[CANCER RESEARCH56, 4965-4969, November 1. 19961
A C4887A Polymorphism in Exon 7 of Human CYP1AJ: Population Frequency,
Mutation Linkages, and Impact on Lung Cancer Susceptibility1
Ingoif Cascorbi,2Jurgen Brockmöller,and Ivar Roots
Institute
of Clinical
Pharmacology,
University
Clinic
Charite,
Humboldt
University
of Berlin,
ABSTRACT
This study reports a C—+Atraasversion at position 4887 in exon 7 of
cytochrome
P4501A1 (CYPJAJ), resulting in a threonine—iisparagine
exchange in codon 461. The polymorphism
is located directly beside the
known codon 462-ile/Val mutation (m2) near the heme binding region.
Schumannstrasse
20,2!,
D-ltX198
Berlin,
Germany
so that we could suggest a classification of CYPJA 1 alleles. Moreover,
the role of m4 in lung cancer susceptibility has been evaluated.
SUBJECTS
AND METHODS
The C4887A mutation leads to the loss of a BsaI cleavage site, which
allows analysis. No linkage of this mutation, termed m4, with other
mutations such as ml (MspI polymorphism in the 3'-flanking region) or
m2 was observedon the sameDNA strand. Systematicmolecular genetic
analyses of mutation linkages revealed that mutation m2 is in strict
linkage disequilibrium with ml. To distinguish the different CYPJAJ
alleles and genotypes, mutation linkages were considered.
Frequency of the m4-contalning allele, termed CYPJAJ*4, among 880
Subjects. A group of 880 individuals [588 males (median age, 57 years)
and 292 females (median age, 61 years)] composed of 304 healthy volunteers
and 576 patients recruited from the Departments of Internal Medicine (Uni
versity Clinic Benjamin Franklin, Free University of Berlin, and University
unrelated
urological diagnoses. The previously studied 142 lung cancer patients (7) were
Caucasian
individuals
was
2.95%
(95%
confidence
limits,
2.21%, 3.86%). ml was found in 7.73%, and m2 in 2.67% of alleles. No
case of African black-specific mutation m3 was detected. The allele fre
quency of CYPJA1°4among 157 lung cancer patients was 2.87% (95%
confidence limits, 1.32%, 5.37%); it was 2.87% (95% confidence limits,
1.71%, 4.49%) in 314 controls matched by age and sex. Thus, the novel
m4-mutatlon may not represent a susceptibility factor for lung cancer.
Clinic
Virchow,
Humboldt
University
of Berlin),
Pneumology
(Lungenklinik
Heckeshorn), and Urology (Krankenhaus Neukölln)in Berlin, Germany. Most
patients were hospitalized because of nonmalignant lung diseases, such as
chronic obstructions, because of cardiovascular diseases, or with various
supplemented by 15 cases [altogether, 124 males (median age, 66 years) and
33 females (median age, 65 years)]. Each lung cancer patient was matched
with two reference patients by sex and age (±2 years). All patients and healthy
volunteers
were
selected
in the years
1991—1995; they gave
their
informed
consent, and the study was approved by the Ethics Committee of the University
Clinic Charité.To avoid confounding
by ethnicity, only subjects of German
extraction were included.
Genotyping
INTRODUCTION
Cytochrome P.450 1A1 (CYP1A1) is a key enzyme in carcinogen
metabolism. By virtue of its polymorphic regulation, CYP1A1 proved
a promising genetic biomarker for susceptibility to certain malignan
cies, particularly lung cancer (1). The mutation detected first (called
ml) was a T—C transition 1194 bp downstream of exon 7, creating
a new MspI cleavage site (2, 3; Fig. 1). This mutation was found to be
overrepresented among lung cancer patients in Japan (4). Reports in
Caucasians could not confirm this finding (5—8),perhaps due to the
lower allele frequency of7.3% (7) compared to 33.2% in Japanese (9).
The A—@Gtransition at nucleotide 4889 (m2) is a rare trait in
Caucasians but occurs in about 20% of Japanese (10). The mutated
enzyme showed enhanced activity (10). Indeed, m2 was overrepre
sented in Japanese lung cancer patients (9) and also in a German study
(7), whereas only a trend was observed in Finland (11). We reevalu
ated two apparently homozygous carriers of m2 from our own study
(7) with the newly available restriction enzyme BsrDI. This led to the
discovery that binding of previously applied primers for allele-spe
cific PCR had been parfly diminished. DNA sequencing of these
samples uncovered a novel mutation (named m4), 2 bases upstream of
the m2 mutation site (Fig. 1) in the heme-binding region of the
enzyme.
On the basis of an improved PCR/RFLP methodology, the fre
quency ofall known CYPJAJ mutations, including m4 and the African
black-specific m3-mutation (12), was determined among 880 unre
lated individuals. Mutations were systematically checked for linkages
Received6120/96;accepted9/3196.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18U.S.C.Section1734solelyto indicatethis fact.
I This
work
was
supported
in part
by Grant
01
EC
9408/0
from
the German
Federal
Ministry of Education, Science, Research, and Technology.
2 To
whom
requests
for
reprints
should
be
addressed,
Institut
für Klinische
Phar
DNA was extracted from leukocytes by a stand
tions were performed with a Perkin-Elmer
cycler
(Weiterstadt,
Germany)
Applied Biosystems 9600 thermo
or with a Biometra
Trio thermoblock
(Gdttin
gen, Germany). For determination of ml and m3, an 899-bp DNA fragment
was amplified using 1 unit Taq polymerase (Perkin-Elmer Applied Biosys
tems, Weiterstadt, Germany), 10 @moI/liter
of primers M3F 5'-GGCFGAG
CAATCTGACCCTA and P80 5'-TAGGAGTCITGTCTCATGCCT, 0.2
mmol/liter
deoxynucleotide
triphosphate
(Boehringer
Mannheim,
Mannheim,
Germany), and 2.4 mmol/liter MgCI2 in a total volume of 50
@l.PCR
conditions were 35 cycles of 0.5 mm at 94°C, 1 mm at 63°C,and I mm at
72°C.The PCR product (12.5 gil) was digested with 50 units MspI (New
England Biolabs, Schwalbach, Germany), generating smaller fragments in case
of the mutation (Table 1). Fragments were evaluated on an ethidium bromide
stained
2.5%
agarose
gel (Fig. 2). m2 was checked
by amplifying
a 204-bp
fragment with primers M2F 5'-CTGTCTCCCTCl'GCITFACAGGAAGC and
M2R 5'-TFCCACCCGTTGCAGCAGGATAGCC (reagent concentrations as
above; PCR conditions were 35 cycles of 0.5 mm at 94°C,
0.5 mm at 63°C,
and
0.5 mm at 72°C).
The product was digested with BsrDI (New England Biolabs;
0.5 units, 65°Covernight). Mutation m4 could be determined from the same
204-bp fragment but using BsaI (New England Biolabs; 2.5 units, 55°C
overnight). Both cleavage sites were lost in the case of the mutations and were
evaluated on a 3% NuSieve 3:1 agarose gel (Fig. 2).
DNA Sequencing. Two hundredfour-bpPCR fragmentsof exon 7 were
processed using a Taq DyeDeoxy terminator sequencing kit (Perkin-Elmer
Applied Biosystems). After oligonucleotides were purified from unbound dye
terminators by chloroform/phenol extraction, they were analyzed with an
Applied Biosystems 373A automated sequencer on a denaturing 6% polyacryl
amide
gel. Sequences
were compared
with the published
CYPIAJ
sequence
(14—16)using ABI sequence navigator software.
Mutation Linkage Analysis. Linkage of ml with m2 was checked after
amplification
of 2047-bp
fragments
containing
both
mutations
(Table
2).
Cycling conditions were 35 cycles of 0.5 mm at 94°C,1 mm at 63°C,and 2
mm at 72°C.
The PCR product (12.5 p3) was digested first with 0.5 units BsrDI
overnight
at
Procedure.
aid procedure (13). CYPJAJ mutations were characterized by RFLP after PCR.
All primers were obtained from TIB Molbiol (Berlin, Germany); PCR reac
at 65°C and then with
100 units MspI
at 37°C, again
overnight.
Fragments were separated on a 2% agarose gel.
makologie, Universitatsklinikum Charité,Humboldt-UniversitAt Berlin, Schumannstrasse
20/21, D-10098 Berlin, Gennany. Phone: 49-30-2802-3635; Fax: 49-30-2802-5153; E
mail: [email protected].
Hypothetical
fragments
linkage
were digested
of ml
with m4 was evaluated
with MspI and separated
as follows:
on a 2% agarose
4965
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
2047-bp
gel. The
C4887A POLYMORPHISM
OF HUMAN
CYPIA!
m4
m2
m3
ml
T6235C
C4887A
A4889G T5639C
(Thr/Asn)461 (Ue/VaI)462
1
1000
2000
400@@1@
3000
I5'
—r@;i
@000
6000
(
•i••@
3'
Exon 1
Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7
Fig. I. Localization of currently known mutations of the human CYPIA1 gene. Mutation numbers are in chronological order of the discovery. Mutations ml and m3 gain a new
MspI cleavage site, m2 leads to the loss of a BsrDI site, and m4 can be detected by BsaI digestion.
Table 1 Restriction fragment length patterns of CYPIAI mutations
Primers for amplification and binding
Restriction
Recognition
positions (nt)―
enzyme
sequence
M3F (5542—5561)IP80(6420—6440)
Mspl
(bp)ml;
Mutation
6235 T—@C
m2; 4889 A—@G
M2F (4739—4763)/M2R (4918—4942)
Cut position
C'CG_G
BsrDI
GCAATG.NN'
Mspl
C'CQG
Fragment
(nt)―
Wild type
Mutation
Wild type
length
899
693, 206
149, 55
6234
4887
Mutation
m3: 5639 T—@C
M3F/P80
m4; 4887 C—@A
204a
nt,
nucleotide.
Nucleotide
M2FIM2R
positions
refer
to
the
Bsal
beginning
of
exon
1 (Fig.
GGTCTCN'NNN_
were
digested
with
BsrDI
followed
by ethanol
precipitation
significance of odds ratios was calculated by the exact Fisher's test. Odds
ratios are given with 95% confidence limits.
RESULTS
to
Identification
remove any enzyme (recognition sites of BsrDI and BsaI overlap by I base).
After resolubilization, DNA was digested with BsaI (Table 2).
Statistics.
Weinberg
(1
p2 + 2pq + q2) from the allele frequencies.
and
heterozygous
Statistical
revealed
(m2/wild
I
phoresis of CYPJAJ DNA fragments stained with
@
ethidium bromide. Fragment lengths are given in
bp. Lanes I and 12, lOO-bp DNA ladder; Lanes
898
2—5,
802
digestion
evaluating
ml
ofm4.
DNA
sequencing
of samples
when
performing
at nucleotide
BsrOI
2 3 4 5 6 7 8
a BsrDI
4887
in codon
digestion
461
BsaI
9 10 11 12
and
m3
[ho-
mozygous wild-type ml (wild type/wild type), het-
\.@
692
“
206
—
erozygous mutant ml (wild type/mutant), het
erozygous mutant m3 (wild type/mutant), and
@
@
@
homozygous mutant ml (mutant/mutant)]; Lanes
6—8,BsrDI digestion testing m2; Lanes 9—11,
BsaI digestion testing m4. A sample carrying m3
was drawn from a United States black.
204
96
mutationsMutation
(bp)ml
linkage
and m2
ml and m4
m2 and m4
@
a
5'-AlTAGGG'VFAGTGGGAGGGACACG;
[i.e.,
gel electro
@
MspI
type)
a C—@A transversion
MspI
Fig. 2. 3% 3: 1 NuSieve-agarose
Frequency
that were apparently homozygously m2 mutant when using allele
specific primers (two cases from a previous publication; Ref. 7) but
Expected genotype frequencies were calculated by the Hardy-
equation
899
802, 97
139, 65
5638
4877
1).
l841-bp band containing ml was cut out using a sterile scalpel, frozen at
—20°C
for 1 h, and thawed at 37°Cfor 5 mm. The gel pieces were transferred
to a 0.45-p.m filter (UFC3, Millipore, Eschborn, Germany) and eluted as
recommended by the manufacturer. The DNA was digested with S units BsaI
and separated on a 2% agarose gel.
A possible linkage of m2 and m4 was tested as follows: 204-bp PCR
fragments
204
Wild type
Mutation
Wild type
Mutation
.
—
I 39
Table 2 Analysis of hypothetical linkages of CYPIAJ
Amplification with primers
Procedure
P7l'@/P80
Sequentialdigestionwith
Mspl and BsrDl
P7la1P80
Mspl
M2F/M2R
binding
position,
nucleotides
digestion
excision
Fragment length
of the
Linked
Nonlinked
Linked
1600, 1553, 253, 241, 206
1806, 1347, 253, 241, 206
448,
1393
1841-bp fragment, and
subsequent Bsal digestion
Nonlinked
448, 484, 909
Sequential digestion with
BsrDl and Bsal
Linked
Nonlinked
204, 139, 55, 10
149, 139, 65. 55
4394-4417.
4966
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2
C4887A POLYMORPHISM OF HUMAN CYPJAI
bp upstream of m2 (Fig. 3)1. This mutation, called m4, led to the loss
of a BsaI restriction site, and this was used for determination. The
population frequency of m4 was studied in 880 individuals. m4
occurred in 2.95% of the 1760 alleles tested (Table 3). This preva
lence was slightly higher than that of m2 (2.67%) but lower than that
of ml (7.73%). The African mutation m3 was not detected in our
CD/@G
GGTGAGAM1@0RTTGCCCG
sample.
Linkage of Mutations. Mutation linkage analysis allowed the
allocation of the mutations to different alleles. m2 was strictly linked
to ml as tested in 42 reference individuals and 12 lung cancer patients.
Among all cases heterozygous for ml and for m4 (two of the controls
and one of the lung cancer patients), the mutations were not linked.
Moreover, three individuals presenting with m2 and m4 (one control
individual and two lung cancer patients) carried these mutations on
different alleles, as could be proven by subsequent digestions with
5'-Sequencing
i/@c@@rr
BsrDI and BsaI. Thus, m4 always appeared without any linkage.
CYPIAJ alleles are defined (Table 3) with reference to the different
mutation linkages. Alleles with 6235C (ml) were called *2A. m2 was
CGGG@AAYGKTCTC@%CC
in strict
@
@/@VI IL@U : j14
3-Sequencing (reverse)
Fig. 3. DNA sequencing electropherogram of a sample heterozygous for m2 (A4889G)
and m4 (C4887A).
(AU)
The m2 mutation
in codon 462 leads to an exchange
linkage
disequilibrium
with ml;
therefore,
this variance
of isoleucine
to valine (GTT); m4 in codon 461 leads to an exchange of threonine (ACC) to
asparagine(AAC). Double-strandsequencingwas performedusing fluorescentdye ter
minators with forward (M2F) and reverse (M2R) primers.
I mutations in 880 unrelated Caucasian individuals and allocation tospecified
Table 3Frequency offour CYPIAallelesNucleotide
frequency―95%
at position (nt)Allele
6235Allele(m4)
confidence4887 4889
5639
(m2)
(m3)
(ml)
A
T
T
157289.3287.78,
A
G
A
T
T
C
C
C
T
895.064.08,
472.671.97.
00.000.00,
T
T
522.952.21,
n%limitsCYP1A1°1
90.72CYPJA1*2AC
(wild type)C
6.19CYPIAI*2BC
3.54CYPIAJ*3C
0.21CYPIAJ°4A
3.86Mutation
136%2.95 frequency (n)°52
7.732.5%
6.5297.5%
confidence limit2.21
confidencelimit3.86
9.07a
Referred
47
2.67
1.97
0
0.00
0.00
3.54
0.21
to 1760 alleles.Table
individualsGenotypeObservedExpected―95%
4 Frequency of CYPIAI genotypes among 880 unrelated Caucasian
confidenceof
limits%*11*1701
CYPIAIn
%
76.85,82.2779.78*J/*2A81
_______
a@I,@4
79.67
11.39.03*J/*2B42
6.404.77*J/*447
3.95,7.045.28*2frJ@2A1
0.630.26*2,AJP2B4
1.160.27*2@4J*42
0.820.30*2B/@2B—
9.20
4.77
5.34
0.1 1
0.45
0.23
7.38,
3.46,
0.420.07*2@9J*4I
0.00
0.00,
0.630.16*4/*4I
0.11
0.00,
0. 11
0.00, 0.630.09
Total _________________
frequencies
are calculated
880 ______________
from the allele
frequencies
0.00,
0.12,
0.03,
100.00
100.00
in Table 3 according
of
*2A was termed *2B The allele providing the novel, nonlinked
mutation m4 was called *4
Frequency of CYPJAJ Genotypes in Controls and in Lung
Cancer. Within the 880 individuals tested, all observed CYPJAJ
genotype frequencies matched exactly the expected percentages as
calculated from the allele frequencies (Table 4). Because mutated
alleles were relatively rare, homozygous carriers occurred only in a
few cases. One individual provided a homozygous genotype
CYP1A1*4fI@4 (0.11%).
CYPJAI*4 was equally distributed among 157 lung cancer patients
(2.87%; 95% confidence limits, 1.32%, 5.37%) and the 314 matched
reference patients (2.87%; 95% confidence limits, 1.71%, 4.49%;
Table 5). Stratification by gender and smoking habits failed to reveal
to the Hardy-Weinberg
law.
4967
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C4887A POLYMORPHISM
OF HUMAN
CYPIAI
Table5 Stratification
consumptionSample
of CYPIAI*4allelefrequencyin lungcancercasesand matchedcontrolsbysexand cigarette
size(n)
limitsTotal157
Cases
2.56Male124
2.73Female
8.44Nonsmoker6
Smoking―33
23.41—20
PY28
6.8721—50
1.78>50PY82
4.55a
PY41
Odds
@
b
ratios
@y =
314
248
66
Cases
2.87
3.23
1.52
C
are calculated
consumption
Frequency(%)
Controls
Controls
Odds
ratio―
2.87
3.02
2.27
1.00
1.07
0.66
0.42,
0.39,
0.01,
3.08
1.91
0.41
1.29
0.68,
0.35,
0.05,
0.24,
8.33
5.36
1.22
3.66
95% Confidence
from the ratio of mutated to nonmutated alleles in the specified subgroup of cancer patients compared to the ratio in the controls.
of one
pack
of 20 cigarettes
per day
for
I year.
CThe total control group was taken for calculation of odds ratios.
a statistically significant difference (Table 5). There was only a trend
of increased CYPJAJ*4 frequency among the nonsmokers and the
moderately smoking patients. Tumor histology had no impact on m4
frequency (data not shown).
DISCUSSION
Improvement of Genotyping CYPJAJ. The introduction of the
restriction enzyme BsrDI for evaluation of nucleotide 4889 (m2)
instead of an allele-specific set of primers led indirectly to the dis
covery of the new C4887A transversion. Whereas the allele-specific
amplification revealed homozygous mutants in two lung cancer pa
tients, the BsrDI RFLP showed only heterozygosity. DNA sequencing
and subsequent linkage analysis showed clearly that both samples
provided one *2B allele and one *4 allele (Fig. 3). The reason for the
mistyping using allele-specific primers was probably the diminish
ment of the annealing capability of the wild type-specific primer
caused by the neighboring 4887A mutation. Difficulties with such
genotyping techniques are well known and have already been com
municated for CYPJAJ with a different strategy to avoid these prob
lems (17, 18).
Characteristics ofthe Novel C4887A Mutation. The significance
of threonine exchanged to asparagine in codon 461 is still unknown.
Both are hydrophilic amino acids. Interestingly, an exchange of the
adjacent hydrophobic amino acid residues, isoleucine to valine, results
an increase of enzyme activity (10). m4 is not in linkage disequilib
rium with another mutation, or at least our sample, totaling 2074
alleles, did not reveal such connections. We checked all cases of m4
paired with ml or m2 by the procedure given in Table 2. Thus, the
genotypes with CYPJAJ*4 in Table 4 are experimentally confirmed.
Linkage analysis showed unambiguously that m2 is always linked
to ml. This finding conforms with initial observations of Hayashi et
a!. (16) and Hirvonen
et a!. (11). Thus, the proposed definition
of
alleles is based on experimental and epidemiological analysis of
mutation linkage. However, we would not exclude the very rare
occurrence of solitary m2 in the Far Eastern population as a result of
recombinant events. The nomenclature follows the recent rules as
applied, e.g., to CYP2D6 alleles (19).
Lung Cancer Risk Due to CYPJAJ Mutations. The strongest
evidence for the role of m2 in lung cancer was found in the Japanese
(10). Due to the relatively high prevalence of the m2-carrying allele
(19.8%) in this population, an increase to 27.5% in lung cancer
patients was of high statistical significance (odds ratio, 1.53;
P = 0.001). In Caucasians, however, m2 represents a rare trait. The
Prevalence of ml is increased in lung cancer (7, 9) as a consequence of
linkage by m2. The role of genotypes containing only nonlinked ml (i.e.,
the CYPJAJ*2A allele) is not clear, at least not in Caucasians. Interest
ingly, ml has been associated with a high degree ofinducibility (21). The
novel mutation m4, however, was equally distributed among lung cancer
patients and controls. With our sample size, an odds ratio 2.8 could be
excluded by power analysis (type-I error 5%, type-Il error 20%) for m4
in heterozygous configuration. Thus, the change to asparagine in codon
461 does not appear to modulate lung cancer susceptibility.
In those studies in which allele-specific PCR was used, some of
the data on m2 should be rechecked because of the potential
disturbance by the m4 mutation to the primer-binding conditions.
However, our previous results on the overrepresentation
of m2 in
lung cancer patients (7) are still valid, despite two lung cancer
cases, who were apparently homozygous for m2 but now show
heterozygosity at nucleotides 4887 and 4889. The newly calculated
odds ratio of m2 in 157 cases and 314 controls is 3.01 (95%
confidence limits, 1.29, 7.26; P
0.004; data not shown). Hence,
CYPJAJ allele *2B has been confirmed as an important host factor
of lung cancer in Caucasians. Our further work aims at the enzy
matic properties, inducibility, and membrane topology of the
CYPJAJ allele *4 product.
ACKNOWLEDGMENTS
As the DNA source of m3, venous blood samples from United States blacks
were kindly donated by Dr. Helmut F. Cascorbi, Department of Anesthesiol
ogy, University Hospitals, Cleveland, Ohio. We thank H. Maszynski and P.
Pietsch
for excellent
technical
assistance.
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C4887A POLYMORPifiSM OF HUMAN CYPJAJ
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A C4887A Polymorphism in Exon 7 of Human CYP1A1:
Population Frequency, Mutation Linkages, and Impact on Lung
Cancer Susceptibility
Ingolf Cascorbi, Jürgen Brockmöller and Ivar Roots
Cancer Res 1996;56:4965-4969.
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