Pulmonary arterial hypertension (PAH) is a rare and debilitating disease characterised by elevated pulmonary arterial pressure and extensive vascular remodelling in the lungs, with a lack of effective curative options. The identification of genetic variations in genes encoding components of the bone morphogenetic protein pathway in individuals with heritable PAH has opened up promising avenues for therapeutics, with encouraging results from preliminary clinical trials targeting this pathway. To deepen the understanding of the molecular mechanisms underpinning this rare condition, detect additional causative genes, and identify novel potential therapeutic strategies, a comprehensive study involving whole-genome sequencing was conducted on a cohort of over 1000 PAH patients. Within this cohort, rare causative heterozygous variants in the Aquaporin-1 (AQP1) gene were identified among a small subgroup of PAH patients. AQP1 is a water channel protein critical for maintaining endothelial cell integrity and fluid homeostasis.

The primary aim of this thesis is to provide insight into the potential pathobiological significance of these rare AQP1 variants in PAH. Computational structural analysis using Pymol was employed to gain preliminary insights into the mutational effects of the identified variants. Subsequently, mutant AQP1 proteins were overexpressed in Hek293T cells, then subjected to confocal microscopy and membrane biotinylation followed by biotin-based ELISAs, to investigate whether the mutant proteins are able to traffic to the cellular membrane. These studies suggested normal membrane trafficking for the Arg126Cys, Val176Glu, Leu181Phe, and Arg195Trp AQP1 mutants, with comparable levels to that of wild-type AQP1. In contrast, the Trp213Term mutant is predicted to display dysfunctional trafficking, although further validation regarding this nonsense mutant is required.

In addition, water and solute permeability through the wild-type and mutant AQP1 proteins was assessed using stable transfection of MDCK cells and transient transfection of Hek293T cells, respectively, followed by calcein self-quenching assays. None of the PAH-causative AQP1 mutants exhibited permeability to glycerol or urea, suggesting a retained ability for strict solute exclusion. Interestingly, water permeability was significantly reduced in the Arg126Cys AQP1 mutant, near to or completely abolished in the Val176Glu, Arg195Trp, and Trp213Term AQP1 mutants, but unaltered in the Leu181Phe AQP1 mutant.

Finally, due to the recurrent nature of the Arg195Trp mutation, identified in 5 of 9 unrelated PAH patients with causal AQP1 variants, an Aqp1 R195W knock-in mouse model was generated to investigate its phenotypic effects in vivo and ex vivo. Surprisingly, neither heterozygous nor homozygous R195W mice displayed haemodynamic measurements suggestive of PH, either when exposed to normoxia or hypoxia. Nevertheless, the mutant mice exhibited significantly reduced urine osmolarity and signs of inflammation. Samples for future assessment of vascular remodelling through protein, mRNA, and immunohistochemistry analysis have been collected.

In conclusion, this project has provided preliminary insights into the role of rare AQP1 variants in PAH and established crucial experimental protocols and reagents for further investigations. Ultimately, these findings may pave the way for the development of precision medicine approaches tailored to patients with specific AQP1 mutations, offering new hope for improved management and treatment outcomes in PAH.