Yonago Acta medica 2007;50:89–97

Association of Functional Gene Polymorphisms ofInterleukin-1 and Transforming Growth Factor- 1 with
the Progression of Liver Fibrosis in Japanese Patientswith Hepatitis C Virus-Related Chronic Liver Disease
Mari Mandai, Yoshikazu Murawaki, Kinya Okamoto, Hideyuki Ohtani, Naoto
Maeda and Isao Yuasa*
Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 and *Division of
Legal Medicine, Department of Social Medicine, School of Medicine, Tottori University Facultyof Medicine, Yonago 683-8503 Japan

Interleukin-1 (IL-1 ) and interleukin-1 receptor antagonist (IL-1RN) are key cytokines
in an inflammatory response, and transforming growth factor 1 (TGF- 1) promotes
hepatic fibrogenesis. The association of polymorphisms in the genes for these cytokines
with liver fibrosis is controversial. The aim of this study was to examine the possible
association of IL-1 , IL-1RN and TGF- 1 polymorphisms with the progression of liver
fibrosis in the Japanese population using cross-sectional and longitudinal study designs.
We examined 183 patients with hepatitis C virus (HCV)-related chronic liver disease (93chronic hepatitis and 90 with cirrhosis). Some of the chronic hepatitis cases were divided into progressive fibrosis and non-progressive fibrosis. IL-1 –31T/C, IL-1RN variable
number of tandem repeats (VNTR) and TGF- 1 +869 T/C polymorphisms were analyzed using a polymerase-chain reaction-based assay. In the cross-sectional study, there
were no significant differences in the genotype distributions of IL-1 , IL-1RN and TGF1 between chronic hepatitis and liver cirrhosis. No significant differences were foundamong Child-Pugh grades in cirrhosis patients. In the longitudinal study, there were no
significant differences in the genotype distributions of IL-1 , IL-1RN and TGF- 1 between progressive fibrosis and non-progressive fibrosis. No significant differences in the
speed at which liver fibrosis develop were found among the genotypes of IL-1 , IL-1RN
and TGF- 1. In disagreement with other studies, the functional gene polymorphisms of
IL-1 , IL-1RN and TGF- 1 were not associated with the progression of liver fibrosis inJapanese patients with HCV-related chronic liver disease.
Key words: interleukin-1 ; interleukin-1 receptor antagonist; liver fibrosis; polymorphism;
transforming growth factor- 1

In hepatitis C virus (HCV)-related chronic liverdisease, the progression of liver fibrosis is a common problem, and the rate of progression varies

markedly between patients (Marcellin et al., 2002;
McCaughan and George, 2004; Feld and Liang,
2006; Massard et al., 2006). Some patients with

Abbreviations: AST, L-asparate aminotransferase; ECM, extracellular matrix; HCV, hepatitis C virus; IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; RFLP, restriction fragment length polymorphism; TGF- 1, transforming growth factor- 1; VNTR, variable number of tandem repeats89

M. Mandai et al.

the progression of liver fibrosis in Japanese patients with HCV-related chronic liver disease.

chronic hepatitis C rapidly develop significant
fibrosis and cirrhosis, while others remain asymptomatic without progression. In general, an older
age at infection, excessive alcohol consumption,
male gender, overweight and immunodeficiency
are known to have an association with a more
rapid progression of fibrosis in chronic hepatitis C.
In addition, evidence indicates that genetic factors
influence the natural course of chronic hepatitis
C. Since fibrosis of the liver is a highly dynamic
process, a number of polymorphisms in functional
genes as host genetic factors influence its progression in patients with chronic hepatitis C, contributing to inter-individual variability (Bataller et al.,2003). Among these polymorphisms, especiallythe proinflammatory or profibrogenetic cytokine
gene polymorphisms are considered to strongly
influence the progression of fibrosis (Bataller etal., 2003). Indeed, the capacity to produce cytokines in individuals largely depends on genetic
polymorphisms. Several studies have examined
the possible association of gene polymorphismsof proinflammatory cytokines, interleukin-1(IL-1 ) and interleukin-1 receptor antagonist (IL1RN), and profibrogenetic cytokine, transforming
growth factor- 1 (TGF- 1), with the progressionof liver fibrosis in chronic hepatitis C (Powell etal., 2000; Gewaltig et al., 2002; Bahr et al., 2003;
Suzuki et al., 2003; Tanaka et al., 2003; Wang etal., 2005), but these results are controversial. In
this study, we investigated cross-sectionally andlongitudinally the association of functional polymorphisms in the genes for these cytokines with

Materials and Methods
Subjects
One hundred and eighty-three patients with HCVrelated chronic liver disease were enrolled in thisstudy (Table 1). All the patients were Japaneseand positive for HCV-RNA. The diagnosis of liver disease was confirmed by laboratory tests and/or histological examination. All patients were
negative for hepatitis B surface antigen and human immunodeficiency virus antibody. The subjects of this study were 93 patients with chronic
hepatitis (mean age 63.6 ± 10.8 years, 53 men and40 women) and 90 patients with liver cirrhosis(mean age 66.4 ± 9.4 years, 45 men and 45 women). Among the chronic hepatitis patients, we haveselected 43 who were followed up for 5 years, anddivided them into the progressive fibrosis group(n = 26, 13 men and 13 women, mean age 55 ± 9
years) and a non-progressive fibrosis group (n =17, 9 men and 8 women, mean age 63 ± 10 years)
according to the 5-year change in the FibroIndex(Koda et al., 2007) which was determined basedon the platelet count, L-asparate aminotransferase(AST) level and -globulin level, as an index forassessing fibrotic stage at both one point and follow up: 1.738 – 0.064 (platelets × 10 4 /mm 3) +0.005 (AST IU/L) + 0.463 ( -globulin g/dL). This

Table 1. Characteristics of patients

Age(year)
Gender(male/female)
AST(IU/mL)ALT(IU/mL)
Platlet(× 104/mL)
HCV RNA(kIU/mL)

Whole patients[183]

Patients withchronic hepatitis [93]Patients withliver cirrhosis [90]64.9 ± 10.2
98/85
68.7 ± 41.8
68.9 ± 52.6
14.8 ± 14.7
359 ± 294

63.6 ± 10.8
53/40
57.0 ± 38.3
68.8 ± 60.4
18.6 ± 17.1
462 ± 309

66.4 ± 9.4
45/45
81.4 ± 42.3
69.2 ± 42.4
10.7 ± 10.0
217 ± 193

ALT, L-alanine aminotransferase; AST, L-asparate aminotransferase; HCV, hepatitis C virus.
[ ], number of patients.

90

Cytokine genotypes and liver fibrosis

10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT,
500 ng/mL bovine serum albumin and 50% glycerol, pH 7.4), 1 μL of Alu I (10 units/µL) (Takara
Shuzo) and 12 μL of sterile H2O was incubatedat 37˚C overnight. The mixture was loaded ontoa polyacrylamide gel (total concentration 10%,concentration of cross-linker 3%, 1× TBE buffer:
89 mM Tris, 89 mM borate and 2.2 mM EDTA).
Electrophoresis was performed at 200V for 70
min at 20˚C using 1× TBE buffer. Bands werevisualized by silver staining (Fig. 1a).

study was approved by the Committee for the Ethics of Medical Experiments on Human Subjectsof the Medical Faculty of the Tottori University,and written-informed consent was obtained fromeach subject before blood was collected.
In addition, the rate at which fibrosis progressed was examined in some chronic hepatitis
patients identified at the exact same time point of
HCV-infection by careful anamnesis. The rate ofprogression per year was estimated as the fibrosis
stage (score 0 to 4) by New Inuyama classification(Ichida et al., 1996) at the time of biopsy divided
by the duration of infection with HCV or as thedifference in the fibrosis stage between the firstand second biopsy divided by the time interval
between the 2 biopsies.

Analysis of the IL-1RN VNTR genotype
The IL-1RN variable number of tandem repeats(VNTR) genotype was analyzed by amplification
followed by staining of the PCR products directly
according to Wang et al. (2003). The PCR was
carried out in a final volume of 25 μL: 2 μL (50ng) of genomic DNA, 0.2 μL of forward primer(5'-CCCCTCAGCAACACTCC-3'), 0.2 μL of reverse primer (5'-GGTCAGAAGGGCAGAGA-3'),2.0 μL of each nucleotide, 2.5 μL of MgCl2 , 1
unit of Taq DNA polymerase (Takara Shuzo), and18 μL of sterile H2O. The conditions for amplification were: initial denaturation at 95˚C for 10
min, followed by 5 cycles of denaturation at 94˚C
for 45 s, annealing at 65˚C for 30 s, and extension
at 72˚C for 30 s, then 30 cycles of denaturation at
94˚C for 30 s, annealing at 60˚C for 30 s, extension at 72˚C for 30 s, and finally 5 cycles of denaturation at 94˚C for 30 s, annealing at 55˚C for 30 s,
and extension at 72˚C for 30 s. The PCR products
were loaded directly onto a polyacrylamide gel(total concentration 10%, concentration of crosslinker 3%, 1× TBE buffer: 89 mM Tris, 89 mM
borate and 2.2 mM EDTA). Electrophoresis was
performed at 200 V for 120 min at 20˚C using
1× TBE buffer. Bands were visualized by silver
staining. Five alleles were assigned based on amplicon size: allele 1 (4 repeats) 442 bp, allele 2 (2repeats) 272 bp, allele 3 (3 repeats) 357 bp, allele
4 (5 repeats) 532 bp and allele 5 (6 repeats) 627
bp (Fig. 1b).

DNA extraction
Genomic DNA was extracted from peripheral
white blood cells using a DNA extracting kit (DNAQuick II: Dainippon Pharmaceutical, Osaka,
Japan) according to the manufacturer’s instructions.
Analysis of the IL-1 –31T/C genotype
The IL-1 –31T/C genotype was analyzed using the PCR with mutagic primers, followed by
the restriction fragment length polymorphism(RFLP) method according to Wang et al. (2003).
The PCR was carried out in a final volume of
25 μL: 2 μL (50 ng) of genomic DNA, 10 pmolof forward primer (5'-AGAAGCTTCCACCAATACTC-3'), 10 pmol of reverse primer (5'-AGC
ACCTAGTTGTAAGGAAG-3'), 200 μM of eachnucleotide, 1.5 mM MgCl2, 1 unit of Taq DNA
polymerase (Takara Shuzo, Kusatsu, Japan), and18 μL of sterile H2O. The conditions for amplification were as follows: initial denaturation at 95˚C for 1 min, followed by 36 cycles of denaturation
at 94˚C for 45 s, annealing at 54˚C for 50 s, andextension at 72˚C for 1 min. RFLP for IL-1 wascarried out. In brief, a mixture of 5 μL of PCR
product, 2 μL of 10× NE buffer (300 mM NaCl,
91

M. Mandai et al.

Fig. 1. Analysis for gene polymorphisms of IL-1 , IL-1RN and TGF- 1. a: IL-1 –31 T/C genotypes. b: IL-1RN
VNTR genotypes. c: TGF- 1 +869 T/C genotypes. IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist;
TGF- 1, transforming growth factor- 1.

at 200 V for 150 min at 10˚C using 1× TBE buffer
as a tank buffer. Bands were visualized with silver
staining (Fig. 1c).

Analysis of the TGF- 1 +869 T/C genotype
The TGF- 1 genotype was analyzed by PCR followed by RFLP according to Wood et al. (2000).
The PCR was carried out in a final volume of
40 μL: 3 μL of genomic DNA, 0.32 μL of forward primer 5'-TTCCCTCGAGGCCCTCCTA-3',
0.32 μL of reverse primer 5'-GCCGCAGCTTGGACAGGATC-3', 2.4 μL of each nucleotide,
4.0 μL of 25 mM MgCl2, 0.8 units of Taq DNA
polymerase (Takara Shuzo) and 29.8 μL of sterile H2O. The conditions for amplification were:
initial denaturation at 96˚C for 10 min, followed
by 30 cycles of denaturation at 96˚C for 60 s, annealing at 62˚C for 60 s, extension at 73˚C for 60 s
and 73˚C for 5 min. The RFLP analysis for TGF1 was carried out with a mixture of 10 μL of the
PCR product, 2.0 μL of 10× NE buffer (50 mM
NaCl, 10 mM Tris-HCl, 10 mM MgCl2 and 1 mM
dithiothreitol, pH 7.9), 0.2 μL of bovine serum albumin, 0.25 μL of MspA1-I (20 units/mL) (Takara
Shuzo) and 7.55 μL of sterile H2O. The solution
was incubated at 37˚C overnight. The mixtures
were loaded onto a polyacrylamide gel (total concentration 15%, concentration of cross linker 3%,
1× TBE buffer: 89 mM Tris, 89 mM borate and2.2 mM EDTA). Electrophoresis was performed

Plasma IL-1 concentration
The plasma concentration of IL-1 was measured
with an ELISA kit (BioSource Europe S.A.,
Nivelles, Belgium).
Statistical analyses
Values were expressed as the mean ± SD. Differences in genotype distributions were tested withthe 2 test. The differences of data between the
2 groups were assessed with a t test. A P value of< 0.05 was regarded as statistically significant.

Results
Genotype distributions and allelic frequencies of IL-1 –31 T/C, IL-1RN VNTR and TGF1 +869 T/C
Genotype distributions and allelic frequencies of
IL-1 –31, IL-1RN and TGF- 1 +869 are shownin Table 2. The frequency of each genotype was92

Cytokine genotypes and liver fibrosis

Table 2. Genotype distributions and allelic frequencies in patients with HCV-related chronic liverdiseaseGenotype distribution
IL-1 –31T/C		Allele frequency

Chronic hepatitis [92]Liver cirrhosis
[85]

T/T		
27%		
23%		

T/C		
49%		
46%		

C/C
P value
24%
0.539
31%		

T
0.51
0.46

C
0.49
0.54

P value
0.488

IL-1RN VNTR		
Chronic hepatitis [92]Liver cirrhosis
[91]

1/1
86%
87%

1/4
1%
2%

1/3
P value
1%
0.824
0%		

Allele 2
0.06
0.11

Others0.94
0.89

P value
0.999

TGF- 1 +869T/C		
Chronic hepatitis [91]Liver cirrhosis
[90]

T/T		
29%		
35%		

T
0.51
0.59

C
0.49
0.41

P value
0.228

1/2
11%
10%

2/4
1%
1%

T/C		
43%		
47%		

C/C
28%
18%

P value
0.211

For technical reasons, genotyping was not possible in all patients.
HCV, hepatitis C virus; IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; TGF- 1, transforming growth factor1; VNTR, variable number of tandem repeats.
[ ], number of patients.

in the Hardy-Weinberg equilibrium ( 2 = 1.597,
P = 0.450 for IL-1 ; 2 = 0.035, P = 0.983 for IL1RN; 2 = 0.938, P = 0.626 for TGF- 1). No
significant differences in genotype distribution or
allelic frequency were observed between chronic
hepatitis and liver cirrhosis, although the TGF- 1+869 C/C homozygote with higher transcriptional
activity tended to more often be in the chronic
hepatitis group. When the cases of cirrhosis weredivided into grade A and grade B + C according to the Child-Pugh classification (Pugh et al.,1973), there was no significant difference in genotype frequencies between the 2 groups grade A
and grade B + C groups at each gene (Table 3), although the TGF- 1 +869 C/C homozygote tended
to be high in grade A cirrhosis but not significant(P = 0.067).

Table 3. Genotype distributions and the ChildPugh classification in patients with liver cirrhosis
Genotype
IL-1 –31 T/C
T homozygote
C carrier
IL-1RN VNTR
Allele 2
OthersTGF- 1 +869T/C
T carrier
C homozygote

Child-PughA
B+C

P value

33 (79%)9 (21%)

24 (71%)
10 (29%)0.424

5 (11%)
40 (89%)4 (11%)
32 (89%)0.999

33 (77%)10 (23%)34 (92%)3 ( 8%)

0.067

IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; LC, liver cirrhosis; TGF- 1, transforming growth
factor- 1.

changes of FibroIndex did not differ between the
genotypes in the progressive or non-progressive
group.

Difference in genotype frequency of IL-1 ,IL-1RN and TGF- 1 between progressive
and non-progressive fibrosis

Speed at which liver fibrosis developed according to genotypes for IL-1 , IL1-RN and
TGF- 1

The chronic hepatitis patients were divided into
those with progressive fibrosis and those with
non-progressive fibrosis according to the 5-yearchange of FibroIndex (Table 4). No significant
differences in genotype frequency were observed
between the 2 groups. In addition, the 5-year

Among the chronic hepatitis patients in whom
the rate at which liver fibrosis developed wasestimated by careful anamnesis, comparisons93M. Mandai et al.

Table 4. Genotype frequency in the progressive and non-progressive fibrosis groups
Non-progressive group
FibroIndex		 Genotype(Δ)		 frequency

Genotype

Progressive group
FibroIndex		 Genotype(Δ)		 frequency

P value		IL-1–31T/CT homozygote
C carrier–0.311 ± 0.266–0.235 ± 0.146

6 (24%)
16 (76%)

0.433 ± 0.504
0.392 ± 0.424

3 (17%)
15 (83%)

0.832

IL-1RN
VNTR

Allele 2
Others–0.153 ± 0.159–0.284 ± 0.197

3 (11%)
24 (89%)0.344 ± 0.279
0.407 ± 0.444

2 (11%)
16 (89%)0.999

TGF- 1+869T/C

T carrier
C homozygote–0.234 ± 0.192–0.284 ± 0.197

18 (67%)9 (33%)

0.427 ± 0.502
0.407 ± 0.444

12 (71%)5 (29%)

0.999

Δ, increment variable; IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; TGF- 1, transforming growth factor- 1;
VNTR, variable number of tandem repeats.

Table 5. The speed at which liver fibrosis developed estimated by careful anamnasis among
genotypes
Liver fibrosis speed(stage/infection duration)

Genotype(mean ± SEM)

Table 6. The speed at which liver fibrosis developed estimated by follow-up liver biopsies
among genotypes

P
value

Liver fibrosis speed(stage/infection duration)P(mean ± SEM) value

Genotype

IL-1–31T/CT homozygote [ 6]C carrier[16]

0.117 ± 0.038
0.167 ± 0.050

0.560

IL-1
–31T/CT homozygote [ 4]C carrier[ 8]

0.046 ± 0.030
0.091 ± 0.126

0.811

IL-1RN
VNTR

Allele 2
Others[ 1][23]

0.108 ± 0.000
0.159 ± 0.036

0.772

IL-1RN
VNTR

Allele 2
Others[ 2] –0.240 ± 0.023[11] 0.027 ± 0.113

0.858

TGF- 1 T carrier[21]+869 T/C C homozygote [ 3]0.162 ± 0.040
0.094 ± 0.007

0.534

TGF- 1 T carrier[ 6]+869 T/C C homozygote [ 6]0.121 ± 0.169
0.032 ± 0.028

0.613

IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; TGF- 1, transforming growth factor- 1; VNTR, variable number of tandem repeats.
[ ], number of patients.

IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; TGF- 1, transforming growth factor- 1; VNTR, variable number of tandem repeats.
[ ], number of patients.

were made according to genotype (Table 5). No
significant differences in clinical findings suchas age, gender, infection duration, and laboratory
data were observed (Table 1). Although the C/Chomozygote of TGF- 1 tends to develop fibrosis
at a slower speed than the T carrier, there were no
significant differences in terms of each gene.

ed with the follow-up liver biopsies, the speed ofthe disease’s progression was examined according
to genotypes (Table 6). There were no statistically significant differences between the genotypes,
although the TGF- 1 C/C homozygote tended tohave a slower progression than the T carrier.
Plasma IL-1 concentration according to
genotype of IL-1 and IL-1RN

Speed of liver fibrosis estimated by follow
up liver biopsies among genotypes of IL-1 ,IL1-RN and TGF- 1

We examined the relationship of plasma IL-1
concentration with the genotypes of IL-1 and IL1RN, and found no significant differences in the
IL-1 concentrations between IL-1 –31C carrier

Among chronic hepatitis patients in whom therate at which liver fibrosis developed was estimat94

Cytokine genotypes and liver fibrosis

Table 7. Plasma IL-1 concentration according
to genotype

and T homozygote, and between IL-1RN allele 2
and the others (Table 7).

Discussion

Genotype

In the present cross-sectional and longitudinalstudies, we found no close association of functional gene polymorphisms of IL-1 –31T/C, IL-1RN
VNTR and TGF- 1 +869T/C with the progressionof liver fibrosis in Japanese patients with HCVrelated chronic liver disease, although the TGF- 1
C/C homozygote tended to have a slower progression of liver fibrosis.
IL-1 is a key cytokine in the inflammatory
response and its biological activities promotes various diseases including chronic hepatitis, in which
an increase in IL-1 stimulates the progressionof liver fibrosis. In fact, serum levels and hepatic
mRNA levels of IL-1 are reported to be elevatedin patients with chronic hepatitis C (Tilg et al.,
1992; Gramantieri et al., 1999). The IL-1 genefamily at 2q13-14 encodes 3 proteins. In this gene,some polymorphisms have seen reported: one isin the promoter region at position –511 and –31,
representing C T and T C transitions, which
are in near total linkage disequilibrium (El-Omar
et al., 2000). The IL-1 –31 C allele (–511 T)
has higher transcriptional activity than the IL-1
–31 T allele (–511 C) (Bataller et al., 2003) suggesting that –31C allele carriers may overproduce
the IL-1 protein, resulting in a more aggressive
necroinflammation and in a rapid progression ofliver fibrosis.
Takamatsu et al. (2000) have reported thatthe carriers of the –511 allele 2 (–31C) were significantly more common in Japanese patients withalcoholic cirrhosis than in those with noncirrhotic
alcoholic liver disease. By contrast, Bahr et al.
(2003) have reported that IL-1 –511T/C genotype
distribution was similar between HCV-induced
chronic hepatitis and cirrhosis in the German population. Tanaka et al. (2003) have also reported
that IL-1 –511C/T genotype distributions did not
differ between F0-F2 and F3-F4 in HCV-related

Plasma IL-1
(ng/mL)(mean ± SD)

P value

Il-1 –31T/C

T homoC carrier12.4 ± 14.2
13.5 ± 13.7

0.757

IL-1RN VNTR

Allele 2
Others12.6 ± 4.4
14.0 ± 14.1

0.753

IL-1 , interleukin-1 ; IL-1RN, interleukin-1 receptor antagonist; TGF- 1, transforming growth factor- 1; VNTR, variable number of tandem repeats.

chronic liver disease in the Japanese population.
In our study, we found no significant difference of
IL-1 –31T/C genotype frequency between HCVrelated chronic hepatitis and cirrhosis in Japanese.
Taken together, the IL-1 –31T/C genotype does
not influence the rate of progression of liver fibrosis in patients with HCV-related chronic liverdisease. In fact, we found no significant differencein the plasma IL-1 concentration in patients with
HCV-related chronic liver disease according to
IL-1 –31T/C genotypes or IL-1RN VNTR genotypes.
IL-1 RN, an anti-inflammatory molecule
which competes for binding with the IL-1 receptor, is encoded by the IL-1RN gene (Santtila etal., 1998). This gene has a penta-allelic 86-bp
tandem repeat polymorphisms VNTR in intron 2.
IL-1RN allele 2 has been shown to be associatedwith enhanced IL-1 production in vitro (Santtila et al., 1998) and in vivo (Hwang et al., 2002).
Bahr et al. (2003) have reported that IL-1RN
allele 2 is significantly more prevalent in HCVrelated cirrhosis as compared with HCV-related
chronic hepatitis. In the Japanese population,
Tanaka et al. (2003) have reported that IL1RNVNTR genotype distributions did not differ between F0–F2 and F3–F4 in HCV-related chronic
liver disease. In our study, we also found no significant difference of IL-1RN VNTR genotype
between HCV-related chronic hepatitis and cirrhosis in Japanese. As the reason for the differ95

M. Mandai et al.

Gewaltig et al. (2002) examined the association of TGF- 1 polymorphisms with the rate at
which fibrosis developed in 26 patients who underwent repeatable liver biopsy, and showed thatthe presence of proline at codon 10 and/or 25 was
associated with a faster progression of fibrosis:+869 C carriers and +915G/C heterozygote developed fibrosis faster. By contrast, our longitudinal
study, in which disease progression was estimatedbased on the duration of HCV infection or thechange of stage in follow-up biopsies, has shownthat the TGF- 1 +869 C/C homozygote had aslower rate of progression than T carriers, but thedifference was not significant. We can not presently explain this difference, but race, the evaluation system, and patient background may all have
had an effect. Large-scale, well-designed studies
are required to clarify this issue.
In conclusion, we did not find a close association of IL-1 , IL-1RN and TGF- 1 genotype
distributions with the progression of liver fibrosis
in HCV-related chronic liver disease in crosssectional and longitudinal studies.

ence between Caucasian and Japanese, the allelicfrequency itself has been attributed to be different
between the both populations. In fact, allele 2/2
of IL-1RN was observed in 10 to 30% of the Caucasian control population (Bahr et al., 2003), but
only 0 to 2% of the Japanese control population(Tanaka et al., 2003; Wang et al., 2003).
TGF- 1 is a pluripotent cytokine that promotes hepatic fibrogenesis by stimulating thesynthesis of extracellular matrix (ECM), inhibiting the degradation of ECM, and activation ofhepatic stellate cells. Indeed, the plasma TGF- 1
concentration is increased in patients with chronic
hepatitis, and correlated with the degree of liver
fibrosis (Murawaki et al., 1998; Thushima et al.,1999). In the human TGF- 1 gene, which is located at chromosome 19q13, eight polymorphisms
are presently known including –800G/A, –509C/T,+869T/C and +915G/C (Gewaltig et al., 2002).
The +869T homozygote, the +915G homozygote
and the –509T carrier are reported to have higher
levels of TGF- 1 (Grainger et al., 1999).
There are several studies about the association of TGF- 1 gene polymorphisms with theprogression of liver fibrosis in patients with HCVrelated chronic liver disease, but the results arecontroversial. Powell et al. (2000) have shownthat the TGF- 1 +915G/G homozygote with hightranscriptional activity was more often in stage
III/IV in Australian HCV-related chronic liverdisease patients, whereas TGF- 1 –509C/T and+869T/C did not differ among the stages of fibrosis. In contrast with Powell’s data, Wang et al.
(2003) have shown that the TGF- 1 +869 T carrier and +915 G/C heterozygote are more often
in stage III/IV in German HCV patients. Suzuki
et al. (2003) have shown no association of TGF1 +869T/C polymorphism with the progressionof liver fibrosis in Japanese HCV patients. Since
the TGF- 1 +915 G/C polymorphism was notobserved in Japanese (Suzuki et al., 2003), we
analyzed only the TGF- 1 +869T/C genotype inthis study. In agreement with Suzuki’s results,
we found no significant association of TGF- 1+869T/C with the progression of liver fibrosis in
HCV-related chronic liver disease.

Acknowledgments: This study was supported by a Grantin-Aid for Scientific Research (C) from the Ministry of
Education, Culture, Sports, Scientice and Technology ofJapan (No. 17590641).

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Received September 21, 2007; accepted October 25, 2007
Corresponding author: Yoshikazu Murawaki, MD

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