The prevalence of folate-remedial MTHFR enzyme variants in humans

Abstract

Studies of rare, inborn metabolic diseases establish that the phenotypes of some mutations in vitamin-dependent enzymes can be suppressed by supplementation of the cognate vitamin, which restores function of the defective enzyme. To determine whether polymorphisms exist that more subtly affect enzymes yet are augmentable in the same way, we sequenced the coding region of a prototypical vitamin-dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), from 564 individuals of diverse ethnicities. All nonsynonymous changes were evaluated in functional in vivo assays in Saccharomyces cerevisiae to identify enzymatic defects and folate remediability of impaired alleles. We identified 14 nonsynonymous changes: 11 alleles with minor allele frequencies <1% and 3 common alleles (A222V, E429A, and R594Q). Four of 11 low-frequency alleles affected enzyme function, as did A222V. Of the five impaired alleles, four could be restored to normal functionality by elevating intracellular folate levels. All five impaired alleles mapped to the N-terminal catalytic domain of the enzyme, whereas changes in the C-terminal regulatory domain had little effect on activity. Impaired activity correlated with the phosphorylation state of MTHFR, with more severe mutations resulting in lower abundance of the phosphorylated protein. Significantly, diploid yeast heterozygous for mutant alleles were impaired for growth, particularly with lower folate supplementation. These results suggested that multiple less-frequent alleles, in aggregate, might significantly contribute to metabolic dysfunction. Furthermore, vitamin remediation of mutant enzymes may be a common phenomenon in certain domains of proteins.

Fig. 1.

Effects of folinic acid supplementation on growth rate of fol3Δ cells and cellular activity of human MTHFR. (a) Growth of fol3Δ MET13 haploid yeast was measured in 96-well plates as described in Materials and Methods. Media was supplemented with folinic acid at the indicated concentrations. The curve labeled FOL3 (FOL3 MET13) was from growth in medium without folinic acid. (b) Growth of fol3Δmet13Δ haploid yeast transformed with phMTHFR in media lacking methionine and supplemented with folinic acid at the indicated concentrations. Three independent transformants were tested at each folinic acid concentration to test reproducibility. The curve labeled met13Δ represented a single isolate of cells, which were transformed with empty vector and grown at 50 μg/ml folinic acid.

Fig. 2.

Functional impact and folate remediability of nonsynonymous MTHFR population variants. (a) Six MTHFR variants were tested for the ability to rescue fol3Δ met13Δ cells in media lacking methionine at three different folinic acid concentrations. The M110I allele and the M110I A222V doubly substituted allele were tested only at 50 and 25 μg/ml folinic acid. The curve labeled Major corresponds to the most common MTHFR allele in the population. Each curve is from a pool of three to six independent transformants. (b) Schematic of the MTHFR protein (656 aa) divided into a N-terminal catalytic domain and a C-terminal regulatory domain of nearly equal size (25). Positions of all nonsynonymous changes are indicated. Benign changes are in green. Changes numbered 1–4 represent folate-remedial alleles indicated in increasing order of severity. Change 5 (R134C) was nearly loss-of-function and not designated as folate remedial (see Results) but was somewhat folate augmentable.

Fig. 3.

Enzyme activity of MTHFR variants. Crude yeast extract from cells transformed with the indicated MTHFR constructs was prepared and assayed for MTHFR activity as described in Materials and Methods. Heat treatment for the indicated times was done on reactions before addition of radiolabeled substrate. Measurements were averages of two independent sets of triplicate assays; error bars indicate standard deviations for the six data points.

Fig. 4.

Heterozygote phenotypes for MTHFR variants as recapitulated in yeast. Homozygosity or heterozygosity of MTHFR alleles was recreated in diploid yeast for the major, R134C, and A222V alleles as described in Materials and Methods. Diploids were obtained from the mating of haploid strains that each expressed a single allele of MTHFR integrated in the genome. Growth as a function of folinic acid supplementation was assayed exactly as for haploids.

Fig. 5.

Immunoblot of human MTHFR variants expressed in yeast. (a) Extracts were made from yeast cells carrying different MTHFR alleles and detected with anti-HA antibody as in Materials and Methods. A222V M110I was a doubly substituted allele; Major indicates the most common MTHFR allele in the population. The two rightmost lanes were, side-by-side, the major allele and the nonphosphorylatable T34A mutant (26). (b) The ratio of signal intensities of the unphosphorylated lower band to the phosphorylated upper band for all variants of MTHFR identified in this study plotted as a function of increasing severity of functional impact. Alleles on the x axis were classified as benign or rank ordered with respect to activity. All benign alleles (including the Major allele and all regulatory domain changes) were plotted and show nearly identical ratios of the two MTHFR species, thus the symbols overlapped.