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The prevalence of folate-remedial MTHFR enzymevariants in humans
Nicholas J. Marini*
†
, Jennifer Gin*, Janet Ziegle
‡
, Kathryn Hunkapiller Keho
‡
, David Ginzinger
‡§
, Dennis A. Gilbert
‡
,and Jasper Rine*
†
*Department of Molecular and Cellular Biology, California Institute for Quantitative Biosciences, Stanley Hall, University of California,Berkeley, CA 94720-3220; and
‡
Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, CA 94404Communicated by Bruce N. Ames, University of California, Berkeley, CA, March 24, 2008 (received for review November 20, 2007)
Studies of rare, inborn metabolic diseases establish that the phe-notypes of some mutations in vitamin-dependent enzymes can besuppressed by supplementation of the cognate vitamin, whichrestores function of the defective enzyme. To determine whetherpolymorphisms exist that more subtly affect enzymes yet areaugmentable in the same way, we sequenced the coding region ofa prototypical vitamin-dependent enzyme, methylenetetrahydro-folate reductase (MTHFR), from 564 individuals of diverse ethnici-ties. All nonsynonymous changes were evaluated in functional
invivo
assays in
Saccharomyces cerevisiae
to identify enzymaticdefects and folate remediability of impaired alleles. We identified14 nonsynonymous changes: 11 alleles with minor allele frequen-cies
<
1% and 3 common alleles (A222V, E429A, and R594Q). Fourof 11 low-frequency alleles affected enzyme function, as didA222V. Of the five impaired alleles, four could be restored tonormalfunctionalitybyelevatingintracellularfolatelevels.Allfiveimpaired alleles mapped to the N-terminal catalytic domain of theenzyme,whereaschangesintheC-terminalregulatorydomainhadlittle effect on activity. Impaired activity correlated with the phos-phorylation state of MTHFR, with more severe mutations resultingin lower abundance of the phosphorylated protein. Significantly,diploid yeast heterozygous for mutant alleles were impaired forgrowth, particularly with lower folate supplementation. Theseresults suggested that multiple less-frequent alleles, in aggregate,might significantly contribute to metabolic dysfunction. Further-more, vitamin remediation of mutant enzymes may be a commonphenomenon in certain domains of proteins.
nutrigenetics
polymorphism
vitamin
T
he ability of enzyme cofactors to remedy metabolic disease-causing mutations has been well documented over the last fourdecades(foroverviewsofthefield,seerefs.1and2).Inmanycases,the etiological lesion results in a missense change in a cofactor-dependent enzyme, resulting in disruption of a single metabolicstep.Subsequentadministrationoftherapeuticdosesofthecognatecofactor restores some activity of the mutant enzyme, leading toclinical improvement. Cofactor-remedial mutations occur in manydifferentenzymesthatusevariouscofactors(2).Forexample,thereare numerous vitamin B
6
(pyridoxine)-responsive disorders thatresult from mutations in specific B
6
-dependent enzymes (2, 3). Inaddition, the phenylalanine hydroxylase (PAH) cofactor tetrahy-drobiopterin (BH
4
) can be used to treat phenylketonuria caused byseveral different missense mutations in PAH (4). The molecularmechanisms that underlie clinical remediation are the ability of elevatedlevelsofcofactortoeitherovercomebindingdefectsofK
m
mutants (2) or to serve as a chemical chaperone to improve foldingand/or stability of mutant enzyme variants (2–5).Perhaps the best-studied case is the common folate-remedialpolymorphism (677C
3
T; A222V) of 5,10-methylenetetrahydrofo-late reductase (MTHFR; ref. 6). For this enzyme, folate is not acofactorinthetraditionalsensebutinsteadcontrolsthelevelofthesubstrate 5,10-methylenetetrahydrofolate. The A222V change re-duces MTHFR activity and increases its thermolability, which canlead to lower levels of serum folate, increased levels of plasmahomocysteine, and genomic DNA hypomethylation, particularly inthose with a diet low in folate (reviewed in ref. 7). Biochemically,the A222V variant may be less tightly bound to its flavin cofactorandmorepronetodissociationintomonomersbutcanbestabilizedby reduced folates (8, 9).Because the A222V variant can affect clinical biomarkers, suchas serum homocysteine, and can be modulated by folate levels, thisallele has been the subject of many epidemiological disease studiesfor which these biomarkers may be risk factors and for which folateis thought to be chemopreventive or therapeutic [e.g., neural tubedefects(NTDs)(10),cardiovasculardisease(ref.11,butseeref.12),and colon cancer (13)]. However, genetic association of the A222Vallele to disease within all these clinical settings has not beenconsistent (10–14). Among the issues that can confound association studies (15), thegene–nutrientinteractionbetweenA222Vactivityandfolatestatusmay affect the outcome and conclusions of such studies. Indeed, A222V is a risk factor for colon cancer when dietary folate intakesare low but may actually be protective when folate intakes areelevated (13). A second issue relevant to our work is the recentappreciation of the importance of low frequency (allele frequency
1%) nonsynonymous variants as potential genetic determinantsin common diseases such as cardiovascular disease and obesity(16–18). However, the potential confounding influence of lowfrequency variants to disease susceptibility has not been addressedin most association studies.We have used MTHFR as a prototype vitamin-dependent en-zyme to answer several questions about the occurrence and bio-chemical impact of low-frequency variation in coding regions, andmostimportantly,theprevalenceoffolate-remediationoffunction-allyimpairedalleles.Tothisend,wesequencedtheMTHFRcodingregionfrom
500individualsofdiverseethnicitiesanddeterminedthebiochemicalimpactofallnonsynonymouschangesasafunctionof intracellular folate status by complementation in the yeast
S. cerevisiae
. The results established that many nonsynonymous sub-stitutions decreased the function of the enzyme and that folate-remediation of impaired alleles was surprisingly common. Further-more, because cells heterozygous for MHTFR variants displayedquantitative defects, the aggregate frequencies of individually rarealleles may contribute to common phenotypes.
Results
MTHFR Variants in Humans.
The entire coding region of humanMTHFR was sequenced by amplifying the coding portion in each
Author contributions: N.J.M., D.A.G., and J.R. designed research; N.J.M., J.G., J.Z., andK.H.K.performedresearch;J.Z.,K.H.K.,D.G.,andD.A.G.contributednewreagents/analytictools;N.J.M.,J.G.,J.Z.,K.H.K.,D.G.,D.A.G.,andJ.R.analyzeddata;andN.J.M.andJ.R.wrotethe paper.The authors declare no conflict of interest.
†
To whom correspondence may be addressed. E-mail: nmarini@berkeley.edu or jrine@berkeley.edu.
§
Present address: Linkage Biosciences, 2503 Bush Street, San Francisco, CA 94115.This article contains supporting information online atwww.pnas.org/cgi/content/full/ 0802813105/DCSupplemental.© 2008 by The National Academy of Sciences of the USA
www.pnas.org
cgi
doi
10.1073
pnas.0802813105 PNAS
June 10, 2008
vol. 105
no. 23
8055–8060
G E N E T I C S
of 11 exons from 564 individuals of diverse ethnicities. The lengthsof the coding regions, the number of alleles interrogated, and allnonsynonymous substitutions are listed in Table 1. In all, weanalyzed 2,081,106 bp of coding DNA and sampled every exon toa depth of
1,000 alleles. These data revealed 14 nonsynonymouschanges, 11 of which show a minor allele frequency (MAF)
1%, with seven alleles seen only once. Some low-frequency alleles wereseen previously (dbSNP entry in Table 1). The number of low-frequency nonsynonymous substitutions was in good agreement with other studies that sampled deeply into random populations(19–21). In addition, three well studied common substitutions wereobservedthatdisplayedtheexpectedglobalpopulationfrequencies(A222V, 29.3%; E429A, 23.6%; R594Q, 4.4%). Asaquality-controlcheckontheaccuracyofthebase-calling,wereanalyzed eight variants (including four singletons) by TaqManallelic-discriminationassaysin100samplesthatwereindependentlyPCR amplified and saw 100% concordance of the data. Further-more, population genotyping data from the Environmental Ge-nome Project (www.niehs.nih.gov/envgenom/) and Perlegen, whichboth used Coriell Institute Cell Repository (Camden, NJ) samplesthat overlap some in this study (dbSNP build 128) were in concor-dance in 814 of 817 (99.6%) genotype calls. For two of the threediscordant loci, our sequence data were unambiguous and ap-peared correct.We obtained complete coding region sequence for 480 individ-uals. Eighteen (4%) were carriers of a low-frequency nonsynony-mous variant. Significantly, the combination of the three commonpolymorphisms(A222V,E429A,andR594Q)withtherangeofthelow frequency changes led to a great deal of individual heteroge-neity. We observed 28 different nonsynonymous genotypes in thisgroup whose haplotype, in most cases, could not be deduced fromthe data. The genotypes of individual Coriell samples at all non-synonymous variant loci are included insupporting information(SI) Dataset S1.
MTHFR-FolateInteraction
In Vivo
.
Becausetheclinicalsignificanceof genetic variants lies in their functional consequence, we tested allnonsynonymous changes for their effect on MTHFR function, andimportantly, whether impaired alleles displayed folate responsive-ness. The assay is based on complementation of
met13
, whichencodes yeast MTHFR, by human MTHFR (22) but with amodification that allowed us to study the gene–nutrient interactionbetween MTHFR and folate. Thus, we introduced folate auxotro-phy (
fol3
) into a
met13
strain, allowing titration of intracellularfolate concentrations by varying folinic acid in the growth media.Folinicacid(5-formyl-tetrahydrofolate)canbemetabolizedinyeastto methenyl-tetrahydrofolate, which in turn can be converted toother folate coenzymes (23). In this way, we measured humanMTHFR functionality (growth in the absence of methionine) as afunction of increasingly limiting cellular folate status.Under these conditions, folinic acid supplementation
50
g/mldidnotconferanysignificantgrowthadvantage(Fig.1
a
).However,at concentrations
50
g/ml, growth clearly correlated with avail-ablefolinicacidinthemedium.Thusintracellularfolatelevelswereratelimitinginthisrange.Whencomparedtogrowthof
FOL3
cells,folinic acid supplementation did not completely compensate forlack of endogenous folate biosynthesis. However, this gap wasmostly reflected in the density at which cells entered stationaryphase rather than growth rate, perhaps reflecting limitations infolinic acid uptake or in the utilization of folinic acid as the solefolate source.The ability of human MTHFR to complement
fol3 met13
cells was a function of folinic acid supplementation in the media (Fig.1
b
). As for folate supplementation, expression of human MTHFRfrom the
GAL1
promoter did not completely compensate for lossof Met13p (compare Fig. 1
b
with
fol3 MET13
cells at equivalentfolatedosesinFig.1
a
).Thus,whenfolinicacidwas
50
g/ml,bothfolate and MTHFR were rate limiting for growth, allowing evensubtle changes in MTHFR activity to be reflected in the growthreadout. Note that folinic acid supplementation of
50
g/ml didnot confer a significant growth advantage to cells expressing eitherthe endogenous yeast MTHFR (
MET13
; Fig. 1
a
) or the majorhuman allele (Fig. 1
b
) but was beneficial for impaired alleles of MTHFR (see below).
Functional Impact of MTHFR Variants.
Five nonsynonymous allelestested over a range of folate concentrations illustrated the range of functional effects observed (Fig. 2
a
). There was nearly completerestoration of function of the A222V variant at 100
g/ml folinicacid and significantly less activity (relative to the major allele) at a
Table 1. Spectrum of nonsynonymous MTHFR alleles observed from sampling
>
500 unselectedindividuals of diverse ethnicities
Exon Length, bpAllelessequenced Variant Occurrences* dbSNP rs no.1 236
†
1,070 None2 239 1,016 M110I 1 Previously unrecognizedR134C 1 455501333 111 1,068 None4 194 1,050 A222V 308 1801133H213R 1 Previously unrecognizedD223N 1 Previously unrecognized5 251 1,056 D291N 1 Previously unrecognized6 135 1,042 None7 181 1,062 E429A 251 1801131G422R 3 455717368 183 1,058 None9 102 1,072 R519C 2 45496998R519L 2 Previously unrecognized10 120 1,072 M581I 1 4559083611 219
†
1,076 R594Q 47 2274976T653M 4 35737219Q648P 1 Previously unrecognized
†
For exons 1 and 11, only the length of the coding portion of the exon is given.
8056
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cgi
doi
10.1073
pnas.0802813105 Marini
et al.
4-fold-lowerlevelofsupplementation(25
g/ml).Thus,undertheseconditions we recapitulated the known folate remediability of the A222V defect. The exact intracellular concentrations of reducedfolates in yeast under these conditions was unknown. Nevertheless,the behavior of the A222V allele effectively calibrated the intra-cellular concentrations in yeast and human cells. The A222Venzyme has
50% the intrinsic activity of common allele (19, 24),and we observed a 50% reduction in growth rate at 50
g
/ml folatesupplementation.Furthermore,weobservedthesame50%dropin A222V enzyme activity in cell-free assays from cells grown at 50
g/ml folinic acid (Fig. 3). Thus, the behavior of A222V in yeastrecapitulated its behavior in human cells.Four low-frequency alleles were tested in the same way (Fig. 2
a
).R519Cappearedbenignbecausegrowthwasunaffectedatallfolateconcentrations. R134C was severely impaired at all folate concen-trations, although activity was somewhat folate-responsive. TheD223N and M110I alleles displayed folate-remedial activity similarto A222V (although less severely impaired) in that growth wassimilar to the major allele at
50
g/ml folinic acid, but functionedpoorly at
50
g/ml folinic acid.The MTHFR enzyme has an N-terminal catalytic domain anda C-terminal regulatory domain, which binds the allostericinhibitor
S
-adenosylmethionine (AdoMet; ref. 25). Of the sixalleles that fell within the catalytic domain (M110I, R134C,H213R, A222V, D223N, and D291N), only H213R was benign(Fig. 2
b
). M110I, A222V, D223N, and D291N displayed folate-remedial behavior in that these enzyme variants were similar tothe major allele at higher concentrations of folate supplemen-tation (50–200
g/ml folinic acid) but were considerably weak-ened as folate became more rate limiting. The R134C variantnever approached the capacity of the major allele to supportgrowth at any level of folate supplementation and hence wasclassified as a responsive but not a remedial allele. All substi-tutions within the regulatory domain (from G422R throughT653M) behaved similarly to the major allele (Fig. 2
b
).
Synergistic Interactions Between Amino Acid Substitutions.
The dis-tribution of variants implied the existence of compound alleles
ba
00.10.20.30.40.50.601020304050607080
25
µ
g/ml Folinic Acid12
µ
g/ml Folinic Acid50
µ
g/ml Folinic Acid
met13
∆
50
µ
g/ml Folinic Acid
time (h)
A 5 9 5
0.00.20.40.60.801020304050607080time (h)
A 5 9 5
1.0
100
µ
g/ml50
µ
g/ml25
µ
g/ml12
µ
g/ml6
µ
g/ml3
µ
g/ml1.5
µ
g/ml0
µ
g/ml
FOL3FOL3MET13fol3MET13fol3met13fol3 met13
phMTHFRvector
Fig. 1.
Effects of folinic acid supplementation on growth rate of
fol3
cellsand cellular activity of human MTHFR. (
a
) Growth of
fol3
MET13
haploidyeast 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 withoutfolinic acid. (
b
) Growth of
fol3
met13
haploid yeast transformed withphMTHFRinmedialackingmethionineandsupplementedwithfolinicacidatthe indicated concentrations. Three independent transformants were testedat each folinic acid concentration to test reproducibility. The curve labeled
met13
represented a single isolate of cells, which were transformed withempty vector and grown at 50
g/ml folinic acid.
Catalytic DomainRegulatory Domain1
342
5
b
00.10.20.30.40.50 10 20 30 40 50 60
MajorR134CA222VD223NR519C
0 10 20 30 40 50 60100
µ
g/ml Folinic Acid 50
µ
g/ml Folinic Acid 25
µ
g/ml Folinic Acidtime (h)
A 5 9 5
a
00.10.20.30.40.5
MajorR134CA222VD223NR519CM110IM110I A222V
0 10 20 30 40 50 6000.10.20.30.40.5
MajorR134CA222VD223NR519CM110IM110I A222V
N 3 2 2 D N 1 9 2 D V 2 2 2 A I 0 1 1 M C 4 3 1 R
Fig. 2.
FunctionalimpactandfolateremediabilityofnonsynonymousMTHFRpopulationvariants.(
a
)SixMTHFRvariantsweretestedfortheabilitytorescue
fol3
met13
cellsinmedialackingmethionineatthreedifferentfolinicacidconcentrations.TheM110IalleleandtheM110IA222Vdoublysubstitutedalleleweretestedonly at 50 and25
g/mlfolinicacid.The curve labeledMajor corresponds tothe most commonMTHFRallele inthe population.Eachcurve is from a poolof three tosixindependenttransformants.(
b
)SchematicoftheMTHFRprotein(656aa)dividedintoaN-terminalcatalyticdomainandaC-terminalregulatorydomainofnearlyequalsize(25).Positionsofallnonsynonymouschangesareindicated.Benignchangesareingreen.Changesnumbered1–4representfolate-remedialallelesindicatedinincreasingorderofseverity.Change5(R134C)wasnearlyloss-of-functionandnotdesignatedasfolateremedial(see
Results
)butwassomewhatfolateaugmentable.
Major R134C
A222V
D223N R519C100
200
300
p m o l e s / m g p r o t e i n / h
no heat55
o
, 10'55
o
, 20'
Fig. 3.
Enzyme activity of MTHFR variants. Crude yeast extract from cellstransformed with the indicated MTHFR constructs was prepared and assayedforMTHFRactivityasdescribedin
MaterialsandMethods
.Heattreatmentforthe indicated times was done on reactions before addition of radiolabeledsubstrate.Measurementswereaveragesoftwoindependentsetsoftriplicateassays; error bars indicate standard deviations for the six data points.
Marini
et al.
PNAS
June 10, 2008
vol. 105
no. 23
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