an increased risk for pulmonary edema induced by pulmonary vasodilators; and, as Hadinnapola et al report, these patients have a poorer chance to survive than IPAH patients; and early referral for lung transplantation must be considered.

Article summary by: Jason M. Elinoff, MD, Assistant Clinical Investigator, Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland.

Expert commentary by: C. Gregory Elliott, MD, Chair, Department of Medicine, Intermountain Medical Center, Murray, Utah and Professor of Medicine, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah; Mark W. Dodson, MD, PhD, Division of Pulmonary and Critical Care Medicine, Intermountain Medical Center, Murray, Utah; and D. Hunter Best, PhD, ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT, and Departments of Pathology and Pediatrics, University of Utah School of Medicine, Salt Lake City, UT.

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