Mutations causing premature termination codons discriminate and generate cellular and clinical variability in HHT

For monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we sub-categorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense mediated decay. In three pre-phenotyped patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC patients, PTC-containing RNA transcripts persisted at low levels (8-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni p<0.05 in HHT+/PTC BOECs clustered significantly only to generic protein terms (‘isopeptide-bond’/‘ubiquitin-like conjugation’) and pulse chase experiments detected subtle protein maturation differences, but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of ‘invariant’ housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor (ATF)4 which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modelled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other “less rare” ENG nonsense variants, but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues.