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Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation
Warren van Loggerenberg1, Shahin Sowlati-Hashjin2, Jochen Weile1
1Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, ON M5G 1X5, Canada; Department of Computer Science, University of Toronto, Toronto, ON M5S 2E4, Canada.
Researchers developed a new method to assess the function of hydroxymethylbilane synthase (HMBS) variants, aiding in the diagnosis of acute intermittent porphyria (AIP). This approach helps distinguish between harmful and harmless HMBS gene mutations for better patient outcomes.
Area of Science:
- Biochemistry and Molecular Biology
- Genetics and Genomics
- Medical Diagnostics
Background:
- Defects in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), a neurological disorder.
- A significant portion of HMBS variants are missense, often classified as variants of uncertain significance (VUS), complicating diagnosis.
Purpose of the Study:
- To develop a high-throughput functional assay for HMBS variants.
- To generate comprehensive variant effect maps for HMBS.
- To improve the classification of HMBS missense variants.
Main Methods:
- Applied saturation mutagenesis and en masse selection to both HMBS isoforms.
- Utilized a multiplexed validated assay to determine functional impact scores for over 84% of possible amino acid substitutions.
- Employed molecular dynamics simulations to investigate active site dynamics.
Main Results:
- Generated variant effect maps for HMBS with high coverage of possible substitutions.
- Demonstrated that HMBS can function as a monomer and identified key residues in active site dynamics.
- The variant effect maps successfully differentiated pathogenic from benign HMBS variants.
Conclusions:
- The developed assay and variant effect maps provide a powerful tool for functional assessment of HMBS variants.
- This approach can aid in the definitive diagnosis of AIP by clarifying the significance of VUS.
- The findings offer predictive power for unobserved clinical HMBS missense variants.
