Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

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Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of interests F.P.R. is an investor in Ranomics, Inc., and is an investor in and advisor for SeqWell, Inc., BioSymetrics, Inc., and Constantiam Biosciences, Inc., and has accepted conference travel support from Illumina, Inc. L.F., A.C., and R.N. are employed by and invested in Invitae. R.J.D. has received both a grant and royalties and has also served as a consultant for Alnylam Pharmaceuticals.