Cytokine single nucleotide polymorphisms in patients’with gallstone: dose TGF-b gene variants affect gallstoneformation?

Padideh Ebadi • Saeed Daneshmandi •

Abbas Ghasemi • Mohammad Hossein Karimi

Received: 11 February 2013 / Accepted: 14 September 2013 / Published online: 27 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Gallstone is a common biliary disorder with

several risk factors. Immune responses and inflammatory

cytokines are important in this disease; as a result, some

cytokines can be detected in bile fluid. In this research,

cytokine gene polymorphisms were studied, and their

effects on gallstone formation were evaluated. On 158

gallstone patients and 254 normal subjects, by PCR- RFLP

method, IL-4-C590T polymorphism and by ARMS-PCR

method, IFN-c T?874A, TNF-a-A308G, IL-6 G-174C and

TGF-b T?869C variants were studied. Pathologic evalua-

tions were done on surgical specimens. There were no

significant differences in distribution of evaluated poly-

morphisms between patient group and normal control

group (P [ 0.05), except TGF-b ?869T allele (P = 0.04,

OR = 1.23, 95 % CI = 1–1.79) which was higher in

patients with gallstone. Although the pro-inflammatory

cytokines such as TNF-a and IL-6 may promote gallstone

formation, in this study no significant correlation between

TNF-a and IL-6 polymorphisms and gallstone formation

was seen. It is taught that TGF-b may affect gallbladder

cells to promote gallstone formation and higher producer

TGF-b ?869T allele can be a risk factor of gallstone dis-

ease, so further studies would be more elucidative.

Keywords Gallstones � Cytokine � TGF-beta �Polymorphism

Introduction

Gallstone is a common but an important disorder leading to

the complicated treatment or surgery. Both environmental

and genetic factors contribute towards susceptibility to the

disease [1]. Congenital, biological, and behavioral factors

are the risk factors for this problem [2]. Nutrition, obesity,

rapid weight gain or loss, and exercise are some of the

behavioral factors [3], while other risks of gallstone for-

mation are associated with sex, age, family history and

ethnic background of the individuals in the population [4,

5]. Different genetic factors related to the risk of gallstone

formation are believed to be the result of ethnic differences

[6]. Gallstone formation associated genes, Lith1 and Lith2,

have been recognized in mice [7]. Various human gene

polymorphisms such as LDL receptor-associated protein,

apo B, apo A1, LDL receptor, and apo E genes are related

to gallstone formation [1, 8], although, these genetic factors

are not specific for gallstone formation. Another factor

having role in gallstone formation is cytokine. Cytokines

are low molecular weight protein mediators that have been

shown to be produced by various immune cells, as well as,

hepatocytes and gallbladder epithelium [9]. It has been

shown that biliary tract and gallbladder epithelial cells

(GBEC) produce cytokines, including IL-6, IL-8, TNF-a,

MCP-1, and express IL-6 and TNF-a receptors [10, 11].

Some investigations indicated that cytokines can modify

gallbladder epithelial cells functions [11]. For instance,

P. Ebadi (&)

Biochemistry Department, School of Medicine, Kazerun Branch,

Islamic Azad University, Kazerun, Iran

e-mail: Padideh_ebadi@yahoo.com

S. Daneshmandi

Department of Immunology, Faculty of Medical Sciences,

Tarbiat Modares University, Tehran, Iran

A. Ghasemi

Alborz Hospital, Social Organization, Karaj, Iran

M. H. Karimi

Transplant Research Center, Shiraz University of Medical

Sciences, Shiraz, Iran

123

Mol Biol Rep (2013) 40:6255–6260

DOI 10.1007/s11033-013-2737-6

TNF-a can modify intracellular signal transduction, ionic

channel activities [12], or absorption/secretion functions

[13]. Other cytokines also play some roles in the gall-

bladder and its epithelial cells [13]. IL-4 is a secretary

cytokine of Th2 cells, whose polymorphism located in

promoter, is related to its production. IFN-c and TGF-bpolymorphism are suited in ?874 and -894 regions,

respectively. It is believed that TGF-b polymorphism is

relevant to its production. TGF-b ?869 T allele is asso-

ciated with higher production of TGF-b1 [14]. TNF-a and

IL-6 as pro-inflammatory cytokines normally affect local

and systemic events. Thus, cytokines associating to dif-

ferent aspects of a disease would be the targets of most

treatment. Since the amount of cytokine protein synthesis

or its function is determined by some polymorphisms in

their genes [15]; studying the cytokine gene polymor-

phisms might be helpful to clarify the starting or progress

of the disease, efficacy of treatment and disease outcome.

Previously, it was reported that polymorphisms at these

positions mentioned cytokine are associated with cytokine

production or function [14, 16–18]. Vishnoi et al. [19]

showed that TNF-a-308 (G/A) and IL-6-174 G/C were not

significantly different in GBC patients in comparison to

healthy controls. Also, in another study, he showed that

there is not any significant correlation between IL-10 gene

polymorphism and risk of gallstone disease in Indian

population [20]. In order to evaluate the role of these

polymorphisms in gallstone formation, we analyzed the

genetic variants of TNF-a A-308G, IL-6G-174C, TGF-bT?896C, IFN-c T?874A, IL-4 C-590T, and their associ-

ation with gallstone formation.

Materials and methods

Study populations

A total of 158 patients with that admitted to Karaj Alborz

Hospital, Karaj, Iran were enrolled and underwent surgery,

consecutively recruited from 2009 to 2011. Their ages

ranged from 26 to 79 years. The histology of gallstone was

confirmed by a pathologist. We included age, sex, ethnic-

ity, disease history, medications, and history of gallstone

formation and exist of other diseases in questionnaire form.

We excluded patients that were not match for ethnicity or

patients that had other diseases. A total of 254 genetically

unrelated normal subjects as a control group were matched

for sex and age with the patients. Blood was taken by

venipuncture from these subjects and the questionnaires

were filled. The study protocol was approved by the ethics

committee of our institution, and written informed consent

was obtained from all participants.

DNA extraction

Blood was taken from these patients before surgery and

control group. Genomic DNA was extracted from Buffy

coat, using a QIAamp DNA Mini Kit (Qiagen, Germany)

according to the manufacturer’s instructions.

Determination of cytokine gene polymorphisms

Cytokine gene polymorphisms were evaluated by poly-

merase chain reaction using a thermal cycler (Techne,

Genius, UK). PCR conditions, PCR cycles and primers are

summarized in Table 1 and 2. ARMS-PCR method was

carried out for TNF-a A-308G [16], IL-6 G-174C [17],

TGF-b T?869C [18], and IFN-c T?874A [14] in 25 ll

reaction mixtures. A beta globin gene primer was used as

an internal control. PCR–RFLP method in a final volume of

25 ll was used for determining the IL-4 C-590T gene

polymorphisms [14]. After PCR, the products were diges-

ted by Ava II (Fermentas, Lithuani) restriction enzyme and

the amplified products were monitored by agarose gel

electrophoresis and ethidium bromide staining (Fig. 1).

Statistical analysis

Allele and genotype frequencies were calculated in patient

and control subjects by direct gene counting. Statistical

evaluation was carried out using the Statistical Package for

the Social Sciences (SPSS), version 16. The frequencies of

the alleles/genotypes were compared in cases and controls

by Chi Square test and Fisher’s exact test. Odds ratios and

95 % confidence intervals (CIs) for relative risks were

calculated. A probability value of P \ 0.05 was considered

Table 1 The PCR conditions for the IL-6, TNF-a, TGF-b, IL-4 and

IFN-c

Locus PCR conditions

TNF-a-A308G 10 cycle: 94 �C 30 s, 61 �C 50 s, 72 �C 40 s

20 cycle: 94 �C 20 s, 56 �C 50 s, 72 �C 40 s

50 ng DNA, 200 lmol dNTPs, 0.7 mM MgCl2

IL-6 G-174C 10 cycle: 94 �C 30 s, 61 �C 50 s, 72 �C 40 s

20 cycle: 94 �C 20 s, 56 �C 50 s, 72 �C 40 s

50 ng DNA, 200 lmol dNTPs, 0.7 mM MgCl2

TGF-b T?869C 10 cycle: 94 �C 30 s, 64 �C 50 s, 72 �C 40 s

20 cycle: 94 �C 20 s, 59 �C 50 s, 72 �C 40 s

50 ng DNA, 200 lmol dNTPs, 3 mM MgCl2

IL-4 C-590T 35 cycle: 94 �C 20 s, 53 �C 50 s, 72 �C 50 s

50 ng DNA, 200 lmol dNTPs, 3 mM MgCl2

IFN-c T?874A 10 cycle: 94 �C 30 s, 62 �C 50 s, 72 �C 40 s

20 cycle: 94 �C 20 s, 56 �C 50 s, 72 �C 40 s

50 ng DNA, 200 lmol dNTPs, 0.7 mM MgCl2

6256 Mol Biol Rep (2013) 40:6255–6260

123

as statistically significant and all the reported p-values were

two-tailed. Arlequin 3.0 software was used to evaluate

deviations from expected Hardy–Weinberg genotypic

proportions.

Results

Among consecutive patients and control 26 and 32 % were

male, respectively (aged from 26 to 79 years) and 74 and

68 % were female (aged from 32 to 78 years). The mean age

of the patients and control group were 41.22 ± 13.53 and

49 ± 11.4, respectively. Male to female ratio (M/F) was

0.35(41/117) in patients and (82/172) 0.47 in the control

group.

Patient’s demography and the indications for gallstone

disease are summarized in Table 3 and the frequencies of

cytokine gene polymorphisms in patients and normal sub-

jects are shown in Table 4. Allele frequencies of TGF-bT?869C variants were significantly different between gall-

stone patients and normal subjects. The frequency of TGF-b?869T allele was significantly higher than that of TGF-b?869T allele (P = 0.04, OR = 1.33, 95 % CI = 1–1.79,

study power = 52 %) in normal subjects. Regarding to

TNF-a A-308G and IL-6 G-174C pro inflammatory cytokine

and, IL-4 C-590T Th2 and IFN-c T?874A Th1 cytokine

markers gene polymorphisms, the differences in distribution

of genotype and allele between patients and normal group

were not significant (P [ 0.05). In addition, after categori-

zation of patients according to their gender the IL-6 in male

group have significant differences between patients and

control (P = 0.034). All polymorphisms were not fitted to

Hardy–Weinberg equilibrium. Armitag’s trend test was used

to check the association of genotypes with acute rejec-

tion whenever the Hardy–Weinberg equilibrium did not

meet.

Table 2 The Primer, PCR methods and product size for the IL-6, TNF-a, TGF-b, IL-4 and IFN-c

Locus Primers Product size Method

TNF-a-A308G Common: 50-AAGAATCATTCAACCAGCGG-30 273 bp ARMS-PCR

G allele: 50-ATAGGTTTTGAGGGGCATCG-30

A allele: 50-ATAGGTTTTGAGGGGCATCA-30

IL-6 G-174C Common: 50-GAGCTTCTCTTTCGTTCC-30 190 bp ARMS-PCR

C allele: 50-CCCTAGTTGTGTCTTGCC-30

G allele: 50-CCCTAGTTGTGTCTTGCG-30

TGF-b T?869C Common:50-TCCGTGGGATACTGAGACACC-30 241 bp ARMS-PCR

C allele: 50-GCAGCGGTAGCAGCAGCG-30

T allele: 50-AGCAGCGGTAGCAGCAGCA-30

IL-4 C-590T Forward: 50-TAAACTTGGGAGAACATGGT-30 TT genotype (195 bp),

CC genotype (175, 20 bp),

CT genotype (195, 175, 20) bp

Ava II based RFLP

Reverse: 50-TGGGGAAAGATAGAGTAATA-30

IFN-c T ? 874A Common: 50-TCAACAAAGCTGATACTCCA-30 262 bp ARMS-PCR

A allele: 50-TTCTTACAACACAAAATCAAATCA-30

T allele: 50-TTCTTACAACACAAAATCAAATCT-30

Beta globin Forward: 50-ACACAACTGTGTTCACTAGC-30 110 bp Internal control

Reverse: 50-CAACTTCATCCACGTTCACC-30

Fig. 1 TGF-b gel electrophoresis. Line 1 marker (100 bp), Line 2,3

CC patient, Line 4,5 CT patient

Table 3 Patient’s demography and the indications for gallstone

disease

Demographic characteristics Patients Controls

Age (year) 41.22 ± 13.53 39.2 ± 11.3

Male/female ratio (N/N) 0.35 (41/117) 0.47 (81/173)

Surgery Positive Negative

Positive familial history of gallstone 18 % Negative

Ethnicity Persian Persian

Cholesterol pigment in pathology Positive Negative

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123

Discussion

The role of cytokines in gallbladder and their probable

influences on gallstone formation are poorly understood [2,

10]. According to the importance of gallstone treatment and

control of biliary tract disorders, we tried to investigate the

impress of cytokines contributing the inflammations or the

symptoms and outcomes of disorders. The liver Kupffer cells

and biliary tract secrete several mediators and cytokines [9].

Gallbladder epithelial cells produce IL-6, IL-8, TNF-a,

TGF-b and several other cytokines as well as some receptors

of these mediators [10, 11]. TNF-a as an inflammatory

cytokine, directly affect the absorption, secretion, and

functions of gallbladder epithelial cell as well as intracellular

signal transduction regulating the ionic channel activities of

these cells [12, 13]. Previous observations suggested that

mucin overproduction is critical factor in the pathogenesis of

gallstones [21]. Among pro-inflammatory cytokines, TNF-ahas been reported to modify the expression of MUC5AC and

MUC2 genes controlling the mucin secretion in murine

intrahepatic biliary epithelial cells [22]. Increased expres-

sion of these genes and TNF-a is now believed to be asso-

ciated to gallstone disease [21]. Also, bacterial infection can

enhance gallbladder cytokine production and gallstone for-

mation [23]. As in gallbladder epithelial cells exposed to

bacterial LPS, an increase in TNF-a mRNA and its secretion

was illustrated [24]. IL-6 is another pro-inflammatory cyto-

kine produced by gallbladder epithelial cells and has the

same functions as TNF-a. It is produced by macrophage

lineage and its production increased by exposure of GBECs

to LPS [24]. Gallstone formation can be due to several fac-

tors relating to the mechanisms of cholesterol increase.

Gallbladder had several important roles. Apart from its role

in cholesterol metabolism, it makes cholesterol and pigment

to be discarded and this may leads to gallstone formation.

Other factor is gallbladder cell absorption and secretion,

inflammation and dysmotility of gallbladder, bacterial LPS

and mucin production [25]. Pro-inflammatory cytokines

Table 4 Results of IL-6 G-174C, TNF-a G-308C, TGF-b ?869C, IL-4 C-590T and IFN-c T?874 A single nucleotide polymorphism

determined in 154 patients with gallstone and 254 control

Gene Genotype Gallstone N (%) Normal N (%) P value OR 95 % CI Study power

IL-6 G-174C CC 20 (12.66 %) 35 (13.78 %) 0.75 1.1 0.59–2.07 5

GC 55 (34.82 %) 76 (29.93 %) 0.30 0.8 0.50–1.25 18

GG 83 (52.54 %) 143 (56.30 %) 0.45 1.16 0.77–1.77 11

G allele 221 (69.94 %) 362 (71.26 %) 0.68 1.07 0.77–1.47 6

C allele 95 (30.07 %) 146 (28.75 %)

TNF-a G-308A GG 98 (62.03 %) 163 (64.18 %) 0.65 1.1 0.7–1.69 6

GA 32 (20.26 %) 48 (18.90 %) 0.73 0.90 0.54–1.56 5

AA 17.73 (28 %) 43 (16.93 %) 0.83 0.95 0.54–1.65 4

G allele 228 (72.16 %) 374 (73.63 %) 0.64 1.08 0.78–1.49 7

A allele 88 (27.85 %) 134 (26.38 %)

TGF-b T?869C CC 41 (25.95 %) 83 (32.68 %) 0.14 1.39 0.87–2.21 31

CT 62 (39.25 %) 102 (40.16 %) 0.85 1.04 0.68–1.59 4

TT 55 (34.82 %) 69 (27.17 %) 0.099 0.70 0.44–1.10 37

C allele 144 (45.57 %) 268 (52.76 %) 0.04* 1.33 1–1.79 53

T allele 172 (54.44 %) 240 (47.25 %)

IL-4 C-590T CC 85 (53.80 %) 145 (57.09 %) 0.51 1.14 0.75–1.74 10

CT 52 (32.92 %) 68 (26.78 %) 0.18 0.75 0.47–1.18 26

TT 21 (13.30 %) 41 (16.15 %) 0.43 1.26 0.69–2.30 12

C allele 222 (70.26 %) 358 (70.48 %) 0.94 1.01 0.73–1.39 3

T allele 94 (29.75 %) 150 (29.53 %)

IFN-c T?874A TT 47 (29.75 %) 71 (27.96 %) 0.96 0.92 0.58–1.45 6

AT 60 (37.98 %) 94 (37.01 %) 0.80 0.96 0.62–1.48 4

AA 51 (32.28 %) 89 (35.04 %) 0.56 1.13 0.73–1.76 8

A allele 162 (51.27 %) 272 (53.55 %) 0.52 1.10 0.82–1.48 9

T allele 154 (48.74 %) 236 (46.46 %)

N absolute number, CI confidence interval, OR odds ratio

* Considered significant with P value threshold of 0.05. In genotypes, each P value is the result of comparing corresponding row with the sum of

other rows

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such as TNF-a and IL-6 affect these gallbladder cells

absorption and secretion. They also influence the inflam-

mation and dysmotility. The effect of LPS in production of

these cytokines is obvious. Enhancement of inflammatory

cytokines such as, IL-1a and PGE2 were shown to affect the

epithelial cell absorptive function associated to bacterial

LPS level [13]. Gallstone could induce and increase

inflammatory cytokines, and chronic inflammatory condi-

tions in gallstone disease and thus may lead to gallbladder

cancer. Secreted inflammatory cytokines could increase the

risk of carcinoma by several factors; for example, inducible

expression of nitric oxide synthase (iNOS) cause high gen-

eration of NO. Also, increased production of cytokines

including IL -1b, IFN-c and TNF-a leads to DNA damage

[26]. These mechanisms maybe common in gallstone for-

mation and subsequent outcome and disorders. However,

another investigation indicated that circulation levels of pro-

inflammatory cytokines such as IL-6, 8, 18, and TNF-a in

gallstone-induced acute pancreatitis was not significantly

different from normal group [27]. It should be mentioned that

pro-inflammatory cytokines are not harmful for billary tract.

In IL-6 deficient mice, it was shown that IL-6 has an impact

on maintenance and integrity of hepatocyte mass during

chronic injury [28]. The amount of cytokine production is

important in gallstone disease; for instance, IL-4 deficiencies

can accelerate gallstone formation in mice. This study also

indicated that IL-4 don’t impress the cholesterol metabolism

[29], Unlike IL-4, IFN-c increased in gallstone patients [26,

29]. It is also clarified that IL-4 has an anti-tumor role against

human biliary tract carcinoma. These data indicate some

beneficial task of IL-4 and dangerous function of IFN-c in

gallstone and biliary disorders [30]. IL-4 and maybe other

anti-inflammatory cytokines regulate gallstone formation,

gallbladder inflammatory conditions and subsequent disor-

ders such as gallbladder carcinoma. In case of other anti-

inflammatory cytokines, TGF-b is a multifunctional cyto-

kine. Biliary epithelial cells contain TGF-b1 having the

ability to attenuate alloantigen-specific immune responses

[31]. Animal model and immunohistochemical studies

indicate that TGF-b1 expression increases according to GBC

progression and strongly influences angiogenesis and mac-

rophage infiltration [32]. It was also demonstrated that TGF-

b1 induces the morphogenesis of gallbladder epithelial cells

[33]. TGF-b1 modulates transitional events such as epithe-

lial- mesenchymal transition of human premalignant epi-

thelial cells and is up-regulated in the gallbladders of patients

with cholelithiasis compared to gallbladders removed

patients without gallstones [34, 35]. According to the roles of

these cytokines in gallbladder and roles in gallstone forma-

tion, in this study we evaluated the importance of gene

variations mentioned cytokines. Any way our results indi-

cate that T allele of TGF-b ?869 SNP is associated with

gallstone formation. TGF-b ?869 T allele is associated with

higher production of TGF-b1 [14]. According to the role of

TGF-b1 mentioned in previous, it may be a genetic risk

factor for gallstone formation [34, 35]. TGF-b1 affect gall-

bladder cells transition and functions and in agreement with

previous studies it’s up-regulated in gallstone disorders [34,

35]; TGF-b production from GBECs increase by exposure to

bacterial LPS [36]. Some studies demonstrated that cytokine

genetic variation is associated to production and gallstone

formation. For example IL-1a production by gallbladder

epithelial cell and its increase in gallstone patients was

shown [13]. Also, it was explained that a CC genotype of IL-

1 and haplotype 1/C of IL-1RN and IL-1b are related to

gallbladder cancer in patients with gallstone [37]. Vishnoi

et al. [38] also reported the association of TGF-b-509 CT

genotype with gallbladder cancer. As a conclusion, we can

conclude that inflammatory cytokines such as TNF-a and IL-

6 are important cytokines to increase the inflammation of

gallbladder and may contribute to accelerate gallstone for-

mation, these functions are the same as IFN-c but in contrast

to IL-4, can protect gallstone formation and some other

gallbladder disorders. TGF-b as a multifunctional cytokine

may modulate the inflammation of biliary tract; however,

elevated level of TGF-b contributes to gallbladder fibrosis

and promotion of disease. Aside from the role of cytokines

for gallstone formation and the impression of genetic back-

ground, in this study we could highlighted the TGF-b ?869 T

allele as a high producer. This is unlike the other cytokines

with no association to gallstone formation. For more eluci-

dations, further studies such as evaluation of gene poly-

morphisms associated to serum level of cytokines or other

mediators are needed.

Acknowledgments The authors are grateful to the Department of

Immunology of Tarbiat Modares University and Islamic Azad Uni-

versity, Kazerun branch for financial support.

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