Journal of Inherited Metabolic Disease

, Volume 40, Issue 2, pp 297–306 | Cite as

Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system

  • Patricie Burda
  • Terttu Suormala
  • Dorothea Heuberger
  • Alexandra Schäfer
  • Brian Fowler
  • D. Sean FroeseEmail author
  • Matthias R. BaumgartnerEmail author
Original Article


5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22–122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.


Missense Mutation Flavin Adenine Dinucleotide AdoMet Flavin Adenine Dinucleotide Patient Fibroblast 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Seraina Lutz for technical support with performing MTHFR enzyme analysis. This work was supported by the Rare Disease Initiative Zurich (radiz), a clinical research priority program for rare diseases of the University of Zurich, Switzerland and the Swiss National Science Foundation (SNSF 31003A_138521 and 31003A_156907).

Compliance with ethical standards

This article does not contain any studies with human or animal subjects performed by any of the authors.

Conflict of interest


Supplementary material

10545_2016_9987_MOESM1_ESM.doc (1 mb)
ESM 1 (DOC 1053 kb)


  1. Birnbaum T, Blom HJ, Prokisch H et al (2008) Methylenetetrahydrofolate reductase deficiency (homocystinuria type II) as a rare cause of rapidly progressive tetraspastic ity and psychoiss in a previously healthy adult. J Neurol 255:1845–1846Google Scholar
  2. Burda P, Schafer A, Suormala T et al (2015) Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients. Hum Mutat 36:611–621CrossRefPubMedGoogle Scholar
  3. Froese DS, Huemer M, Suormala T, et al (2016) Mutation update and review of severe MTHFR deficiency. Hum Mutat 37:427-38. doi: 10.1002/humu.22970Google Scholar
  4. Frosst P, Blom HJ, Milos R et al (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 10:111–113CrossRefPubMedGoogle Scholar
  5. Goyette P, Rozen R (2000) The thermolabile variant 677C–> T can further reduce activity when expressed in cis with severe mutations for human methylenetetrahydrofolate reductase. Hum Mutat 16:132–138CrossRefPubMedGoogle Scholar
  6. Goyette P, Sumner JS, Milos R et al (1994) Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification. Nat Genet 7:195–200CrossRefPubMedGoogle Scholar
  7. Goyette P, Frosst P, Rosenblatt DS, Rozen R (1995) Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency. Am J Hum Genet 56:1052–1059PubMedPubMedCentralGoogle Scholar
  8. Goyette P, Christensen B, Rosenblatt DS, Rozen R (1996) Severe and mild mutations in cis for the methylenetetrahydrofolate reductase (MTHFR) gene, and description of five novel mutations in MTHFR. Am J Hum Genet 59:1268–1275PubMedPubMedCentralGoogle Scholar
  9. Guenther BD, Sheppard CA, Tran P, Rozen R, Matthews RG, Ludwig ML (1999) The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat Struct Biol 6:359–365CrossRefPubMedGoogle Scholar
  10. Homberger A, Linnebank M, Winter C et al (2000) Genomic structure and transcript variants of the human methylenetetrahydrofolate reductase gene. Eur J Hum Genet 8:725–729CrossRefPubMedGoogle Scholar
  11. Huemer M, Mulder-Bleile R, Burda P, et al (2015) Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency. J Inherit Metab Dis 39:115-24. doi: 10.1007/s10545-015-9860-6Google Scholar
  12. Jacques PF, Bostom AG, Williams RR et al (1996) Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 93:7–9CrossRefPubMedGoogle Scholar
  13. Kluijtmans LA, Wendel U, Stevens EM, van den Heuvel LP, Trijbels FJ, Blom HJ (1998) Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency. Eur J Hum Genet 6:257–265CrossRefPubMedGoogle Scholar
  14. Lee MN, Takawira D, Nikolova AP et al (2009) Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli. Biochemistry 48:7673–7685CrossRefPubMedPubMedCentralGoogle Scholar
  15. Litzkas P, Jha KK, Ozer HL (1984) Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension. Mol Cell Biol 4:2549–2552CrossRefPubMedPubMedCentralGoogle Scholar
  16. Marini NJ, Gin J, Ziegle J et al (2008) The prevalence of folate-remedial MTHFR enzyme variants in humans. Proc Natl Acad Sci U S A 105:8055–8060CrossRefPubMedPubMedCentralGoogle Scholar
  17. Martin YN, Olson JE, Ingle JN et al (2006) Methylenetetrahydrofolate reductase haplotype tag single-nucleotide polymorphisms and risk of breast cancer. Cancer Epidemiol Biomarkers Prev 15:2322–2324CrossRefPubMedGoogle Scholar
  18. Matthews RG, Vanoni MA, Hainfeld JF, Wall J (1984) Methylenetetrahydrofolate reductase. Evidence for spatially distinct subunit domains obtained by scanning transmission electron microscopy and limited proteolysis. J Biol Chem 259:11647–11650PubMedGoogle Scholar
  19. Melenovska P, Kopecka J, Krijt J et al (2015) Chaperone therapy for homocystinuria: the rescue of CBS mutations by heme arginate. J Inherit Metab Dis 38:287–294CrossRefPubMedGoogle Scholar
  20. Molloy AM, Daly S, Mills JL et al (1997) Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates: implications for folate intake recommendations. Lancet 349:1591–1593CrossRefPubMedGoogle Scholar
  21. Pavlikova M, Sokolova J, Janosikova B et al (2012) Rare allelic variants determine folate status in an unsupplemented European population. J Nutr 142:1403–1409CrossRefPubMedGoogle Scholar
  22. Pejchal R, Sargeant R, Ludwig ML (2005) Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction. Biochemistry 44:11447–11457CrossRefPubMedGoogle Scholar
  23. Pejchal R, Campbell E, Guenther BD, Lennon BW, Matthews RG, Ludwig ML (2006) Structural perturbations in the Ala –> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation. Biochemistry 45:4808–4818CrossRefPubMedPubMedCentralGoogle Scholar
  24. Rady PL, Szucs S, Grady J et al (2002) Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, G1793A. Am J Med Genet 107:162–168CrossRefPubMedGoogle Scholar
  25. Shan X, Wang L, Hoffmaster R, Kruger WD (1999) Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae. J Biol Chem 274:32613–32618CrossRefPubMedGoogle Scholar
  26. Sibani S, Leclerc D, Weisberg IS et al (2003) Characterization of mutations in severe methylenetetrahydrofolate reductase deficiency reveals an FAD-responsive mutation. Hum Mutat 21:509–520CrossRefPubMedGoogle Scholar
  27. Sorensen JT, Gaustadnes M, Stabler SP, Allen RH, Mudd SH, Hvas AM (2016) Molecular and biochemical investigations of patients with intermediate or severe hyperhomocysteinemia. Mol Genet Metab 117:344–350CrossRefPubMedGoogle Scholar
  28. Sumner J, Jencks DA, Khani S, Matthews RG (1986) Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine. J Biol Chem 261:7697–7700PubMedGoogle Scholar
  29. Suormala T, Gamse G, Fowler B (2002) 5,10-Methylenetetrahydrofolate reductase (MTHFR) assay in the forward direction: residual activity in MTHFR deficiency. Clin Chem 48:835–843PubMedGoogle Scholar
  30. Tran P, Leclerc D, Chan M et al (2002) Multiple transcription start sites and alternative splicing in the methylenetetrahydrofolate reductase gene result in two enzyme isoforms. Mamm Genome 13:483–492CrossRefPubMedGoogle Scholar
  31. van der Put NM, van den Heuvel LP, Steegers-Theunissen RP et al (1996) Decreased methylene tetrahydrofolate reductase activity due to the 677C–> T mutation in families with spina bifida offspring. J Mol Med (Berl) 74:691–694CrossRefGoogle Scholar
  32. van der Put NM, Gabreels F, Stevens EM et al (1998) A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 62:1044–1051CrossRefPubMedPubMedCentralGoogle Scholar
  33. Watkins D, Rosenblatt DS (2012) Update and new concepts in vitamin responsive disorders of folate transport and metabolism. J Inherit Metab Dis 35:665–670CrossRefPubMedGoogle Scholar
  34. Watkins D, Rosenblatt DS (2014) Inherited disorders of folate and cobalamin transport and metabolism. In: Beaudet AL, Vogelstein B, Valle D et al (eds) The online metabolic and molecular bases of inherited disease. McGraw-Hill, New YorkGoogle Scholar
  35. Weisberg IS, Jacques PF, Selhub J et al (2001) The 1298A–> C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis 156:409–415CrossRefPubMedGoogle Scholar
  36. Wu BM, Tomatsu S, Fukuda S, Sukegawa K, Orii T, Sly WS (1994) Overexpression rescues the mutant phenotype of L176F mutation causing beta-glucuronidase deficiency mucopolysaccharidosis in two Mennonite siblings. J Biol Chem 269:23681–23688PubMedGoogle Scholar
  37. Yamada K, Chen Z, Rozen R, Matthews RG (2001) Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci U S A 98:14853–14858CrossRefPubMedPubMedCentralGoogle Scholar
  38. Yano H, Nakaso K, Yasui K et al (2004) Mutations of the MTHFR gene (428C > T and [458G > T + 459C > T]) markedly decrease MTHFR enzyme activity. Neurogenetics 5:135–140CrossRefPubMedGoogle Scholar

Copyright information

© SSIEM 2016

Authors and Affiliations

  • Patricie Burda
    • 1
  • Terttu Suormala
    • 1
  • Dorothea Heuberger
    • 1
    • 2
  • Alexandra Schäfer
    • 1
  • Brian Fowler
    • 1
  • D. Sean Froese
    • 1
    • 3
    Email author
  • Matthias R. Baumgartner
    • 1
    • 3
    Email author
  1. 1.Division of MetabolismUniversity Children’s HospitalZurichSwitzerland
  2. 2.Division of Surgical ResearchUniversity HospitalZurichSwitzerland
  3. 3.Radiz – Rare Disease Initiative Zurich, Clinical Research Priority Program for Rare DiseasesUniversity of ZurichZurichSwitzerland

Personalised recommendations