Genetic Polymorphism of CCR6-Rs2301436
Increases Susceptibility of HCC and Associates
with Tumor Severity in HCV-G4 Infected Egyptian
ABSTRACT
Objective: To provide a novel information of CCR6-
rs2301436 gene polymorphism effects on susceptibility
and severity of HCC among HCV related cirrhotics,
comparatively to CCR2-64Ile and CCR5-Δ32 allelic
variants.
Methods: 120 patients with HCC on background of HCV-
related liver cirrhosis; 120 cirrhotics without HCC and 240
healthy controls were recruited in this study. All subjects
were genotyped for: CCR2-64Ile, CCR5-Δ32 and CCR6-
rs2301436.
Results: There was increased risk of having HCC among
heterozygotes of +/64Ile of CCR2, +/∆32 of CCR5 and
G/A of CCR6-rs2301436 after adjusting for non-matched
variables compared with wild genotypes when HCC
patients were tested against either cirrhotics without HCC
or healthy controls.
Only carriers of CCR6-rs2301436 (G/A and A/A) were
significantly associated with increased risk of 4.1 folds,
4.1 folds and 2.7 folds for lymphatic permeation, distant
metastasis and advanced TNM staging of HCC,
respectively, after adjusting for confounders. We found no
significant association between those gene polymorphisms
and any of serum markers of liver injury.
Conclusion: CCR6-rs2301436 gene polymorphism is
involved in development and severity of HCC in HCV
related Egyptian cirrhotics. This contribution could be not
induced by liver injury.
Keywords- CCR6-rs 2301436 gene polymorphism,
hepatocellular carcinoma, HCV genotype 4, CCR2-64Ile,
CCR5-Δ32 and Egyptian cirrhotics.
INTRODUCTION
Beyond 170 million people worldwide are infected with
hepatitis C virus (HCV) [1], the mainstream of whom will
create chronicity with long term impediment such as
cirrhosis, liver failure and hepatocellular carcinoma (HCC)
[2]. Chemokines umpire the recruitment of inflammatory
cells into tissues and back into the lymphatics and
peripheral blood. Hence, they are requisite to the spatial
rescue of regulatory immune cells [3]. Also, chemokines
made by malignant cells aid survival of the malignant cells
[4]. CCR2 and CCR5 are two of a cluster of six
chemokine receptor-genes mapped to 3p21 [5]. Ligands
for CCR2 include CCL2, CCL7, CCL8 and CCL13, all of
which are expressed in the hepatic environment. CCR2 has
a crucial role in innate and adaptive immunity, T helper
(Th1) inflammation and hepatic fibrosis [6].
The ligands for CCR5 comprise CCL3 (macrophage
inflammatory protein-1α; MIP-1α), CCL5 (regulatory
upon activation, normal T-cell expressed; RANTES),
which is always released by thrombin-stimulated platelets.
Yet CCR5 associated chemokines direct recruitment of
effector T cell to the portal tracts [7]. CCR6 is another C-
C chemokine receptor which is encoded by a gene located
in the long arm of chromosome 6 (6q27) [8], CCL20
(previously known as MIP-3α) [9]. It was found that
CCL20-CCR6 axis showed a significant up-regulation in
HCC tissues [10], [11]. Several allelic variants of these
chemokine receptors have been reported. A substitution
mutation in the open reading frame of the CCR2, resulting
in a valine (Val) to isoleucine (Ile) at amino acid 64
designed CCR2(64Ile), and a 32-bp deletion in the CCR5
gene leading to a non-functional protein and CCR5
promoter single nucleotide polymorphism (SNP) designed
CCR5-Δ32 were identified [12], [13]. Both CCR
mutations have been reported to be associated with
delayed disease progression in human immunodeficiency
virus-1 (HIV-1) infected individuals [14], [15]. Also,
several studies have mentioned the role of those
polymorphisms in HCV infection with contrastive results
[16]–[20].
Although no clear associations between both CCR2-64Ile
or CCR5-Δ32 mutation and development of HCC were
initially reported [21], [22] , a recent study suggested that
CCR2-64Ile gene polymorphism is an important factor for
the susceptibility of HCC but it might not influence the
tumor progression[23]. Currently, SNP in CCR6 with a
reference sequence (rs) of 2301436 has been reported [24].
This SNP was detected in different diseases [24]-[26],
however, no expression data are currently available
concerning its role in HCC.
Taken in account all these facts, we hypothesized that the
CCR6 gene polymorphism could influence the
susceptibility of HCC. Thus, the aim of this study was to
provide a novel information of CCR6-rs 2301436 gene
polymorphism effects on susceptibility and severity of
HCC among Egyptian patients with HCV- genotype
4(G4)-related liver cirrhosis comparatively to CCR2-64Ile
and CCR5-Δ32 allelic variants.
2. MATERIALS AND METHODS
2.1 Eligible Subjects
This prospective, hospital- based and case-controlled study
was conducted in Internal Medicine Department at Minia
University Hospital, Egypt between December 2012 and
March 2014. A series of patients with HCC on background
of HCV-related liver cirrhosis was enrolled consecutively
from the attendants of the outpatient clinics. The group of
HCC patients was compared with two other groups:
patients with HCV-related liver cirrhosis and healthy
controls.
2.2 Hepatocellular Carcinoma Group
They were 79 (65.8%) male, 41 (34.2%) female. The
diagnosis of HCC was based on the characteristic criteria
of the National Guidelines for HCC [27]. The diagnosis
was frequently confirmed histologically [28].
2.3 Liver Cirrhosis Group
This group comprised 120 patients: 77 (64.2%) were
males and 43 (35.8%) were females with HCV-related
liver cirrhosis. Liver cirrhosis was defined histologically
[29], or non-histologically by evidence of portal
hypertension in the presence of chronic liver disease.
2.4 Control Group
A total of 240 healthy individuals were randomly selected
from medical and paramedical staff of the same hospital as
a control group. They were 162 (67.5%) male, 78 (32.5%)
female. Patients and controls were unrelated and
prospectively matched for race, ethnicity and
demographics.
2.5 Informed Consent
The present study was approved by the Institutional Ethics
Committee of School of Medicine, Minia University,
Egypt. The guidelines and regulations of the 1975 Helsinki
Declaration and International Conference on
Harmonization Guidelines for Good Clinical Practice were
followed. All study subjects gave informed written
consent to participate in this study.
2.6 Laboratory Evaluation
The whole blood specimens collected from the study
subjects, were placed in tubes containing EDTA and
immediately centrifuged and stored at -80°C until used for
genomic DNA extraction and genotyping. Data for age,
sex, race, ethnicity, residential area, alcohol consumption,
tobacco smoking and current history of type2 diabetes
mellitus (T2DM) that was diagnosed according to the
American Diabetes Association Classification criteria [30]
were obtained. Other clinico-pathological characteristic
such as hepatitis B surface antigen (HBsAg), antibodies to
HCV (anti-HCV) and human immunodeficiency virus
(anti-HIV), polymerase chain reaction (PCR) for HCV-
RNA, HCV genotypes, status of liver function in terms of
Child-Pugh class, AFP, total bilirubin, aspartate
aminotransferase (AST), alanine aminotransferase(ALT),
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serum albumin, prothrombin time (PT), international
normalized ratio (INR), platelet count, stage of HCC and
imaging studies were examined. Staging of HCC was
assessed by tumor-nodes- metastasis (TNM) clinical
staging system [31].
Anti-HCV was detected by second generation ELISA kits
(Ortho-Clinical Diagnostics Co. Inc, Tokyo, Japan) and
HCV RNA was measured in serum using a standardized
automated qualitative real time – PCR (Abbot M 2000,
USA), with lower detection limit of 12 IU of HCV
RNA/ml. The HCV genotypes were determined using
reverse hybridization (Innolipa; Inngenetics, Genetics.
Gent, Belgium). All patients were tested positive for anti
HCV-G4.
HBs-Ag was assessed by radioimmunoassay kits
(Lumipulse II HBs Ag , Fujirebio Co. Inc, Tokyo, Japan).
Exclusion criteria included patients who co-infected with
HBV and/or HIV, those who had other causes of liver
disease, other cancers by an initial screening examination,
in addition to those who had a history of alcohol intake of
≥ 80 gm/ day for longer than 10 years.
2.6.1 DNA Extraction
Genomic DNA was extracted from the whole blood by
Thermoscientific Gene Jet Genomic DNA Purification
Kits (Sigma, UK) according to the manufacturer’s
instructions.
2.6.2 CCR2 Genotyping
For characterization of the CCR2 polymorphism, sequence
specific primer PCR was used (SSP-PCR). The primers
used were CCR2- 440,441 and 442, Table 1.
Amplification was done using kits from Thermo Scientific
Dream Taq Green PCR Master Mix (2x) (Sigma, UK)
according to manufacturer’s instructions.
2.6.3 CCR5 Δ32 Genotyping
CCR5Δ32 genotype was determined by sizing PCR
amplicons that include the entire region of the deletion
sense and antisense primers, Table 1. Amplification was
determined by using Thermoscientific Dream Taq Green
PCR Master Mix (2x) kits (Sigma, UK) according to
manufacturer's instructions.
Table 1 Primers used for CCR2-64Ile, CCR5-Δ32 and CCR6-rs 2301436 DNA typing.
Polymorphism PCR Primers
CCR2- 64Ile
CCR2 440 5' -TGGGCAACATGCTGGTCA-3'
CCR2 441 5'-CCAAAGACCCACTCATTTG- 3'
CCR2 442 5'-GTGGGCAACATGCTGGTCG-3'
CCR5- Δ32 5'-TGTTTGGGTCTCTCCCAG-3'
5ʹ-CACAGCCCTGTGCCTCTT-3
CCR6-rs 2301436 5'-CCATTCTGGGCAGTGAGTCA-3'
5'-AGCAGCATCCCGCAGTTAA-3'
2.6.4 CCR6-rs 2301436 Genotyping
CCR6- rs 2301436 genomic variants were detected using
PCR, followed by a restriction fragment length
polymorphism (RFLP) assay. The presence of the G
nucleotide of position rs2301436 of CCR6 gene creates a
recognition site for the Hpa II enzyme. Primers used are
shown in Table 1. Amplification was determined using
Thermoscientific Dream Taq Green PCR Master Mix (2x)
kits (Sigma, UK) according to manufacturer’s instructions.
2.7 Statistical Analyses
Supported on the gene frequencies, expected phenotype
frequencies were designed according to the Hardy-
Weinberg equation and contrasted with the practical
frequencies via the χ2 test. Results were analyzed using
SPSS (Chicago, IL), version 21. Categorical variables
were described as frequency and percentage. Continuous
variables were presented as mean+ standard deviation
(SD). Logarithmic transformation was performed for
variables that were non-normally distributed. Categorical
variables were compared using χ2 –test or Fisher's exact
test when appropriate. Student′s-t test and one-way
ANOVA followed by Post Hoc test were used to compare
continuous variables. The odds ratios (ORs) and adjusted
odd ratios (AOR) with their 95% confidence interval (CI)
were predictable by simple logistic regression
investigation, whilst the accustomed odds ratios (AORs)
with their 95%CI were predictable by various logistic
regression models following calculating for other
confounders. A p- value<0.05 was considered significance.
3. RESULTS
3.1 Characteristics of the study subjects
A total of 480 subjects were enrolled in the present study:
120 patients with HCC on background of HCV-related
liver cirrhosis (group I); 120 patients with HCV-related
liver cirrhosis not complicated by HCC (group II), and 240
healthy controls (group III). Their initial characteristics are
displayed in Table 2. The 3 study groups were matched for
age, sex, residential area and exposure to smoking. Group
I and II were matched for platelet count, serum levels of
total billirubin, albumin, INR, AST, viral load and
functional status of liver in terms of Child-Pugh class
whereas group I had statistically increased proportion of
T2DM (59(49.2%) vs. 29(24.2%), p=<0.001) and serum
ALT (102.1±73.47 IU/L vs. 68.5±32.5 IU/L, p=0.05)
compared with group II. When the patients groups were
compared with controls (group III), each showed
statistically significant increased serum values of total
billirubin, AST, ALT and INR and showed significantly
decreased values of platelet count and serum albumin.
3.2 The Genotypic Distribution and Allelic
Frequency of CCR2-64Ile, CCR5-∆32 and
CCR6-rs2301436 Gene Polymorphisms in the
Study Groups
In our recruited control group, the frequencies of 64Ile of
CCR2 (p > 0.05, χ
2
value = 0.01); ∆32 of CCR5 (p > 0.05,
χ
2
value= 0.05) and G/A of CCR6-rs 2301436 (p> 0.05, χ
2
value =0.03) were in Hardy-Weinberg equilibrium,
respectively.
The OR and AOR with their 95%CI of genotypic
distributions of CCR2-64Ile, CCR5-∆32 and CCR6-
rs2301436 compared with wild genotypes in the group of
HCC and the group of liver cirrhosis without HCC are
shown in Table 3. For heterozygotes of CCR2-64Ile
(+/64Ile), CCR5-∆32 (+/∆32) and CCR6-rs2301436 (G/A),
they had a significant risk of 7.6 folds (95% CI=4.1-8.1,
p=<0.001); 5.7 folds (95% CI=1.63-6.1, p=0.01) and 4.9
folds (95% CI=2.4-9.8, p=<0.001) to have HCC,
respectively, compared to subjects with wild genotype
after adjusting for T2DM and other SNPs. Also,
individuals with homozygotes of CCR2-64Ile (64Ile/64Ile),
CCR5-∆32 (∆32/∆32) had 6 folds risk (95% CI=1.2-9.8,
p=0.03) and 5.3 folds risk (95% CI=1.1-5.8,p=0.04),
respectively on the susceptibility of HCC, although there
was not a significance, after adjusting for T2DM and other
SNPs. No A/A homozygous carriers could be detected
among patients of either group I or II.
The relationship between genotypic distributions of the 3
gene polymorphisms compared with wild genotypes in
HCC patients and healthy controls are shown in Table 4.
Heterozygotes of CCR2-64Ile (+/64Ile), CCR5-∆32
(+/∆32) and CCR6-rs2301436 (G/A) had a significant risk
of 6.2 folds (95% CI=3.9-7.7, p=<0.001); 7 folds (95%
CI=3.2-7.5, p=<0.001) and 9.98 folds (95% CI=9.7-16.3,
p=<0.001) to have HCC, respectively, compared to
subjects with wild genotype after adjusting for T2DM and
other SNPs. None of the healthy controls exhibited
homozygosity of any of the studied gene polymorphisms.
Analysis of frequency of 64Ile, ∆32 and A alleles in the
studied groups revealed that the presence of those alleles
had a significantly higher risk of occurrence of HCC after
controlling for T2DM and other SNPs when compared
with wild alleles in HCC patients and either cirrhotics
without HCC (AOR=5.9, 95%CI= 3.2-10.8, p=<0.001;
AOR= 3.3, 95%CI= 1.8-5.8, p=<0.001 and AOR= 8.1,
95%CI= 2.1-9.1, p= 0.002, respectively) or controls
(AOR= 5.2, 95%CI= 4.9-8.4, p=<0.001; AOR= 10.3,
95%CI= 1.2-19.5, p=0.04 and AOR=8.5, 95%CI= 7.9-9.8,
p=<0.001, respectively), Tables 3 and 4.
3.3 The Relationship between Genotypic
Frequencies of the Studied Gene
Polymorphisms and Clinical Pathological
Characteristics in HCC Patients
Due to the small number of homozygotes for CCR-64Ile,
CCR5-∆32 and CCR6-rs 2301436 alleles among the study
groups, they were pooled with heterozygotes into a single
subgroup to be compared with wild homozygotes. Except
for CCR5-∆32 carriers who were older than wild type
homozygotes (57.2 ± 5.7 vs 54.5 ± 4.5, p= 0.01), patients
Table 2 Baseline characteristics of the study groups and the tumor clinicopathological features in hepatocellular
carcinoma (HCC) patients.
Variable
Cirrhotics With
HCC (I)
N=120
Cirrhotics Without
HCC (II)
N=120
Controls
(III)
N=240
P Value
Age: (years)
Range
M ± SD
(43-69)
54.8 ± 4.3
(45-65)
54.1 ± 6.4
(40-60)
54.1 ± 3.1
0.342*
I vs II I vs III II vs III
0.61 0.55 1.0
Sex: n (%)
Male
Female
79 (65.8)
41 (34.2)
77 (64.2)
43 (35.8)
162 (67.5)
78 (32.5)
0.815*
I vs II I vs III II vs III
0.79 0.75 0.53
Residence: n (%)
Urban
Rural
42 (35)
78 (65)
45 (37.5)
75 (62.5) 91 (37.9)
149 (62.1)
0.859*
I vs II I vs III II vs III
0.69 0.59 0.94
Smoking: n (%)
Yes
No
20 (16.7)
100 (83.3)
26 (21.7)
94 (78.3) 45 (18.8)
195 (81.2)
0.61*
I vs II I vs III II vs III
0.33 0.63 0.51
International Journal of Medicine and Medical Sciences, ISSN:2051-5731, Vol.48, Issue.1 1690
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REFERENCES
polymorphism was the 32 base pair deletion CCR5-∆32
[12]. Investigations on the potential association of CCR5-
∆32 gene polymorphism with occurrence of HCC are
scarce. To the best of our knowledge, only Nahon et al
[21], [22] who have studied the role of this gene
polymorphism on the risk of HCC among both alcohol-
related cirrhosis patients as well as HCV- infected patients,
they reported a lack of association of CCR5-∆32 gene
polymorphism with occurrence of HCC among both
alcohol-related cirrhosis patients [21] and HCV- infected
patients [22]. Therefore, the present study is the first to
show a significant association of heterozygotes for CCR5-
∆32 gene polymorphism with development of HCC after
adjusting for other confounders when the group of HCC
patients were tested against either cirrhotics or healthy
controls, Table III and IV. It has been reported that CCR5-
∆32 allelic variant creates a truncated protein that fails to
reach the cell surface [38], however, the mechanism
behind this variant interaction with HCC development has
to be elucidated. In the current study, the carriage of
CCR5-∆32 allele was significantly related to old age and
the severity of liver disease (decreased platelet count),
Table V. Old age and advanced stages of underlying
cirrhosis are major risk factors for HCC [39].
It was reported that the genetic expression of either CCR2
or CCR5 was not associated with tumor severity in HCC
patients including; tumor number, tumor size, lymph node
metastasis, distant metastasis, clinical stage and Child-
Pugh grade, indicating that those genetic polymorphisms
had unapparent influence on its functional level. Although
similar results were reported by Yeh et al., [23] in the case
of CCR2-64Ile allelic carriers, extra studies are desirable.
It is significant to note that CCR2-64Ile and CCR5-∆32
gene polymorphism are in linkage disequilibrium, owing
to next site of CCR2 and CCR5 genes with a chemokine
receptor gene cluster on human chromosome 3p21[40]. In
this regard, we must take into account while considering
statistical involvement among particular polymorphisms
and clinical product, since recognized polymorphism may
presently be a sign related to the other polymorphism that
has a straight consequence on disease outcome. In order to
conquer this obstacle, we attuned for other gene
polymorphisms as we tested the association of a certain
gene polymorphism and risk of HCC.
To the best of our knowledge, this is the first study to
provide novel information about the effects of CCR6-
rs2301436 gene polymorphism on susceptibility and
severity of HCC in HCV-related cirrhosis patients. Our
study revealed that G/A heterozygotes had a significant
increased risk of 4.9 folds (95%CI= 2.4-9.8) to have HCC
compared with wild homozygotes when HCC patients
were studied against patients with liver cirrhosis. This risk
increased to 9.98 folds (95%CI=9.67-16.3) when the
group of HCC patients was studied against healthy
controls. No A/A homozygotes of CCR6- rs2301436 SNP
could be elicited in the studied groups.
By univariate analysis, the demographic characteristics
and liver-related serological markers were not influenced
by the carriage of CCR6-rs2301436 allele, whereas,
carriers had significant increased proportions with
lymphatic permeation, distant metastasis, and advanced
TNM stage compared with non-carriers. Also, in
multivariate analysis, we found that carriers CCR6-
rs2301436 allele had a significant high risk to have
lymphatic permeation, distant metastasis and advanced
TNM stage compared with non-carriers after adjusting for
other SNPs and non-matched confounders including
T2DM.
Northfield et al., [41], reported that CCL20 functions as a
chemoattractant for CCR6+ lymphocytes and DCs, as well;
serum levels of CCL20 are considerably high in CHC and
persist to amplify sophisticated fibrosis and cirrhotic
disease [41]. Appearance of this chemokine is also
obviously improved in hepatoma cells compare directly
with vascular endothelial growth factor (VEGF)
expression and tumor size [42]. High levels of CCR6
mRNA has also been noticed on Th17 cells and regulatory
T cells (Tregs) within the inflammatory tumor
microenvironment [11]. This sturdily implied role of the
CCR6-CCL20 axis in regulating cirrhosis and HCC
associated angiogenesis encouraged us to guess that its
allelic variant must has a position in this story,
nevertheless, the exact mechanism has to be yet clarified.
In the current study, we insisted of including a control
group of liver cirrhosis that was matched with the HCC
group for demographic characteristics and disease stage to
put aside any possibility that the studied polymorphisms
contribute to fibrosis progress and the development of
cirrhosis, rather than directly to the pathogenesis of HCC.
Also, the increased significant occurrence of T2DM
among HCC patients was considered on performing
statistical analysis to guard against its definite role in
pathogenesis of HCC [43].
Besides, no significant correlation between the three
studied chemokine genetic polymorphisms and serum
markers including; AFP, AST and ALT could be found in
the current study. Hence, we considered that the influence
of these genetic variants on susceptibility of HCC could
not through liver injury. Because of the relatively small
size of our cohort and monocentric nature of the study, our
study should be considered an exploratory trial.
5. CONCLUSION
Our results suggest that heterozygotes of CCR2-64Ile,
CCR5-∆32 and CCR6-rs2301436 allelic variants may be
crucial host genetic factors associated with susceptibility
of HCC in Egyptian cirrhotics infected with HCV- G4.
However, only G/A heterozygotes of CCR6-rs2301436
gene polymorphism might influence the tumor severity
and this contribution could be not induced by liver injury.
These results highlight the need of further studies
including functional relevance and clinical implications.
Conflict of Interest
The authors declare that there are no conflicts of interest.
International Journal of Medicine and Medical Sciences, ISSN:2051-5731, Vol.48, Issue.1 1691
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