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" (VUS). 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 expectation. However, the maps showed variants at the dimerization interface to be unexpectedly well tolerated, and suggested residue roles in active site dynamics that were supported by molecular dynamics simulations. Most importantly, these HMBS variant effect maps can help discriminate pathogenic from benign variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

The aim of this study was to evaluate the effect of sitagliptin on glucose tolerance, plasma lipids, energy expenditure and metabolism of brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal muscle in overweight individuals with prediabetes (impaired glucose tolerance and/or impaired fasting glucose).

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.

Monocarboxylate transporter 8 (MCT8) facilitates cellular influx and efflux of the thyroid hormones (THs) T(4) and T(3). Mutations in MCT8 lead to severe psychomotor retardation. Here, we studied the importance of 2 highly conserved residues (Arg445 in transmembrane domain 8 and Asp498 in transmembrane domain 10) for substrate recognition and helix interactions. We introduced single and double mutations (R445A, R445C, R445D, R445K, D498A, D498E, D498N, D498R, R445A+D498A, R445D+D498R, and R445K+D498E) in human MCT8 cDNA and studied the effects on MCT8-mediated TH uptake and metabolism in transfected cells. The impact of these mutations on MCT8 protein expression, dimerization capacity, and subcellular localization was studied by Western blotting and confocal microscopy. We found that mutations in Arg445 or Asp498 that alter the local charge resulted in a near-complete loss of TH uptake capacity, whereas the expression, stability, and subcellular localization of these mutant proteins was similar to those for wild-type MCT8. Given the impaired TH uptake, TH efflux could not be adequately studied. The importance of opposite charges at Arg445 and Asp498 was studied by exchanging these residues (R445D+D498R). In particular, T(4) uptake was less severely reduced by the exchange mutation than by the single mutations. Mutations of Arg445 and Asp498 to equally charged residues (R445K and/or D498E) resulted in TH uptake levels similar to wild-type MCT8. The presence of 2 oppositely charged residues at positions Arg445 and Asp498 that are predicted in close structural proximity is crucial for efficient TH uptake, which may indicate the presence of an, at least transient, charge pair between these residues.