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.

羟甲基胆烷合酶 (HMBS) 缺陷可导致急性间歇性卟啉症 (AIP)，这是一种急性神经系统疾病。尽管基于测序的诊断可以是明确的，但 ~1/3 的临床 HMBS 变异是错义变异，并且大多数临床报告的 HMBS 错义变异被指定为“意义不确定的变异”(VUS)。通过饱和诱变、集体选择和测序，我们对 HMBS 的红细胞特异性和普遍存在的异构体应用了多重验证分析，获得了所有可能的氨基酸取代中 > 84% 的可信功能影响评分。由此产生的变异效应图总体上符合生化预期，并提供了 HMBS 可以作为单体发挥作用的进一步证据。此外，这些图谱暗示特定残基在活性位点动力学中发挥作用，这得到了分子动力学模拟的进一步支持。最重要的是，这些图谱可以帮助区分致病性和良性 HMBS 变异，甚至为尚未观察到的临床错义变异主动提供证据。