Author: Felipe De Jesus Navarro Vela
Navarro Vela, Felipe De Jesus, 2025 A Large AGTR1 Missense Variant Library For High-throughput Pharmacogenomic Screening, Flinders University, College of Medicine and Public Health
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Hypertension is a chronic condition and the leading cause of morbidity and mortality worldwide, affecting over 400 million individuals around the globe. The angiotensin II type 1 receptor (AT1R), encoded by the AGTR1 gene, is a G protein-coupled receptor (GPCR) that plays a central role in regulating vascular constriction and salt-water homeostasis. Genetic variation in AGTR1, particularly missense mutations, can significantly alter receptor sensitivity to angiotensin II (AngII) and modify the intracellular signalling cascade contributing to hypertensive disease. Despite their potential clinical relevance, many naturally occurring AGTR1 missense variants remain functionally uncharacterised, due to the small representation and study of population-specific variants within more representative medical research frameworks.
This research aimed to systematically characterise the functional diversity of AGTR1 missense variants to better understand their impact on receptor activity and hypertension by building a comprehensive variant library. To achieve this, all reported missense substitutions were compiled from the gnomAD database and the scientific literature, yielding 406 unique targets for mutagenesis. A key innovation of this work lies in the establishment of a high-throughput, scalable site-directed mutagenesis (SDM) pipeline to generate an arrayed library of AGTR1 variants, enabled by optimised primer design, in vivo two-fragment assembling, barcoding and long-read sequencing validation using next-generation sequencing. This approach achieved a 99.7% success rate in a single cloning round, demonstrating high accuracy and efficiency. The resulting arrayed AGTR1 variant library covers over 400 validated constructs and represents the largest arrayed resource of its kind to date. Unlike pooled libraries, this arrayed format enables discrete analysis of individual variants, providing clear genotype-phenotype relationships and facilitating large-scale functional screening. Preliminary experiments established proof of concept for receptor activity in HEK293 cells, supporting the feasibility of implementing future high-throughput ligand-response screening. Given the association between AGTR1 genetic variants and hypertensive treatment outcomes and risks, this work not only generated an unprecedented, high-quality AGTR1 variant resource. It also introduced a highly economical and technically robust SDM workflow, broadly applicable to large-scale variant characterisation for improved understanding of variant-specific contributions to disease mechanisms and inter-individual variability in treatment response.
Keywords: GPCR, AGTR1, AT1R, Pharmacogenomic, mutagenesis, library, single nucleotide polymorphism, Hypertension
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Joshua Dubowsky