Carcinogenesis vol.18 no.12 pp.
2307–2311, 1997
ACCELERATED PAPER
Germ line polymorphisms in cytochrome-P450 1A1 (C4887
CYP1A1) and methylenetetrahydrofolate reductase (MTHFR)
genes and endometrial cancer susceptibility
Manel Esteller1,2, Angel Garcia3, Josep Maria Martinez-
Palones4, Jordi Xercavins4 and Jaume Reventos1,5
1Unitatde Recerca Biomedica, Centre d’Investigacions en Bioquimica i
Biologia Molecular Vall d’Hebron and Departments of 3Pathology and
4Gynecology and Obstetrics, Hospitals Vall d’Hebron, Barcelona, Spain
2Currentaddress: Tumor Biology, The Johns Hopkins Oncology Center,
424 North Bond Street, Baltimore, MD 21213, USA
5To
whom correspondence should be addressed at: Unitat de Recerca
Biomedica, Centre d’Investigacions en Bioquimica i Biologia Molecular
Vall d’Hebron, 14th Floor, Hospital Universitari Materno-Infantil Vall
d’Hebron, Pg. Vall d’Hebron 119–129, 08035, Barcelona, Spain
We describe here a case-control study to identify associ-
ations between polymorphisms at the methylenetetrahy-
drofolate reductase (MTHFR) and cytochrome P-450 1A1
(CYP1A1) genes and susceptibility to endometrial cancer.
Accordingly, genotype frequencies in 80 endometrial carcin-
oma patients were compared with frequencies in 60 con-
trols. DNA analysis suggest a significantly increased
endometrial cancer risk with an alanine to valine substitu-
tion at nucleotide 677 of MTHFR gene with an odds ratio
of 2.8 (95% confidence interval: 1.36–6.14, P J 0.002).
Moreover, the tumors from patients with the valine allele
were more undifferentiated (P J 0.03). On the other hand,
a recently described mutation in exon 7 of CYP1A1 gene
(threonine exchanged to asparagine in codon 461) showed
a strong association with endometrial cancer risk with an
odds ratio of 6.36 (95% confidence interval: 1.99–26.5,
P J 0.0004). Thus, this study suggests that polymorphisms
at MTHFR and a novel CYP1A1 variant could influence
susceptibility to endometrial cancer, although larger sample
sizes would be required to corroborate these findings.
Introduction
Classical genetic approaches for identifying susceptibility
genes, although successful for cancer with strong familial
links, have not yet identified corresponding genes for sporadic
cancers. For example, the BRCA1 and BRCA2 genes in breast
cancer and several DNA mismatch repair genes in hereditary
nonpolyposis colon cancer, although strongly associated with
familial cancer, accounts only for ,10% of nonfamilial malig-
nancies (1,2). In addition, phenotype variation between indi-
viduals carrying the same mutation in a high-penetrance gene
has been described, i.e. the severity of duodenal polyposis in
carriers of identical APC gene germ line mutations may be
influenced for a locus on chromosome 1p35-p36 (3).
Such observations have led to a number of studies attempting
to identify those low-penetrance genes that modify an indi-
*Abbreviations: MTHFR, methylenetetrahydrofolate reductase; CYP1A1,
cytochrome P-450 1A1; CYPs, phase I cytochromes P450; GSTs, phase II
glutathione-S-transferases; Thr, threonine; Asn, asparagine; Ala, alanine; Val,
valine; PCR-RFLP, polymerase chain reaction and restriction fragment length
polymorphism; OR, odds ratio; CI, confidence intervals; HNPCC, hereditary
non-polyposis colorectal carcinoma; PAHs, polycyclic aromatic hydrocarbons.
© Oxford University Press
vidual’s risk of cancer. Because sporadic cancers result from
mutations in transforming genes, and carcinogen detoxification
influences the mutational events in these key genes, several
polymorphic carcinogen-metabolism genes are potentially use-
ful candidates. In this sense, three supergene families have
attracted interest: phase I cytochromes P450 (CYPs*) and
phase II glutathione-S-transferases (GSTs) and N-acetyltrans-
ferases. In particular, certain variants at CYP1A1, CYP2D6,
CYP2E1, GSTM1, GSTT1, NAT1 and NAT2 genes have been
related with altered risk of various cancers (4).
Three polymorphisms had been described in the human
CYP1A1 gene: a Msp I RFLP in the 39-noncoding region (5),
an adenine to guanine transition in the heme-binding domain
of exon 7 (Ile .Val exchange in residue 462) (6), and an
African-American-specific RFLP in intron 7 (7). The first two
have been found overrepresented among lung cancer patients
in Japan (8), but reports in Caucasians are inconclusive (4).
Moreover, both have been related with a slightly elevated risk
of colon and postmenopausal breast cancer (9–11). In addition,
the Msp I polymorphism has been found associated with breast
cancer in African-American women (12). Recently, we have
found an enhanced endometrial cancer risk associated with the
39-end and exon 7 CYP1A1 germ line polymorphisms (13).
Finally, a new polymorphism in exon 7 of human CYP1A1
(C4887) has been recently described resulting in a threonine
(Thr) to asparagine (Asn) exchange in codon 461 in the heme
binding region of the protein (14). The significance of this
variant is still unknown, although the exchange of the adjacent
amino acid residues, isoleucine to valine, results in a change
in enzyme activity (15).
On the other hand, germ line polymorphisms in the same
oncogenes and tumor suppressor genes may also account for
the different cancer susceptibility. In this sense, rare alleles of
HRAS1 and p53 may confer an increased risk of certain types
of cancer, including breast, ovarian and endometrial cancer
(13,16–18). A disproportionally high rate of CpG . TpG
transitions is frequently observed in tumor suppressor genes
in various types of carcinomas (19) potentially due to deamin-
ation of 5-methylcytosine. In addition, methyldeficient diets
may cause imbalances in the pools of nucleotide precursors
leading to DNA strand breaks and mutations (20,21). Moreover,
due to methylenetetrahydrofolate reductase (MTHFR) gene
being a critical enzyme in the regulation of folate and methion-
ine metabolism, both of which are important factors in DNA
methylation and synthesis, germ line polymorphisms in the
MTHFR gene could influence susceptibility to cancer. In this
sense, an alanine (Ala) . valine (Val) (nucleotide 677: C .T)
germ line polymorphism of the MTHFR gene been found to
be related to the risk of colorectal cancer (22).
Although endometrial carcinoma is a common female
malignancy, relatively little attention has been given to genetic
susceptibility factors. The present study was undertaken to
examine the recently described CYP1A1 and MTHFR germ
line polymorphisms as potential molecular markers of endomet-
rial carcinoma susceptibility.
2307
M.Esteller et al.
Fig. 1. MTHFR gene polymorphism analyzed by PCR. The polymorphism
at position 677 in the MTHFR gene was studied by PCR followed by Hinf I
restriction enzyme digestion. Lane 1, mol. wt marker (pGEM/Hinf I, Rsa I
and Sin I); lane 2, water control; lane 3, mutant homozygote; lanes 4, 5
and 8, wild-type homozygotes; lanes 6 and 7, mutant heterozygotes.
Positions of the 198- and 175-base pair polymorphic DNA fragments are
shown in the right margin.
Material and methods
Study subjects
We analyzed DNA extracted from 80 unrelated Caucasian patients with
histologically proven diagnosis of endometrial carcinoma recruited in the
Department of Obstetrics and Gynecology at the Hospital Universitari Materno-
Infantil Vall d’Hebron of Barcelona. The protocol was approved by the
Institutional Review Board and informed consent was obtained from all the
patients involved in the study. None of the patients had received radiation
therapy or hormonal treatment prior to surgery. Their ages ranged from 45–
82 years. Only five patients were premenopausal and the remaining 75 were
postmenopausal. The stage distribution of the 80 patients according to the
International Federation of Gynecology and Obstetrics (F.I.G.O.) staging
system was Stage Ia (11 cases), Stage Ib (17 cases), Stage Ic (7 cases), Stage
IIa (14 cases), Stage IIb (11 cases), Stage IIc (6 cases), Stage IIIa (9 cases),
Stage IIIa (3 cases) and Stage IIIc (2 cases). Histologically, 74 of the 80
patients had endometrioid-type carcinomas whereas the remainder were four
clear cell carcinomas and two papillary serous carcinomas. Among all
surgically collected endometrial carcinomas, 40 were well-differentiated (G1),
23 were moderately differentiated (G2) and 17 were poorly differentiated
(G3). The prevalence of the MTHFR and CYP1A1 polymorphisms studied
were compared to those observed in a control group comprising 60 unrelated
women from the same region and with the same ethnic background, attending
to the Hospital Universitari Materno-Infantil Vall d’Hebron of Barcelona in
the annual gynecological cancer screening program. Controls were randomly
selected from those women who were free of clinical or histological malig-
nancy. In addition, none had any personal history of cancer. Their ages ranged
from 44–76 years. No differences were observed between cases and controls
according to age distribution (P . 0.05). All endometrial cancer patients and
control subjects were selected at Vall d’Hebron Hospital of Barcelona from
January 1993 to December 1995. DNA was extracted from fresh endometrial
tissue by proteinase K digestion and phenol/chloroform extraction (23).
Genotyping
The analysis of C.T transversion at position 677 of MTHFR, which results
in the replacement of Ala by Val, was performed using polymerase chain
reaction and restriction fragment length polymorphism (PCR-RFLP) as
described by Frosst and co-workers (24). The Hinf I RFLP analysis for
MTHFR polymorphism is illustrated in Figure 1. A single undigested band at
198 base pair represents a homozygous wild-type allele, two bands at 198-
and 175-base pairs represents the heterozygous genotype and a single band
at 175 base pair represents a homozygous rare mutant allele.
The analysis of C.T transversion at position 4887 in exon 7 of CYP1A1,
which results in the replacement of Thr by Asn at residue 461 in the heme
binding region of the enzyme, was performed using PCR-RFLP as described
by Cascorbi and co-workers (14). The Bsa I RFLP analysis for CYP1A1
polymorphism is illustrated in Figure 2. A single undigested band at 204 base
pair represents a homozygous rare mutant allele, two bands at 204- and 139-
base pairs represent the heterozygous genotype and a single band at 139 base
pair represents a homozygous wild-type allele.
Statistical analysis
The odds ratio (OR) and 95% confidence intervals (CI) were calculated as a
measure of the association between genotypes and endometrial cancer. The
StatXact-Turbo statistical package was used to obtain exact P-values. Expected
genotype frequencies were calculated by the Hardy-Weinberg equation
(15 p2 1 2pq 1 q2) from the allele frequencies.
Homozygous and heterozygous patients for the MTHFR Val variant where
2308
Fig. 2. CYP1A1 gene polymorphism analyzed by PCR. The polymorphism
at position 4887 in exon 7 of the CYP1A1 gene was studied by PCR
followed by Bsa I restriction enzyme digestion. Lane 1, mol. wt marker
(pGEM/Hinf I, Rsa I and Sin I); lane 2, water control; lanes 3, 5, 6, 7 and
8, wild-type homozygotes; lane 4, mutant heterozygote. Positions of the
204- and 139-base pair polymorphic DNA fragments are shown in the right
margin.
Table I. Association between germ line polymorphisms in CYP1A1
(Thr–Val) and MTHFR (Ala–Val) and endometrial cancer
Genotype Case no. (%) Control no. (%)
CYP1A1
Thr/Thr 55 (68.7) 56 (93.3)
Thr/Val 21 (26.2) 4 (6.7)
Val/Val 4 (5) –
ORa 6.36 (1.99–26.5) P 5 0.0004
MTHFR
Ala/Ala 25 (31.2) 34 (56.6)
Ala/Val 43 (53.7) 20 (33.3)
Val/Val 12 (15) 6 (10)
ORa 2.88 (1.36–6.14) P 5 0.002
aHeterozygous and homozygous mutant genotypes combined.
collapsed because both genotypes have significantly lower sp. act., lower
residual activity after heating and enhanced homocysteine levels compared to
the Ala homozygous genotype (24).
Results
Table I shows the frequency of MTHFR and CYP1A1 geno-
types in controls and in patients with endometrial carcinoma
and Table II shows the distribution of both rare alleles according
to clinicopathologic characteristics in endometrial carcinoma.
Within the 60 individuals tested in the control group, all
observed CYP1A1 and MTHFR genotype frequencies matched
exactly the expected percentages as calculated from the allele
frequencies.
The MTHFR Val allele, collapsing the heterozygous and
homozygous categories, was significantly associated with endo-
metrial cancer with an OR of 2.8 (95% confidence interval:
1.36–6.14, P 5 0.002). The MTHFR Val allele distribution in
endometrial cancer patients according to age at onset, F.I.G.O.
stage and histological type of the tumors was not significantly
different (Table II). However, when the results from the
endometrial cancer patients were divided into well-differenti-
ated (G1) versus moderately and poorly differentiated tumors
(G21G3), a statistically significant association between the
MTHFR Val allele and an undifferentiated cellular grade in
endometrial carcinoma was found (P 5 0.03).
Finally, a statistically strong significant association was
found between endometrial carcinoma and replacement of
threonine by asparagine at residue 461 in the heme binding
region of the CYP1A1 gene (nt 4887). The OR and 95% CI
of endometrial cancer risk for the combined genotypes of
heterozygous and homozygous rare mutant alleles was 6.36

Table II. Characteristics of patients with endometrial cancer in relation to polymorphisms of the CYP1A1 and MTHFR genes
CYP1A1 polymorphism Bsa I genotypes
Variables No. of patients w/w
Case 80
Age
ø60 23 13
.60 57 15
Histology
Endometrioid 74 50
Clear cell/UPSC 6 5
F.I.G.O. stage
I 35 24
II 1 III 45 31
Cellular grade
G1 40 31
G21G3 40 24
aP was calculated by χ2 test.
w: wild-type allele; m: mutant allele; NS: Not significant (P .0.05); UPSC: Uterine Papillary Serous Carcinoma.
(CI 1.99–26.5, P 5 0.0004). No significant differences in the
distribution of CYP1A1 rare mutant allele in endometrial
cancer patients according to age at onset, F.I.G.O. stage,
cellular differentiation grade, and histological type of the
tumors were found (Table II). Composite genotype analysis of
the codon 461-Val variant with the codon 462 and Msp I
CYP1A1 gene variants previously studied (13), reveals that
the simultaneous carriers of codon 461-codon 462 variants
possess the higher endometrial cancer risk (P 5 0.001).
In addition, the simultaneous carriers of MTHFR-Val and
CYP1A1-codon 461 Val risk variants show a higher endomet-
rial cancer risk (OR 5 16, CI 95%, 2.61–163.29, P 5 0.0003)
compared with those that possess neither of them.
Discussion
Although endometrial carcinoma is the most frequently dia-
gnosed neoplasm of the female genital tract, little is known
about genetic factors in the etiology of the disease. Several
studies have shown endometrial carcinoma to be a significant
component in a dominantly inherited cancer syndrome known
as hereditary non-polyposis colorectal carcinoma (HNPCC).
The genetic background of HNPCC involves mutations in
several genes including MSH2, MLH1, PMS1 and PMS2 (1).
In addition, epidemiologic studies have shown that a woman
who is the first-degree relative of a patient affected with
endometrial cancer has a significantly increased cancer risk.
For example, analysis of the Cancer and Steroid Hormone
Study data indicated that mothers and sisters of endometrial
cancer patients had 2.7 times the risk for endometrial cancer
than did controls (25). Some may harbor a strong hereditary
predisposition to cancer, although its identification is obscure.
To our knowledge, this is the first report of an association
of a MTHFR germ line polymorphism with endometrial cancer
risk. In this study, we found an association between the C.T
transversion at position 677 of MTHFR gene, which results
in the replacement of alanine by valine, and endometrial cancer
risk with an OR of 2.8 (95% confidence interval: 1.36–6.14,
P 5 0.002). Moreover, expanding our first finding of enhanced
endometrial cancer risk associated with two known CYP1A1
polymorphisms (13), a stronger significant association was
found between endometrial carcinoma risk and a recently
described new genetic polymorphism in CYP1A1 gene (14),
a replacement of threonine by asparagine at residue 461 in the
CYP1A1 and MTHFR genes and endometrial cancer
MTHFR polymorphism Hinf 1 genotypes
w/m 1 m/m Pa w/w w/m 1 m/m Pa
NS
10 6 17 NS
19 38
NS NS
24 22 52
1 4 2
NS NS
11 14 21
14 11 34
NS NS
9 8 32
16 17 23 P 5 0.03
heme binding region in exon 7, with an OR of 6.36 (CI 1.99–
26.5, P 5 0.0004).
Germ line variants of the MTHFR gene could be
involved in endometrial cancer risk by altering DNA
methylation or by influencing the rate of DNA mutation.
The MTHFR mutation studied (677C.T Ala-to-Val)
causes reduced enzyme activity (24). A decrease in the
product of the MTHFR gene, 5-methylenetetrahydrofolate
as (5-methylTHF), could contribute to carcinogenesis
5-methylTHF provides the methyl group for de novo
methionine synthesis and DNA methylation (26). Abnormalit-
ies of DNA methylation are one of the most consistent
molecular changes in human cancers. The alterations consist,
simultaneously, of increased potential capacity for DNA
methylation, widespread genomic hypomethylation, and more
regional areas of hypermethylation; in this sense, selective
growth and transformation of cells can result from DNA
hypomethylation of protooncogenes or hypermethylation of
tumor suppressor genes (27). Particularly for endometrial
cancer, atypical distribution and increased levels of protein
carboxyl methyltransferase (involved in methylation of ras
and other GTP-binding proteins) (28,29) and abnormal
methylation of estrogen receptor gene (30) have been
described.
In addition, an imbalance of the MTHFR substrate, 5,10-
methylenetetrahydrofolate (5,10-methyleneTHF), interferes
with thymidylate biosynthesis, leading to accumulation of
deoxyuridylate in DNA (31). Removal of this abnormal
base might labilize DNA to strands breaks (32) and
chromosome breaks appear to be important in nearly all
human cancers (33.).
Finally, our finding that a recently described CYP1A1
genetic polymorphism is associated with endometrial cancer
risk, could be related with the known involvement of estrogens
in both initiation and promotion of endometrial cancer. In
premenopausal women, persistent anovulation due to the
polycystic ovary syndrome (34) and ovarian neoplasia
(granulosa cell tumor, thecal cell tumor or adrenocortical
hyperplasia) often causes an estrogen-predominant milieu
associated with the occurrence of endometrial cancers; in
addition, adipose tissues contribute to the formation of extra-
glandular estrogen (mainly estrone), which is relevant for
tumor risk factor, especially in perimenopause (35). Due to
2309
M.Esteller et al.
the fact that estrogen metabolism is partially determined by
cytochrome P450 activity and is under the genetic control of
both the CYP1A1 and CYP1A2 genes, the new CYP1A1
genetic polymorphism studied may influence the production
of estrogen 2-hydroxylated metabolites (36) and therefore
individual susceptibility to endometrial cancer.
In a parallel way, the individuals who inherit the rare
CYP1A1 genotypes could suffer alterations in the metabolism
of polycyclic aromatic hydrocarbons (PAHs) (known human
carcinogens found in tobacco, smoke, food and combustion
fumes) to reactive intermediates with mutagenic activity (37),
contributing perhaps also to the higher endometrial cancer risk
detected. In this sense, PAHs have a high capacity for adduct
formation in human cancer cells (38) and benzo[a]pyrene-
DNA adducts have been identified in human endometrium
(39). In addition, PAHs metabolites have been shown to
activate the H-ras protooncogene in vitro (40), a gene closely
related to K-ras, which is found to be mutated in about one
quarter of all sporadic endometrial carcinomas (41) and which
is thought to be altered early in the progression from endomet-
rial hyperplasia to endometrial carcinoma (42,43). Finally, we
should point out that PAHs are lipophilic and stored in adipose
tissue, and obesity is an epidemiological classic risk factor for
endometrial carcinoma (44).
In conclusion, our preliminary data are in agreement with
a genetic susceptibility to endometrial cancer associated with
a MTHFR germ line polymorphism and a recently described
rare CYP1A1 variant. In addition to suggest the contribution
of polymorphisms in DNA methylation-related genes and
carcinogen-metabolism genes to an enhanced cancer risk, this
study could provide a link with the epidemiological association
between estrogen exposure, obesity and endometrial cancer.
Therefore, studies in larger populations of sporadic endometrial
cancer cases, in families with endometrial cancer aggregation
with different phenotypes, and in functional assesment of the
genotypes described, are in progress.
Acknowledgements
This work was supported in part by the Institut Catala de la Salut and Fondo
de Investigaciones Sanitarias (Grant FIS 95/0501). Manel Esteller is a fellow
of Spanish Ministerio de Educacion y Ciencia, Universitat Rovira i Virgili.
We thank Dr Lluis Armadans for his help with the statistical analysis of the
data and Mrs T.Berry for correction of the manuscript.
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Received on March 21, 1997; revised on July 4, 1997; accepted on August

19, 1997

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