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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

Abstract

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.

Keywords

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.

Notes

Acknowledgements

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

None.

Supplementary material

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

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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

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