Shifting landscapes of human MTHFR missense-variant effects.

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.

Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain.

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.

Correction to: Prevalence of hypertension and diabetes after exposure to extracorporeal shock-wave lithotripsy in patients with renal calculi: a retrospective non-randomized data analysis.

The ninth author name was incorrectly published in the original publication. The correct name should read as 'Cédric Poyet'.

Prevalence of hypertension and diabetes after exposure to extracorporeal shock-wave lithotripsy in patients with renal calculi: a retrospective non-randomized data analysis.

PURPOSE: To evaluate the association of shock-wave lithotripsy (SWL) for kidney stones and hypertension or diabetes. METHODS: Patients with urolithiasis treated by SWL were retrospectively identified. To assess whether shock-wave application to the kidney is associated with long-term adverse effects, patients after SWL for kidney stones were selected as the main group of interest. Patients treated with shock waves for distal ureter stones only were chosen as a comparison group. A questionnaire was sent to all patients to assess the prevalence of hypertension and diabetes. The Swiss Health Survey (SHS) dataset was used as an additional comparison group. RESULTS: After a median follow-up of 13.7 years, the odds ratio (OR) to report hypertension [OR 1.30 (95% CI 1.10-1.95)] or diabetes [OR 1.54 (95% CI 1.21-1.97)] was significantly higher in patients treated with SWL compared to the SHS dataset. In comparison with the kidney group, participants in the SHS had a significantly lower OR to report hypertension at follow-up [OR 0.79 (95% CI 0.65-0.95)], while the OR to report hypertension [1.16 (95% CI 0.79-1.70)] was not significantly different in the distal ureter group. For diabetes, a significantly lower [OR 0.60 (95% CI 0.46-0.78)] in the SHS group and a non-significantly lower [OR 0.68 (95% CI 0.38-1.22)] in the ureter group was noted compared to the kidney group. CONCLUSION: Compared to the SHS data set SWL was in general associated with hypertension and diabetes. However, no clear difference between patients after SWL to the kidney compared to SWL to the distal ureter was seen and thus the data do not support a causal relationship.