Heterozygous transcriptional signatures unmask variable premature termination codon (PTC) burden alongside pathway-specific adaptations in blood outgrowth endothelial cells from patients with nonsense DNA variants causing hereditary hemorrhagic telangiectasia

ABSTRACT Frameshift and nonsense DNA variants represent the commonest causes of monogenic inherited diseases. They usually generate premature termination codon (PTC)-containing RNA transcripts that produce truncated proteins in recombinant systems, but endogenously are subject to nonsense mediated decay. To examine native consequences of these variants, we derived cells from pre-genotyped patients. Blood outgrowth endothelial cells (BOECs) were established from individuals with hereditary hemorrhagic telangiectasia (HHT) due to a heterozygous nonsense variant in ACVRL1 , ENG or SMAD4 that each encode an endothelial cell-expressed protein mediating bone morphogenetic protein (BMP)/ transforming growth factor (TGF)-β signalling. RNA sequencing alignments to PTC alleles varied from 8-23% of expected, and differed between same-donor replicates. Differential gene expression analyses were validated by single cell qRT-PCR, and identification of changes in wider, disease-specific BMP/TGF-β pathway components. However, the most differentially expressed genes clustered to disease-independent terms for post translational protein modification (isopeptide bond; ubiquitin-like conjugation). They were the only terms meeting Benjamini significance after clustering Bonferroni-ranked, differentially expressed genes from the 5,013 meeting 10% intraassay coefficients of variation, and significance was robust to normalisation methods. Optimised pulse chase experiments supported perturbed wildtype protein maturation, but no PTC-truncated protein was identified. Unexpectedly, BOEC cultures with highest PTC persistence were discriminated in unsupervised hierarchical clustering of low GINI coefficient ‘invariant’ housekeeper genes, and patterns were compatible with higher cellular stress. The findings support a model whereby PTCs are more of a burden in stressed cells, and lead us to conclude that overlooked and varying PTC burdens contribute to biological variability.