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Resume:
Mol Biol Rep (****) **:*** ***
Association of functional polymorphisms in MMPs genes
with gastric cardia adenocarcinoma and esophageal squamous cell
carcinoma in high incidence region of North China
Yan Li Dong-lan Sun Ya-nan Duan
Xiao-juan Zhang Na Wang Rong-miao Zhou
Zhi-feng Chen Shi-jie Wang
Received: 8 January 2009 / Accepted: 17 June 2009 / Published online: 28 June 2009
Springer Science+Business Media B.V. 2009
Abstract The aim of the present study was to investigate Introduction
the association of single nucleotide polymorphisms (SNPs)
in matrix metalloproteinase (MMPs) with the risk of gastric Esophageal squamous cell carcinoma (ESCC) is the com-
cardia adenocarcinoma (GCA) and esophageal squamous mon type of upper gastrointestinal cancer, with evident
cell carcinoma (ESCC). Genotypes were analyzed by characteristics of geographical distribution about its
polymerase chain reaction-restriction fragment-length development. China is a high incidence country for
polymorphism method in 592 patients and 624 healthy esophageal squamous cell carcinoma; the incidence and
individuals. Signi cant differences in allele and genotype mortality are half of world level. Cixian County and
distributions of MMP-2 -1306C ? T SNP were observed Shexian County of Hebei province lie in the southern foot
between ESCC and controls (P = 0.02 and 0.01, respec- of Taihang Mountain, a border area of Hebei, Henan and
tively). Compared with the C/T ? T/T genotypes, C/C Shanxi provinces, which is one of the high-risk areas for
genotype signi cantly increased the risk of ESCC esophageal cancer in China. Gastric cardia adenocarcinoma
(OR = 1.57, 95% CI = 1.10 2.23), especially in individ- (GCA) as another prevalent tumor is named as adenocar-
uals in smoker group and in the group with positive family cinoma of the oesophago-gastric junction (OGJ) by World
history. The strati cation analysis showed there were risk Health Organization (WHO) [1]. An increased incidence of
changes of GCA for -735C/C genotype carrier in non- GCA was observed in Europe [2]. Epidemiological studies
smoker, for MMP-12 -82G allele and MMP-13 -77A/G have suggested that in China GCA shares very similar
genotype carrier in smoker. Our study indicated that these geographic distribution with ESCC, especially in a popu-
four functional polymorphisms might play roles in devel- lation of high incidence region. Studies have showed that
oping ESCC and GCA in high incidence region of North the trend of the incidence and mortality of ESCC had
China. decreased slightly, on the other hand, GCA showed a sig-
ni cant increase trend in Cixian County and Shexian
Keywords Gastric cardia adenocarcinoma County in Hebei province for the past few years [3]. The
Esophageal squamous cell carcinoma different trends of incidence between ESCC and GCA may
Matrix metalloproteinase (MMPs) have the indication that difference characteristic of
Single nucleotide polymorphism molecular biology may exist. Thus, the comparative study
on ESCC and GCA particularly in a local place is critical in
order to understand the risk factor and pathogenesis of
these diseases.
Matrix metalloproteinase (MMP), a zinc protease,
Y. Li D. Sun Y. Duan X. Zhang N. Wang decomposes the extracellular matrix (ECM) and basal
R. Zhou Z. Chen S. Wang membrane and plays a leading role in process of tumor
Department of Molecular Biology, Hebei Cancer Institute,
invasion and metastasis. Recent studies have demonstrated
The Fourth Hospital of Hebei Medical University,
that MMPs are involved in early tumorigenesis by modulating
Jiankanglu 12, 050011 Shijiazhuang, China
cell proliferation, apoptosis, and host immune surveillance
e-mail: ********@*****.***.**
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198 Mol Biol Rep (2010) 37:197 205
from Cixian County and Shexian County during the endo-
[4]. MMP-2, the major structural component of basement
scopic screening campaign between 2003 and 2006. All the
membrane, also known as 72 kDa gelatinase, primarily
cancer patients and control subjects were unrelated Han
hydrolyzes type IV collagen [5]. In addition, active MMP-2
nationals. Information of sex, age, smoking habit and
degrades insulin-like growth factor binding proteins and
family history was obtained from cancer patients and
releases insulin-like growth factors, which are well known to
healthy controls by an interview following sampling. For
have a strong effect on stimulating cell proliferation and
smoking habit, the former and present smoking status, the
inhibiting apoptosis [4]. MMP-12 (human macrophage me-
number of cigarettes per day, and the time of starting and
talloelastase) and MMP-13 (human collagenase-3) are loca-
quitting were inquired. Individuals who formerly or cur-
ted in the same chromosome region (11q22). MMP-12
rently smoked ve cigarettes/day for at least. Individuals
promotes angiogenesis by cleaving structural components of
with at least one- rst-degree relative or two-second-degree
the extracellular matrix, such as collagen type IV and brin
relatives having esophageal/cardiac/gastric cancer were
[4]. MMP-13 cleaves native collagen but has a higher activity
de ned as having a family history of upper gastrointestinal
on type II collagen than MMP-1. It also acts to degrade var-
cancers (UGIC). Smoking status and family history were
ious extracellular macromolecules including proteoglycans
only available from a subset of cancer patients and healthy
[6]. These activities of MMPs are believed to be linked to both
controls (Table 1). The study was approved by the Ethics
cancer development and progression.
Committee of Hebei Cancer Institute and informed consent
Somatic mutation of the MMPs gene in cancer has not
was obtained from all recruited subjects.
been reported so far, suggesting that the overexpression of
MMPs is probably due to the change of transcriptional and
not gene ampli cation or an activating mutation. Several
DNA extraction
single nucleotide polymorphisms (SNPs) in the MMPs pro-
moter region have been identi ed, functional analysis of these
Venous blood (5 ml) was collected from each subject into
SNPs suggested that modulation the transcriptional activity of
Vacutainer tubes containing EDTA and stored at 4 C. After
MMPs may increase the risk of individual tumor incidence
sampling, genomic DNA was extracted within 1 week by
[7 11]. Previously, we have investigated the association of
proteinase K (Merck, Darmstadt, Germany) digestion fol-
the single nucleotide polymorphism in MMP-1, MMP-3 and
lowed by a salting out procedure according to the previ-
MMP-7 genes promoter with the risk of ESCC and GCA, and
ously described method [20].
the results indicate that the SNPs of MMPs may play different
roles in developing ESCC and GCA [12 14]. MMP-2
-1306C ? T, -735C ? T; MMP-12 -82A ? G and MMP- MMP-2 -1306C ? T, -735C ? T; MMP-12 -82A ? G
13 -77A ? G, were functional polymorphisms have been and MMP-13 -77A ? G genotyping
described that seem to alter transcriptional levels [15 18].
The MMP-2 -1306C ? T, -735C ? T; MMP-12 -82A ? G
Based on our previous nding, in this study, we further
and MMP-13 -77A ? G genotypes were determined by
studied the association of the four polymorphisms with the
risk of ESCC and GCA development. polymerase chain reaction-restriction fragment-length poly-
morphism (PCR-RFLP) assay. The primers for amplifying the
MMP-2, MMP-12, and MMP-13 promoter fragments are
showed in Table 2. The PCR was performed in a 20 ll volume
Materials and methods
containing 100 ng of DNA template, 2.4 ll of 109 PCR
Study participants buffer, 1 U of Taq DNA polymerase (Tiangen Biotech Co.,
Ltd, Beijing, China), 0.4 ll of 10 mmol/l dNTPs and 200 nM
This study included 592 patients (335 with ESCC and 257 of each primer. The PCR cycling conditions were 5 min at
94 C followed by 35 cycles of 45 s at 94 C, 45 s at 58 C for
with GCA) and 624 healthy individuals. The cases were
-1306C ? T, 65.5 C for -735C ? T, 57 C for -82A ? G
outpatients for endoscopic biopsy or inpatients for tumor
and 53 C for -77A ? G, and 45 s at 72 C, with a nal step at
resection in the local tumor hospitals in Cixian County and
72 C for 10 min to allow for the complete extension of all
Shexian County between 2003 and 2006. All patients were
PCR fragments. The 8 ll aliquot of every PCR product was
pathologically con rmed by the local county hospitals.
subjected to digestion at 37 C overnight in a 10 ll reaction
Esophageal carcinomas were all squamous cell carcinomas.
Gastric cardiac carcinomas were all adenocarcinomas with containing 10 U of respective restriction enzyme. After
their epicenters at the gastroesophageal junction, i.e., from digestion, the products were separated on a 4% agarose gel
1 cm above until 2 cm below the junction between the that was stained with ethidium bromide. The length of PCR
end of the tubular esophagus and the beginning of the products, restriction enzymes, and fragments length are
saccular stomach [19]. Healthy subjects were recruited summarized in Table 1.
123
Mol Biol Rep (2010) 37:197 205 199
Table 1 PCR conditions for
Polymorphisms Primers Product Restriction Fragment
MMP-2, MMP-12 and MMP-13
length enzyme length
restriction fragment length
polymorphisms MMP-2
50 -CTTCCTAGGCTG
-1306C [T 193 bp XspI 188 ? 5 bp(C)
GTCCTTACTGA-30 (F)
50 -CTGAGACCTGAAG 162 ? 26 ? 5 bp(T)
AGCTAAAGAGCT-30 (R)
50 -GGATTCTTGGC
-735C [T 391 bp HinfI 391 bp(C)
TTGGCGCAGGA-30 (F)
50 -GGGGGCTGGGTA 338 ? 53 bp(T)
0
AAATGAGGCTG-3 (R)
MMP-12
50 -GAGATAGTCAAG
-82A [G 199 bp PvuII 199 bp
GGATGATATCA-30 (F)
50 -AAGAGCTCCAG 175 bp ? 24 bp
0
AAGCAGTGG-3 (R)
MMP-13
50 -GATACGTTCTTA
-77A [G 445 bp XspI 445 bp
CAGAAGGC-30 (F)
50 -GACAAATCATC 244 bp ? 201 bp
0
TTCATCACC-3 (R)
Table 2 Demographic characteristics in ESCC, GCA patients and healthy controls
Group Controls n ESCC GCA
a
P valuea
n P value n Gender
Male 400(64.1) 225(67.2) 168(65.4)
Female 224(35.9) 110(32.8) 0.343 89(34.6) 0.107
b
0.95b
Mean age (SD) 60.4(8.42) 60.1(9.33) 0.55 60.5(8.30)
Smoking status
Smokers 264(42.3) 133(39.7) 126(49.0)
Non-smokers 360(57.7) 202(60.3) 0.44 131(51.0) 0.07
Family history of UGIC
Positive 221(35.4) 162(48.4) 124(48.2)
0.00c 0.00d
Negative 403(64.6) 173(51.6) 133(51.8)
ESCC esophageal squamous cell carcinoma; GCA gastric cardiac adenocarcinoma; UGIC upper gastrointestinal cancer
a
P value for Chi-square test
b
P value for T test
c
Age, gender and smoking status adjusted odds ratio (OR) = 1.71, 95% con dence interval (CI) = 1.30 2.24
d
Age, gender and smoking status adjusted odds ratio (OR) = 1.70, 95% con dence interval (CI) = 1.27 2.28
Statistical analysis
For a negative control, distilled water was used instead
of DNA in the reaction system for each panel of PCR. The
Statistical analysis was performed using SPSS11.5 soft-
PCR reactions of 10% of the samples were run in duplicate
ware package (SPSS Company, Chicago, Illinois, USA).
for quality control.
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200 Mol Biol Rep (2010) 37:197 205
Hardy Weinberg analysis was performed to compare the 65.4% men) was comparable to that in healthy controls
observed and expected genotype frequencies using the Chi- (64.1% men) (P = 0.34 and 0.11, respectively). The propor-
square test. Comparison of the MMP-2 -1306C ? T, tion of smokers in ESCC, GCA patients (39.7 and 49.0%,
-735C ? T; MMP-12 -82A ? G and MMP-13 -77A ? G respectively), and healthy controls (42.3%) were no signi -
genotype distributions in the study groups was performed cant different (P = 0.44 and P = 0.07, respectively). In
by means of two-sided contingency tables using Chi-square addition, the frequency of positive family history of UGIC in
test. The MMP-2 -1306C ? T and -735C ? T haplotype ESCC (48.4%) and GCA (48.2%) patients was signi cantly
higher than that in healthy controls (35.4%) (P \ 0.001).
frequencies and linkage disequilibrium coef cient were
estimated using the EH linkage software (version 1.2, Thus, family history of UGIC signi cantly increased the risk
Rockefeller University, New York) and 2LD program, of developing ESCC (adjusted OR = 1.71, 95% CI = 1.30
respectively. The odds ratio (OR) and 95% con dence 2.24) and GCA (adjusted OR = 1.70, 95% CI = 1.27 2.28).
interval (CI) were calculated using an unconditional The allele frequencies and genotype distributions of
logistic regression model. A probability level of 5% was MMPs in patients and controls are summarized in Table 3.
The frequencies of MMP-2 -1306C ? T, -735C ? T;
considered signi cant.
MMP-12 -82A ? G and MMP-13 -77A ? G genotypes
in control groups did not signi cantly deviate from that
expected for a Hardy Weinberg equilibrium (P [ 0.05).
Results
Association of MMP-2 -1306C ? T SNP with the risk
Characteristic of subjects
of ESCC and GCA
The relevant characteristics of the study subjects are shown in
Table 2. The mean age of ESCC cases, GCA cases, and The signi cant differences in allele frequencies and genotype
distributions of the MMP-2 -1306C ? T polymorphism were
controls was 60.1 9.3 (range 34 85), 60.5 8.3 (range
37 86), and 60.4 8.4 years (range 31 78), respectively. observed between ESCC and control (P = 0.02 and 0.01,
The gender distribution in ESCC and GCA patients (67.2 and respectively). Compared with the C/T ? T/T genotypes, the
Table 3 Distributions of the
Polymorphisms Controls n ESCC GCA
MMPs SNP genotype/allele in
genotype/allele a
P valuea
ESCC,GCA patients and n P value n healthy controls
MMP-2 -1306 C/T
T/T 6(1.0) 39(0.9) 0.01 4(1.6) 0.41
C/T 137(21.0) 48(14.3) 46(17.9)
C/C 487(78.0) 284(84.8) 207(80.5)
T 143(11.5) 54(8.1) 0.02 54(10.5) 0.56
C 1,105(88.5) 616(91.9) 460(89.5)
MMP-2 -735 C/T
T/T 29(4.6) 13(3.9) 0.78 9(3.5) 0.06
C/T 187(30.0) 100(29.9) 63(24.5)
C/C 408(65.4) 222(66.3) 185(72.0)
T 245(19.6) 126(18.8) 0.66 81(15.8) 0.06
C 1,003(80.4) 544(81.2) 433(84.2)
MMP-12 -82 A/G
A/A 588(94.2) 322(96.1) 0.21 241(94.9) 0.79
A/G 36(5.8) 13(3.9) 16(5.1)
A 1,176 657 0.18 498 0.87
G 36 13 16
MMP-13 -77 A/G
A/A 137(22.0) 76(22.7) 0.99 60(23.3) 0.54
A/G 324(51.9) 170(50.7) 123(47.9)
G/G 163(26.1) 89(26.6) 74(28.8)
Bold values indicate positive
A 598 322 0.95 243 0.81
signi cance
G 650 348 271
a
P value for Chi-square test
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Mol Biol Rep (2010) 37:197 205 201
Association of MMP-12 -82A ? G SNP with the risk
C/C genotype signi cantly modi ed the risk of developing
of ESCC and GCA
ESCC (OR = 1.57, 95% CI = 1.10 2.23) (Table 4). Fur-
thermore, when strati ed for smoking status and family his-
The allelotype and genotype distribution of the MMP-12
tory of UGIC, the C/C genotype signi cantly modi ed the risk
-82A ? G SNP in the overall ESCC and GCA patients were
of developing ESCC in the smoker or family history of UGIC
not signi cantly different from that in healthy controls
groups (adjust OR = 1.96, 95% CI = 1.10 2.23 and
(P [ 0.05). Compared with the A/A genotypes, the A/G
OR = 2.06, 95% CI = 1.19 3.55, respectively) (Table 4).
genotype did not signi cantly modify the risk of developing
Association of MMP-2 -735C ? T SNP with the risk ESCC and GCA (OR = 0.63, 95% CI = 0.32 1.20 and
OR = 1.05, 95% CI = 0.56 1.95, respectively). When
of ESCC and GCA
strati ed by smoking status and family history of UGIC, the
carriers with A/G genotype had a tendency of increasing
There was no signi cant difference in genotype and allelo-
type distributions of the MMP-2 -735C ? T polymor- susceptibility to GCA in smoker (adjust OR = 2.03,
95%CI = 0.90 4.60) (Table 5).
phisms between patients (ESCC and GCA) and control
(P = 0.78, 0.66 and P = 0.06, 0.06, respectively). Com-
Association of MMP-13 -77A ? G SNP with the risk
pared with the C/T ? T/T genotypes, the C/C genotype
of ESCC and GCA
increased the trend of risk of developing GCA (OR = 1.36,
95% CI = 0.99 1.87) (Table 4). In addition, compared with
The allelotype and genotype distribution of the MMP-13
the C/T ? T/T genotypes, the C/C genotype signi cantly
-77A ? G SNP in the overall ESCC and GCA patients
modi ed the risk of developing GCA in nonsmoker (adjust
was not signi cantly different from that in healthy controls
OR = 1.70, 95% CI = 1.07 2.68) (Table 4).
Table 4 Correlation between SNP of MMP-2 and susceptibility to ESCC and GCA
P valuea OR(95% CI)a P valuea OR(95% CI)a
Groups -1306 C/T genotype (cases,%) -735 C/T genotype (cases, %)
C/T ? T/T C/C C/T ? T/T C/C
Overall
Control 137(22.0) 487(78.0) 1.00 216(34.6) 408(65.4) 1.00
1.57(1.10 2.23)b 1.04(0.79 1.38)b
ESCC 51(15.2) 284(84.8) 0.01 113(33.7) 222(66.3) 0.78
b
1.36(0.99 1.87)b
GCA 50(19.5) 207(80.5) 0.41 1.17(0.81 1.67) 72(28.0) 185(72.0) 0.06
Non-smoker
Control 78(21.7) 282(78.3) 1.00 124(34.4) 236(65.6) 1.00
1.37(0.88 2.13)c 1.08(0.75 1.56)c
ESCC 34(16.8) 168(83.2) 0.17 66(32.7) 136(67.3) 0.69
c
1.70(1.07 2.68)c
GCA 26(19.8) 105(80.2) 0.66 1.12(0.68 1.84) 31(23.7) 100(76.3) 0.02
Smoker
Control 59(22.3) 205(77.7) 1.00 92(34.8) 172(65.2) 1.00
1.96(1.09 3.53)c 0.98(0.63 1.51)c
ESCC 17(12.8) 116(87.2) 0.02 47(35.7) 86 (64.7) 0.92
c
1.11(0.71 1.74)c
GCA 24(19.0) 102(81.0) 0.46 1.22(0.72 2.08) 41(32.5) 85(67.5) 0.65
Negative family history
Control 83(20.6) 320(79.4) 1.00 129(32.0) 274(68.0) 1.00
1.29(0.81 2.05)d 0.89(0.61 1.29)d
ESCC 29(16.8) 144(83.2) 0.29 60(34.7) 113(65.3) 0.53
d
1.37(0.88 2.13)d
GCA 25(18.8) 108(81.2) 0.65 1.12(0.68 1.84) 34(25.6) 99(74.4) 0.16
Positive family history
Control 54(24.5) 167(75.6) 1.00 87(39.4) 134(60.6) 1.00
2.06(1.19 3.55)d 1.34(0.87 2.04)d
ESCC 22(13.6) 140(86.4) 0.01 53(32.7) 109(67.3) 0.18
1.28(0.75 2.19)d 1.47(0.92 2.35)d
GCA 25(20.2) 99(79.8) 0.37 38(30.6) 86(69.4) 0.11
Bold values indicate positive signi cance
a
P value, ORs and 95% CIs were calculated by unconditional logistic regression with the CT ? TT as the reference group
b
Adjusted for age, gender, smoking status and UGIC family history
c
Adjusted for age, gender and UGIC family history
d
Adjusted for age, gender and smoking status
123
202
123
Table 5 Correlation between SNPs of MMP-12, MMP-13 and susceptibility to ESCC and GCA
Groups MMP-12 genotype (cases, %) P valuea OR(95% CI)a MMP-13 genotype (cases, %) P valuea OR(95% CI)a P valuea OR(95% CI)a
A/A A/G A/A A/G G/G
Overall
Control 588(94.2) 36(5.8) 1.00 137(22.0) 324(51.9) 163(26.1) 1.00 1.00
b b
ESCC 322(96.1) 13(3.9) 0.21 0.66(0.35 1.26) 76(22.7) 170(50.7) 89(26.6) 0.75 0.95(0.68 1.32) 0.94 0.98(0.67 1.44)b
b b
GCA 241(94.9) 16(5.1) 0.79 1.08(0.59 1.99) 60(23.3) 123(47.9) 74(28.8) 0.45 0.87(0.60 1.25) 0.86 1.04(0.69 1.56)b
Non-smoker
Control 337(93.6) 23(6.4) 1.00 87(24.2) 166(46.1) 107(29.7) 1.00 1.00
ESCC 195(96.5) 7(3.5) 0.15 0.53(0.22 1.25)c 49(24.3) 94(46.5) 59(29.2) 0.98 1.01(0.65 1.55)c 0.93 0.98(0.61 1.57)c
GCA 126(96.2) 5(3.8) 0.28 0.58(0.22 1.56)c 24(18.3) 67(51.1) 40(30.5) 0.16 1.46(0.86 2.50)c 0.30 1.36(0.76 2.42)c
Smoker
Control 251(94.4.) 13(5.6) 1.00 48(18.2) 158(59.8) 58(22.0) 1.00 1.00
ESCC 127(95.5) 6(4.5) 0.86 0.91(0.34 2.55)c 27(20.3) 76(57.1) 30(22.6) 0.57 0.86(0.50 1.48)c 0.80 0.92(0.48 1.75)c
c c
GCA 114 (90.5) 12(9.5) 0.09 2.03(0.90 4.60) 36(28.6) 56(44.4) 34(27.0) 0.01 0.47(0.28 0.80) 0.43 0.78(0.43 1.43)c
Negative family history
Control 379(91.8) 24(7.2) 1.00 85(21.1) 209(51.9) 109(27.0) 1.00 1.00
ESCC 169(97.7) 4(2.3) 0.07 0.37(0.13 1.10)d 44(25.4) 83(48.0) 46(26.6) 0.24 0.77(0.49 1.20)d 0.43 0.82(0.50 1.35)d
d d
GCA 123(92.5) 10(7.5) 1.28(0.60 2.76) 35(26.3) 59(44.4) 39(29.3) 0.13 0.69(0.42 1.12) 0.61 0.87(0.51 1.49)d
Positive family history
Control 209(94.6) 12(5.4) 1.00 52(23.5) 115(52.1) 54(24.4) 1.00 1.00
d d
ESCC 153(94.4) 9(5.6) 0.96 1.03(0.42 2.49) 32(19.8) 87(53.7) 43(26.5) 0.44 1.23(0.73 2.07) 0.40 1.30(0.71 2.35)d
d d
GCA 118(95.2) 6(4.8) 0.81 0.89(0.32 2.42) 25(20.2) 64(51.6) 35(28.2) 0.61 1.16(0.66 2.04) 0.36 1.35(0.71 2.55)d
Bold values indicate positive signi cance
a
P value, ORs and 95% CIs were calculated by unconditional logistic regression with the A/A as the reference group
b
Adjusted for age, gender, smoking status and UGIC family history
c
Adjusted for age, gender and UGIC family history
d
Adjusted for age, gender and smoking status
Mol Biol Rep (2010) 37:197 205
Mol Biol Rep (2010) 37:197 205 203
Table 6 Correlation between the MMPs haplotypes and susceptibility to ESCC and GCA
P valuea P valuea
Haplotype Control(n %) ESCC(n%) OR(95% CI) GCA(n %) OR(95% CI)
1.00a 1.00b
T1306//T735 28(2.2) 17(2.5) 9(1.7)
T1306/C735 116(9.3) 101(15.1) 0.28 1.43(0.74 2.77) 44(8.6) 0.70 1.18(0.52 2.70)
C1306/T735 211(16.9) 80(11.9) 0.16 0.62(0.32 1.20) 75(14.6) 0.80 1.11(0.50 2.45)
C1306/C735 893(71.6) 472(70.5) 0.66 0.87(0.47 1.60) 386(75.1) 0.45 1.35(0.63 2.88)
1.00c 1.00d
A12/G13 632(50.6) 342(51.0) 264(51.4)
A12/A13 579(46.4) 316(47.2) 0.93 1.01(0.83 -1.22) 233(45.3) 0.73 0.96(0.78 1.19)
G12/G13 20(1.6) 6(0.9) 0.21 0.55(0.22 1.59) 10(1.9) 0.65 1.20(0.55 2.59)
G12/A13 17(1.4) 6(0.9) 0.37 0.65(0.26 1.67) 7(1.4) 0.98 0.99(0.40 2.40)
a
P value, ORs and 95% CIs were calculated by unconditional logistic regression with the T1306//T735 as the reference group in ESCC
b
ORs and 95% CIs were calculated by unconditional logistic regression with the T1306//T735 as the reference group in GCA
c
ORs and 95% CIs were calculated by unconditional logistic regression with the A12/G13 as the reference group in ESCC
d
ORs and 95% CIs were calculated by unconditional logistic regression with the A12/G13 as the reference group in GCA
(P [ 0.05). When strati ed by smoking status and family developing GCA in nonsmoker. The MMP-12 -82G allele
history of UGIC, the carriers with the A/G genotype in signi cantly modi ed the risk of developing GCA, in
contract, MMP-13 -77A ? G A/G genotype signi cantly
smoker had lower risk in developing GCA (adjust
OR = 0.47, 95% CI = 0.28 0.80) (Table 5). lowered the risk of developing GCA in smoker.
Previous studied have demonstrated that in the gene
Haplotype of MMP-2 two SNPs with susceptibility promoter region of MMP-2, there are sequence variations
and several functional SNPs. Two transitions (-1306C ? T
to ESCC and GCA
and -735C ? T), located at a core recognition sequence of
The MMP-2 -1306C ? T and -735C ? T polymorphisms Sp1 (CCACC box), lead to a strikingly low promoter
displayed linkage disequilibrium (D0 = 0.58, P = 0.00) activity because of abolishing the Sp1-binding site [15, 16].
and the results are presented in Table 6. We did not Transient transfection experiments showed that reporter
observe a signi cant difference in haplotype frequencies gene expression driven by the C allelic were greater than
between cases and controls. Compared with the haplotype reporter gene expression driven by the T allelic, indicating
of T-1306 T-735, the others did not signi cantly modify the the functional signi cance of these two polymorphisms
risk of developing ESCC and GCA. [15]. Numerous studies have investigated if the -1306C
? T and the -735C ? T polymorphisms are associated
Haplotype of MMP-12 and MMP-13 SNPs with risk of some cancers, and the results obtained in dif-
with susceptibility to ESCC and GCA ferent groups were inconsistent. Studies reported that C
allele of -1306C ? T SNP might be a potential risk factor
The MMP-12 and MMP-13 polymorphisms displayed for cancers, including lung, gastric cardia, oral, breast and
linkage disequilibrium (D0 = 0.51, P = 0.00) and the cervical cancer [21 25]. In contrast, Rollin et al. and Grieu
results are presented in Table 6. We did not observe a et al. [26, 27] found that no difference observed in MMP-2
-1306C ? T genotypes between controls and patients for
signi cant difference in haplotype frequencies between
cases and controls. Compared with the haplotype of MMP- non-small cell lung cancer and breast cancer. About
-735C ? T polymorphism, the studies of lung cancer and
12 A/MMP-13 A, the others did not signi cantly modify
the risk of developing ESCC and GCA. ESCC had shown that -735C/C homozygote could increase
the risk of cancers [21, 26]. In our study, we found that the
-1306C/C increased the risk of developing ESCC. How-
ever, no signi cant differences were observed between
Discussion
GCA and control. Compared with the -735C ? T C/T ?
This study showed that family history of UGIC signi - T/T genotypes, the C/C genotype increased the trend of
cantly increased the risk of developing ESCC and GCA. risk of developing GCA, but no signi cant differences
The MMP-2 -1306C/C genotype signi cantly modi ed the were observed between ESCC and control. When strati ed
risk of developing ESCC in smoker or positive family for smoking status and family history of UGIC, the
-1306C ? T C/C genotype could signi cantly modify the
history of UGIC; on the other hand, the MMP-2
-735C ? T C/C genotype signi cantly modi ed the risk of risk of developing ESCC in smoking groups and positive
123
204 Mol Biol Rep (2010) 37:197 205
family history of UGIC; and the MMP-2 -735C ? T C/C MMP-12 and MMP-13, located at chromosome 11q22,
genotype signi cantly modi ed the risk of developing are two neighboring genes. Our study have found the
GCA in nonsmoker. linkage disequilibrium between MMP-12 and MMP-13
(D0 = 0.51). MMP-12 A/MMP-13 G (50.6%) and MMP-12
The -82A ? G SNP at position -82 in the promoter of
the MMP-12 gene is located at a core recognition sequence A/MMP-13 A (46.4%) were the common haplotype in the
of AP-1. In vitro experiments showed that the A allelic control of the high risk region. Comparing to the haplotype
increased the binding ability of AP-1 to enhance the gene of MMP-12 A/MMP-13 A, the other haplotypes did not
transcription [17]. Kader et al. [28] showed that the MMP- signi cantly modify the risk of developing ESCC and
12 G allelic increased the metabasis of bladder cancer in GCA.
smoking group. Su et al. [29] found that the MMP-12 G In conclusion, our study indicated that the functional
allelic signi cantly modi ed the risk of developing lung polymorphism in MMP-2, -12, -13 genes may be play a
cancer in males group. Our study found that there was no role in developing ESCC and GCA of high incidence
signi cant difference in genotype and allelotype distribu- region of North China. Also traditional epidemiological
tions of the MMP-12 -82A ? G polymorphisms between studies suggested that there may be common environ-
patients (ESCC and GCA) and control of high-risk areas in mental and genetic factors to morbidity of ESCC and GCA,
Cixian County and Shexian County in Hebei province. however, differences in molecular mechanism of ESCC
When strati ed for smoking status, genotypes carrying and GCA development may exist. Thus, the exact etiolo-
MMP-12 -82G allele signi cantly modi ed the risk of gies mechanisms of the two malignancies need closer study
developing GCA in smoking group. It is worthy to mention and explore.
that all smokers are males. These results indicate that the
Acknowledgments We thank the patients and control individuals
high transcription activity of MMP-12 G allelic may
for taking part in this study. We also thank many doctors in the Fourth
increase the risk of cancers in some group. Hospital of Hebei Medical University, and in Cancer Prevention and
The MMP-13 -77A ? G SNP at position -77 in the Control Institute of Shexian County, and in Cancer Prevention and
promoter of the MMP-13 gene is a binding site for the Control Institute of Cixian County, China, for their assistance in
recruiting study subjects. We would like to Dr Nan Mei, for critical
transcription factor, PEA3 (AGGAAG). In vitro experi-
reading of the manuscript. This work is supported by funds for the
ments showed that the SNP altered the gene transcription potentially distinguished scienti c project construction in programme
activity [18]. Yoon et al. [18] found that the MMP-13 in Hebei Universities.
-77A ? G SNP was associate with atherosclerosis in the
Con ict of interest The authors report no con icts of interest. The
abdominal aorta of black men. To the best of our knowl-
authors alone are responsible for the content and writing of the paper.
edge, our study is the rst molecular epidemiologic study
with regard to the association of the MMP-13 polymor-
phism and the risk of cancer development. Our study
demonstrated that the MMP-13 -77A ? G genotypes were
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