Donnelly 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.
Institute Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
Am J Hum Genet. 2023 Oct 5;110(10):1769-1786. doi: 10.1016/j.ajhg.2023.08.012. Epub 2023 Sep 19.
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.
羟甲基胆素合酶（HMBS）缺陷可导致急性间歇性卟啉症（AIP），这是一种急性神经疾病。尽管基于测序的诊断可以明确，但约有三分之一的临床 HMBS 变异是错义变异，且大多数临床报告的 HMBS 错义变异被指定为“意义未明的变异”（VUS）。我们使用饱和诱变、大规模选择和测序，应用一种多重验证的测定法对 HMBS 的红细胞特异性和普遍存在的同工型进行了检测，从而为 >84%的所有可能氨基酸取代获得了可靠的功能影响评分。所得的变异效应图谱通常与生化预期相符，并进一步证明 HMBS 可以作为单体发挥作用。此外，这些图谱提示特定残基在活性位点动力学中具有作用，这进一步得到分子动力学模拟的支持。最重要的是，这些图谱可以帮助区分致病性和良性的 HMBS 变异，甚至可以主动为尚未观察到的临床错义变异提供证据。
