Gene structure of human and mouse methylenetetrahydrofolate
Philippe Goyette, Aditya Pai, Renate Milos, Phyllis Frosst, Pamela Tran, Zhoutao Chen, Manuel Chan,
Departments of Human Genetics, Pediatrics, and Biology, McGill University Health Center, Montreal, Quebec, Canada H3H 1P3
Received: 28 January 1998 / Accepted: 9 April 1998
Abstract. Methylenetetrahydrofolate reductase (MTHFR) cata-
lyzes the conversion of 5,10-methylenetetrahydrofolate to 5-
methyltetrahydrofolate, a co-substrate for homocysteine remeth-
ylation to methionine. A human cDNA for MTHFR, 2.2 kb
in length, has been expressed and shown to result in a catalyti-
cally active enzyme of approximately 70 kDa. Fifteen muta-
tions have been identified in the MTHFR gene: 14 rare mutations
associated with severe enzymatic deficiency and 1 common vari-
ant associated with a milder deficiency. The common polymor-
phism has been implicated in three multifactorial diseases: occlu-
sive vascular disease, neural tube defects, and colon cancer. The
human gene has been mapped to chromosomal region 1p36.3
while the mouse gene has been localized to distal Chromosome
(Chr) 4. Here we report the isolation and characterization of
the human and mouse genes for MTHFR. A human genomic
clone (17 kb) was found to contain the entire cDNA sequence of
2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp.
Intron sizes ranged from 250 bp to 1.5 kb with one exception of
4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5Ј exon 1 and
extend to the end of the coding sequence. The mouse amino acid
sequence is approximately 90% identical to the corresponding
human sequence. The exon sizes, locations of intronic boundaries,
and intron sizes are also quite similar between the two species.
The availability of human genomic clones has been useful in de-
signing primers for exon amplification and mutation detection. The
mouse genomic clones will be helpful in designing constructs
for gene targeting and generation of mouse models for MTHFR
Methylenetetrahydrofolate reductase (MTHFR) plays a major role
in the metabolism of folates. It converts 5,10-methylenetetra-
hydrofolate, a carbon donor in nucleotide biosynthesis, to 5-
methyltetrahydrofolate, a carbon donor in the remethylation of
homocysteine to methionine. Severe and mild deficiencies of
MTHFR have been described, and a wide spectrum of clinical
symptoms has been reported. In severe MTHFR deficiency, with
hyperhomocysteinemia and homocystinuria, clinical features in-
clude peripheral neuropathy, developmental delay, hypotonia, sei-
zures, and thromboses (Rosenblatt 1995). Biochemical findings
and the age of onset of symptoms correlate reasonably well with
the residual enzyme activity (0–20% of control values). A milder
form of MTHFR deficiency, characterized by a thermolabile en-
zyme with reduced specific activity (35%–50% of control values),
is present at high frequency in the general population and is asso-
ciated with mild hyperhomocysteinemia, an independent risk fac-
tor for occlusive arterial disease (Kang et al. 1991; Engbersen et al.
1995; Frosst et al. 1995).
We have previously reported the isolation and expression of a
2.2-kb cDNA encoding human MTHFR (Goyette et al. 1994;
Frosst et al. 1995). We have mapped the human gene to chromo-
somal region 1p36.3 (Goyette et al. 1994) and the mouse gene to
distal Chr 4 (Frosst et al. 1996). We have identified 14 rare mu-
tations in homocystinuric patients with severe MTHFR deficiency
(Goyette et al. 1994, 1995, 1996), and a common polymorphism,
C677T, which converts an alanine codon to valine (Frosst et al.
1995). This common polymorphism, which is present on approxi-
mately 35% of alleles in the North American population, encodes
the thermolabile variant and predisposes to mild hyperhomocys-
teinemia when folate status is low (Frosst et al. 1995; Jacques et al.
1996; Christensen et al. 1997). This genetic-nutrient interactive
effect is believed to be a risk factor for arteriosclerosis (Frosst et
al. 1995; Kluijtmans et al. 1996; Gallagher et al. 1996; Christensen
et al. 1997; reviewed in Rozen 1997) and neural tube defects (van
der Put et al. 1995; Whitehead et al. 1995). In contrast, the mutant
homozygous genotype may decrease the risk for colon cancer
(Chen et al. 1996; Ma et al. 1997).
In this communication, we report the characterization of the
genomic structure for human MTHFR. We also show the corre-
sponding analysis of the mouse gene, with a comparison of the
overall organization of the gene and the amino acid sequences in
these two species.
Materials and methods
Screening of genomic libraries.
Genomic libraries were screened by
standard methods of plaque hybridization (Sambrook et al. 1989). The
2.2-kb human cDNA was radiolabeled and used as a probe in screening
both human and murine genomic libraries. Screening for the human gene
was performed on a phage library of partial EcoRI digestion fragments
from total genomic DNA (ATCC # 37385), and on a phage library of
Chromosome (Chr) 1-specific complete EcoRI digestion fragments (ATCC
# 57738). Screening for the mouse gene was performed on a DASH
library of partial Sau3 A digestion fragments from total genomic DNA of
mouse strain 129SV (obtained from J. Rossant, University of Toronto).
Positive clones were purified by sequential rounds of screening and isola-
tion, and phage DNA was isolated with phage DNA isolation columns
(QIAGEN, Chatsworth, CA). Human clones were digested with EcoRI to
release the inserts, and then with XbaI to facilitate cloning into Bluescript
plasmid (Stratagene, La Jolla, CA). The mouse clones were digested with
SalIorEcoRI, and the inserts were subcloned into Bluescript.
Characterization of mouse cDNA sequences.
Mouse genomic clones
were sequenced (Sequenase kit, Amersham, Oakville, ON) with human
cDNA primers spanning most of the available 2.2-kb cDNA. These se-
quences were then used to generate mouse-specific cDNA primers. The
mouse-specific primers were used in PCR amplification of overlapping
Correspondence to: R. Rozen at McGill University-Montreal Children’s
Hospital Research Institute, 4060 St-Catherine Street West, Room 242,
Montreal, Quebec H3Z 2Z3, Canada
Mammalian Genome 9, 652–656 (1998).
© Springer-Verlag New York Inc. 1998