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.

Department of Human Genetics McGill University Montreal QC H3A 0C7 Canada

Department of Pediatrics and Inherited Metabolic Disorders Charles University 1st Faculty of Medicine and General University Hospital Prague Ke Karlovu 2 12 08 Praha 2 Czech Republic

Division of Metabolism and Children's Research Center University Children's Hospital Zürich University of Zürich CH 8032 Zurich Switzerland

Institute of Biomedical Engineering University of Toronto ON M5S 3G9 Canada

Invitae Corp San Francisco CA 94103 USA

Lunenfeld Tanenbaum Research Institute Sinai Health System Toronto ON M5G 1X5 Canada; The Donnelly Centre University of Toronto Toronto ON M5S 3E1 Canada; Department of Molecular Genetics University of Toronto Toronto ON M5S 3E1 Canada

Lunenfeld Tanenbaum Research Institute Sinai Health System Toronto ON M5G 1X5 Canada; The Donnelly Centre University of Toronto Toronto ON M5S 3E1 Canada; Department of Molecular Genetics University of Toronto Toronto ON M5S 3E1 Canada; Department of Computer Science University of Toronto Toronto ON M5S 2E4 Canada

Zobrazit více v PubMed

van Leeuwen J., Pons C., Boone C., Andrews B.J. Mechanisms of suppression: The wiring of genetic resilience. BioEssays. 2017;39:1700042. PubMed PMC

Chen S., Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. J. Clin. Oncol. 2007;25:1329–1333. PubMed PMC

Cooper D.N., Krawczak M., Polychronakos C., Tyler-Smith C., Kehrer-Sawatzki H. Where genotype is not predictive of phenotype: towards an understanding of the molecular basis of reduced penetrance in human inherited disease. Hum. Genet. 2013;132:1077–1130. PubMed PMC

Hartwell L.H., Szankasi P., Roberts C.J., Murray A.W., Friend S.H. Integrating genetic approaches into the discovery of anticancer drugs. Science. 1997;278:1064–1068. PubMed

Aly A., Ganesan S. BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability. J. Mol. Cell Biol. 2011;3:66–74. PubMed PMC

Noordermeer S.M., van Attikum H. PARP Inhibitor Resistance: A Tug-of-War in BRCA-Mutated Cells. Trends Cell Biol. 2019;29:820–834. PubMed

Richards S., Aziz N., Bale S., Bick D., Das S., Gastier-Foster J., Grody W.W., Hegde M., Lyon E., Spector E., ACMG Laboratory Quality Assurance Committee Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015;17:405–424. PubMed PMC

Nykamp K., Anderson M., Powers M., Garcia J., Herrera B., Ho Y.-Y., Kobayashi Y., Patil N., Thusberg J., Westbrook M., Topper S., Invitae Clinical Genomics Group Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria. Genet. Med. 2017;19:1105–1117. PubMed PMC

Weile J., Roth F.P. Multiplexed assays of variant effects contribute to a growing genotype-phenotype atlas. Hum. Genet. 2018;137:665–678. PubMed PMC

Weile J., Sun S., Cote A.G., Knapp J., Verby M., Mellor J.C., Wu Y., Pons C., Wong C., van Lieshout N. A framework for exhaustively mapping functional missense variants. Mol. Syst. Biol. 2017;13:957. PubMed PMC

Starita L.M., Ahituv N., Dunham M.J., Kitzman J.O., Roth F.P., Seelig G., Shendure J., Fowler D.M. Variant Interpretation: Functional Assays to the Rescue. Am. J. Hum. Genet. 2017;101:315–325. PubMed PMC

Sun S., Weile J., Verby M., Wu Y., Wang Y., Cote A.G., Fotiadou I., Kitaygorodsky J., Vidal M., Rine J. A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase. Genome Med. 2020;12:13. PubMed PMC

Froese D.S., Huemer M., Suormala T., Burda P., Coelho D., Guéant J.-L., Landolt M.A., Kožich V., Fowler B., Baumgartner M.R. Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency. Hum. Mutat. 2016;37:427–438. PubMed

Huemer M., Diodato D., Schwahn B., Schiff M., Bandeira A., Benoist J.-F., Burlina A., Cerone R., Couce M.L., Garcia-Cazorla A. Guidelines for diagnosis and management of the cobalamin-related remethylation disorders cblC, cblD, cblE, cblF, cblG, cblJ and MTHFR deficiency. J. Inherit. Metab. Dis. 2017;40:21–48. PubMed PMC

Huemer M., Baumgartner M.R. The clinical presentation of cobalamin-related disorders: From acquired deficiencies to inborn errors of absorption and intracellular pathways. J. Inherit. Metab. Dis. 2019;42:686–705. PubMed

Huemer M., Diodato D., Martinelli D., Olivieri G., Blom H., Gleich F., Kölker S., Kožich V., Morris A.A., Seifert B., EHOD consortium Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry. J. Inherit. Metab. Dis. 2019;42:333–352. PubMed

Rommer P.S., Zschocke J., Fowler B., Födinger M., Konstantopoulou V., Möslinger D., Stögmann E., Suess E., Baumgartner M., Auff E., Sunder-Plassmann G. Manifestations of neurological symptoms and thromboembolism in adults with MTHFR-deficiency. J. Neurol. Sci. 2017;383:123–127. PubMed

Watkins D., Rosenblatt D.S., Fowler B. Disorders of Cobalamin and Folate Transport and Metabolism. In: Saudubray J.-M., Baumgartner M.R., Walter J., editors. Inborn Metabolic Diseases: Diagnosis and Treatment. Springer; Berlin, Heidelberg: 2016. pp. 385–399.

Casas J.P., Bautista L.E., Smeeth L., Sharma P., Hingorani A.D. Homocysteine and stroke: evidence on a causal link from mendelian randomisation. Lancet. 2005;365:224–232. PubMed

Den Heijer M., Lewington S., Clarke R. Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies. J. Thromb. Haemost. 2005;3:292–299. PubMed

Clarke R., Bennett D.A., Parish S., Verhoef P., Dötsch-Klerk M., Lathrop M., Xu P., Nordestgaard B.G., Holm H., Hopewell J.C., MTHFR Studies Collaborative Group Homocysteine and coronary heart disease: meta-analysis of MTHFR case-control studies, avoiding publication bias. PLoS Med. 2012;9:e1001177. PubMed PMC

van Meurs J.B.J., Pare G., Schwartz S.M., Hazra A., Tanaka T., Vermeulen S.H., Cotlarciuc I., Yuan X., Mälarstig A., Bandinelli S. Common genetic loci influencing plasma homocysteine concentrations and their effect on risk of coronary artery disease. Am. J. Clin. Nutr. 2013;98:668–676. PubMed PMC

Blom H.J., Smulders Y. Overview of homocysteine and folate metabolism. With special references to cardiovascular disease and neural tube defects. J. Inherit. Metab. Dis. 2011;34:75–81. PubMed PMC

Smulders Y.M., Blom H.J. The homocysteine controversy. J. Inherit. Metab. Dis. 2011;34:93–99. PubMed PMC

Fezeu L.K., Ducros V., Guéant J.-L., Guilland J.-C., Andreeva V.A., Hercberg S., Galan P. MTHFR 677C → T genotype modulates the effect of a 5-year supplementation with B-vitamins on homocysteine concentration: The SU.FOL.OM3 randomized controlled trial. PLoS ONE. 2018;13:e0193352. PubMed PMC

Lek M., Karczewski K.J., Minikel E.V., Samocha K.E., Banks E., Fennell T., O’Donnell-Luria A.H., Ware J.S., Hill A.J., Cummings B.B., Exome Aggregation Consortium Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536:285–291. PubMed PMC

Karczewski K.J., Francioli L.C., Tiao G., Cummings B.B., Alföldi J., Wang Q., Collins R.L., Laricchia K.M., Ganna A., Birnbaum D.P. Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv. 2019:531210.

Guenther B.D., Sheppard C.A., Tran P., Rozen R., Matthews R.G., Ludwig M.L. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat. Struct. Biol. 1999;6:359–365. PubMed

Sibani S., Leclerc D., Weisberg I.S., O’Ferrall E., Watkins D., Artigas C., Rosenblatt D.S., Rozen R. Characterization of mutations in severe methylenetetrahydrofolate reductase deficiency reveals an FAD-responsive mutation. Hum. Mutat. 2003;21:509–520. PubMed

Shan X., Wang L., Hoffmaster R., Kruger W.D. Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae. J. Biol. Chem. 1999;274:32613–32618. PubMed

Burda P., Suormala T., Heuberger D., Schäfer A., Fowler B., Froese D.S., Baumgartner M.R. Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system. J. Inherit. Metab. Dis. 2017;40:297–306. PubMed

Yamada K., Chen Z., Rozen R., Matthews R.G. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc. Natl. Acad. Sci. USA. 2001;98:14853–14858. PubMed PMC

Liew S.-C., Gupta E.D. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: epidemiology, metabolism and the associated diseases. Eur. J. Med. Genet. 2015;58:1–10. PubMed

Marini N.J., Gin J., Ziegle J., Keho K.H., Ginzinger D., Gilbert D.A., Rine J. The prevalence of folate-remedial MTHFR enzyme variants in humans. Proc. Natl. Acad. Sci. USA. 2008;105:8055–8060. PubMed PMC

Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. PubMed PMC

Froese D.S., Kopec J., Rembeza E., Bezerra G.A., Oberholzer A.E., Suormala T., Lutz S., Chalk R., Borkowska O., Baumgartner M.R., Yue W.W. Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition. Nat. Commun. 2018;9:2261. PubMed PMC

Pejchal R., Campbell E., Guenther B.D., Lennon B.W., Matthews R.G., Ludwig M.L. Structural perturbations in the Ala--> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation. Biochemistry. 2006;45:4808–4818. PubMed PMC

Benjamini Y., Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B Stat. Methodol. 1995;57:289–300.

Suormala T., Gamse G., Fowler B. 5,10-Methylenetetrahydrofolate reductase (MTHFR) assay in the forward direction: residual activity in MTHFR deficiency. Clin. Chem. 2002;48:835–843. PubMed

Burda P., Schäfer A., Suormala T., Rummel T., Bürer C., Heuberger D., Frapolli M., Giunta C., Sokolová J., Vlášková H. Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients. Hum. Mutat. 2015;36:611–621. PubMed

Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013;8:2281–2308. PubMed PMC

Shapiro S.K., Ehninger D.J. Methods for the analysis and preparation of adenosylmethionine and adenosylhomocysteine. Anal. Biochem. 1966;15:323–333. PubMed

Ueland P.M., Rozen R. CRC Press; 2005. MTHFR Polymorphisms and Disease.

Frosst P., Blom H.J., Milos R., Goyette P., Sheppard C.A., Matthews R.G., Boers G.J.H., den Heijer M., Kluijtmans L.A., van den Heuvel L.P. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Genet. 1995;10:111–113. PubMed

Goyette P., Rozen R. The thermolabile variant 677C-->T can further reduce activity when expressed in cis with severe mutations for human methylenetetrahydrofolate reductase. Hum. Mutat. 2000;16:132–138. PubMed

Rentzsch P., Witten D., Cooper G.M., Shendure J., Kircher M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019;47(D1):D886–D894. PubMed PMC

Kircher M., Witten D.M., Jain P., O’Roak B.J., Cooper G.M., Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 2014;46:310–315. PubMed PMC

Raimondi D., Tanyalcin I., Ferté J., Gazzo A., Orlando G., Lenaerts T., Rooman M., Vranken W. DEOGEN2: prediction and interactive visualization of single amino acid variant deleteriousness in human proteins. Nucleic Acids Res. 2017;45(W1):W201–W206. PubMed PMC

Tavtigian S.V., Greenblatt M.S., Harrison S.M., Nussbaum R.L., Prabhu S.A., Boucher K.M., Biesecker L.G., ClinGen Sequence Variant Interpretation Working Group (ClinGen SVI) Modeling the ACMG/AMP variant classification guidelines as a Bayesian classification framework. Genet. Med. 2018;20:1054–1060. PubMed PMC

Hecht M., Bromberg Y., Rost B. Better prediction of functional effects for sequence variants. BMC Genomics. 2015;16(Suppl 8):S1. PubMed PMC

Auton A., Brooks L.D., Durbin R.M., Garrison E.P., Kang H.M., Korbel J.O., Marchini J.L., McCarthy S., McVean G.A., Abecasis G.R., 1000 Genomes Project Consortium A global reference for human genetic variation. Nature. 2015;526:68–74. PubMed PMC

Keller R., Chrastina P., Pavlíková M., Gouveia S., Ribes A., Kölker S., Blom H.J., Baumgartner M.R., Bártl J., Dionisi-Vici C., individual contributors of the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) Newborn screening for homocystinurias: Recent recommendations versus current practice. J. Inherit. Metab. Dis. 2019;42:128–139. PubMed

Stiffler M.A., Hekstra D.R., Ranganathan R. Evolvability as a function of purifying selection in TEM-1 β-lactamase. Cell. 2015;160:882–892. PubMed

Flynn J.M., Rossouw A., Cote-Hammarlof P., Fragata I., Mavor D., Hollins C., 3rd, Bank C., Bolon D.N. Comprehensive fitness maps of Hsp90 show widespread environmental dependence. eLife. 2020;9:e53810. PubMed PMC

Esposito D., Weile J., Shendure J., Starita L.M., Papenfuss A.T., Roth F.P., Fowler D.M., Rubin A.F. MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect. Genome Biol. 2019;20:223. PubMed PMC