Functional validation ofEIF2AK4(GCN2) missense variants associated with pulmonary arterial hypertension

Abstract

AbstractPulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations ofEIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis.EIF2AK4variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteenEIF2AK4variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that severalEIF2AK4variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1.5 GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification ofEIF2AK4variants and potentially identify hypomorophic alleles receptive to pharmacological activation.