Protective Effect of HLA-DRB1*11 and Predisposition of HLA-C*04 in the Development of Severe Liver Damage in Brazilian Patients with Chronic Hepatitis C Virus Infection.
ABSTRACT The objective of this study was to investigate human leucocyte antigen (HLA) genes in patients chronically infected with hepatitis C virus (HCV) and to analyse the possible role of these genes in the progression of chronic hepatitis C. One hundred and forty-five (145) Brazilian patients infected only with HCV genotype 1 were evaluated. HLA class I (A*, B*, C*) and class II (DRB1*, DQA1*, DQB1*) typing were carried out by PCR-SSO, through Luminex technology. Associations were found with protection against development of liver damage by both DRB1*11 (5.0% versus 18.2%, P = 0.0016, OR = 0.23, CI 95% = 0.09-0.58; Pc=0.0208) and DRB1*11-DQA1*05-DQB1*03 haplotype (4.2% versus 15.3%, P = 0.0032; OR = 0.24, CI 95% = 0.08-0.64). Liver damage was associated with HLA-C*04 in patients with <20 years of infection (38.4% versus 9.1%, P = 0.002, OR = 6.25, CI 95% = 1.97-19.7; Pc=0.0238). It is concluded that HLA alleles can influence the development of liver damage in HCV type-1 chronically infected Brazilian patients.
This is an Accepted Article that has been peer-reviewed and approved for publication in the
Scandinavian Journal of Immunology, but has yet to undergo copy-editing and proof correction.
Please cite this article as an “Accepted Article”; doi: 10.1111/j.1365-3083.2012.02755.x
Received date: 18-April-2012
Accepted date: 09-Jul-2012
Regular Manuscript: PROTECTIVE EFFECT OF HLA-DRB1*11 AND PREDISPOSITION OF
HLA-C*04 IN THE DEVELOPMENT OF SEVERE LIVER DAMAGE IN BRAZILIAN
PATIENTS WITH CHRONIC HEPATITIS C VIRUS INFECTION
Short title: HLA-DRB1*11 and C*04 on liver damage
Amanda Vansan Marangon1, Giovanni Faria Silva2, Camila Fernanda Verdichio de Moraes3, Rejane
Maria Tommasini Grotto3, Maria Inês de Moura Campos Pardini3, Dayse Sousa de Pauli4, Jeane
Eliete Laguila Visentainer, Ana Maria Sell1, Ricardo Alberto Moliterno1.
1Immunogenetics Laboratory, Maringá State University, UEM, Maringá-PR, Brazil.
2Gastroenterology Division, Department of Internal Medicine, Botucatu Medical School, São Paulo
State University, UNESP, Botucatu-SP, Brazil.
3Molecular Biology Laboratory of Blood Transfusion Center, Botucatu Medical School, São Paulo
State University, UNESP, Botucatu-SP, Brazil
4Clinical Hospital of Londrina, Londrina State University, UEL, Londrina-PR, Brazil.
MSc. Amanda Vansan Marangon
Laboratório de Imunogenética, Departamento de Ciências Básicas da Saúde,
Universidade Estadual de Maringá, Av. Colombo, 5790; Maringá, PR, Brasil
Tel: +55 44 3011 4864; Fax: +55 44 3011 4931
Scientific heading: Basic Immunology
The objective of this study was to investigate HLA genes in patients chronically infected with
hepatitis C virus, and to analyze the possible role of these genes in the progression of chronic
hepatitis C. One hundred and forty-five (145) Brazilian patients infected only with HCV genotype 1
were evaluated. HLA class I (A*, B*, C*) and class II (DRB1*, DQA1*, DQB1*) typing were
carried out by PCR-SSO, through Luminex technology. Associations were found with protection
against development of liver damage by both DRB1*11 (5.0% vs. 18.2%, P=0.0016, OR=0.23, CI
95%=0.09-0.58; Pc=0.0208) and DRB1*11-DQA1*05-DQB1*03 haplotype (4.2% vs. 15.3%,
P=0.0032; OR=0.24, CI 95%=0.08-0.64). Liver damage was associated with HLA-C*04 in patients
with less than 20 years of infection (38.4% vs. 9.1%, P=0.002, OR=6.25, CI 95%=1.97-19.7;
Pc=0.0238). It is concluded that HLA alleles can influence the development of liver damage in
HCV type-1 chronically infected Brazilian patients.
The authors wish to thank all the volunteers who were part of this study. We thank Fabiano
Cavalcanti Melo and Marco Antonio Braga for technical performance. We thank Dr. Dennis
Armando Bertolini and Dr. Luiza Tamie Tsuneto for reading the paper.
KEYWORDS: HLA; Fibrosis; Cirrhosis; Liver; Hepatitis C
Hepatitis C virus (HCV) infection is one of the main causes of chronic liver disease that
occurs in approximately 50 to 85% of infected individuals [1,2] The chronic hepatitis C can be
evidenced by histopathologic changes, starting with liver inflammation, frequently associated with
fibrosis, possibly evolving into cirrhosis, and in some cases, hepatocellular carcinoma. . The
mechanisms responsible for the onset and progression of hepatic lesions during chronic hepatitis C
are not fully understood. Factors such as viral genotype, age, gender, concurrent infections, and
alcoholism may influence the disease progression; however, these factors individually cannot
explain why many patients evolve benignly for decades while others rapidly progress towards end-
stage liver disease [4-7].
Hepatitis C Virus seems to have no direct cytopathic effect on the infected hepatocytes as
healthy individuals may host the virus [8,9]. In that case, the disease’s progression and liver
damage may apparently be the result of the host’s immune response against the virus, which
involves a complex interaction between the innate and adaptive immune response [9, 10]. So the
diversity of genes related to the immune response may explain, in part, the immune response
variability to infections. Among the many immune response gene families are the human leucocyte
antigen (HLA) class I and class II genes of the major histocompatibility complex. Several studies
have related HLA variants to the onset of infection or spontaneous viral clearance of HCV [11-15];
however, studies that evaluate the association of HLA with the severity of liver damage in chronic
HCV patients are scarce, particularly in Brazilian populations. In that context, the objective of this
work was to investigate HLA gene polymorphism in Brazilian chronic HCV patients with different
levels of fibrosis/cirrhosis, and analyze the possible role of these polymorphisms in the progression
and severity of chronic hepatitis C.
MATERIALS AND METHODS:
Between September 2004 and January 2009 a total of 761 patients received the diagnostic of
HCV infection at Internal Medicine Department, Gastroenterology Division, Botucatu Medical
School, São Paulo State University, UNESP, Botucatu, SP, Brazil (22º53'09" S; 48º26'42" W).
From those, 145 unrelated patients with infection only by the HCV genotype 1 were selected to be
included in this study. A total of 616 patients were excluded by the following exclusion criteria:
human immunodeficiency virus positive serology, hepatitis B virus co-infection, other hepatic
diseases, presence non-1 HCV genotype, and history of antiviral therapy before liver biopsy.
Clinical data from the patients were collected from their medical records. The studied protocol was
approved by the Ethics Committee for Research Involving Human Beings of the São Paulo State
University, Botucatu, São Paulo, Brazil, and all subjects provided written consent to participate in
The biopsies in all patients were carried out using the technique of Menghini or with Tru-Cut
needle to determine the degree of fibrosis and progression of infection. The fragments were
analyzed when at least eight portal spaces were present and were stained with hematoxylin and
eosin, masson trichrome and reticulin stain. The hepatic biopsies were analyzed by a pathologist
using the METAVIR score: F0 - no fibrosis; F1 - portal fibrosis without septa; F2 - portal fibrosis
with few septa; F3 - numerous septa without cirrhosis; and F4 – cirrhosis .
The patients were stratified into subgroups according to the fibrosis stage: absence of fibrosis
or mild fibrosis (F0-F2, n=85), advanced fibrosis or cirrhosis (F3-F4, n=60), without cirrhosis (F0-
F3), with cirrhosis (F4).
HCV genotyping was defined through reverse line probe assay technique (INNOLIPA®
v.1.0, Innogenetics, Ghent, Belgium), according to manufacturer instructions. This genotyping was
preceded by the extraction of total viral RNA present in patient’s plasma, followed by reverse-
transcription-polymerase chain reaction (RT-PCR), using the Amplicor HCV test version 2.0 kit
(Roche Diagnostic Systems, Branchburg, NJ, USA).
Patient’s genomic DNA was extracted from 250 µL of peripheral venous blood collected into
a tube containing EDTA, using the NeoIsocolumn kit (One Lambda Inc., San Diego, CA, USA).
HLA typing (HLA-A*, B*, C*, DRB1*, DQA1*, and DQB1*) was carried out using the
polymerase chain reaction-sequence specific oligonucleotides technique (PCR-SSO, One Lambda®,
Canoga Park, CA, USA), in which the amplified product was hybridized with microspheres linked
to specific probes for HLA allele groups (One Lambda®, Canoga Park, CA, USA). Reaction
readings were done by flow cytometry using Luminex technology (One Lambda®). Samples were
analyzed through the HLA FusionTM software (One Lambda Inc., San Diego, CA, USA).
HLA specificity frequencies between the groups were compared by 2x2 contingency tables
using the chi-square test with Yates’ correction or Fisher’s Test. Significant P values were corrected
by the number of alleles studied in each locus for associations not previously characterized (Pc;
Bonferroni correction). Odds ratio values with 95% confidence interval were also calculated.
Statistical analyses were performed using the
SISA statistics software program
(http://www.quantitativeskills.com/sisa/). As the gametic phase of alleles from the different loci is
not known – segregation analysis into families was not carried out – haplotypes and the calculation
of their frequency were determined by the likelihood-ratio test, through the Arlequin statistical
software program . The Hardy-Weinberg equilibrium  was achieved by calculating the
expected genotype frequencies and comparing them to the observed values, also using Arlequin
The patients’ characteristics are summarized in Table 1. According METAVIR score, 58.6%
of the patients presented no or mild fibrosis (4.8% were F0, 29.7% F1, and 24.1% F2), and 41.8%
presented advanced fibrosis or cirrhosis (15.2% were F3, and 26.2% F4). The mean age of the
patients with advanced fibrosis or cirrhosis were higher than for the patients without fibrosis or mild
fibrosis (48.6 ± 9.8 years versus 39.8 ± 9.3 years, respectively, P<0.05). The same trend was seen
when comparing F4 group versus F0-F3 group (27.8 ± 9.6 years versus 19.9 ± 7.0 years
respectively, P<0.05). With respect to the possible risk factors to acquire hepatitis C, the use of
intravenous drugs was the main risk factor associated to F0-F2 group (27.1%), compared to only 2
patients (3.3%) of the F3-F4 group (P<0.05%). It also occurred when F0-F3 group was compared
with F4 group (20.5% versus 5.3%, P<0.05).
HLA Class II Group of alleles
To evaluate the role of HLA specificities in disease progression, the patients were firstly
stratified into individuals with absence of fibrosis or mild fibrosis (F0-F2, n=85), and individuals
with advanced fibrosis or cirrhosis (F3-F4, n=60). Next, the patients with cirrhosis (F4 stage, n=38)
were separated and compared with patients without cirrhosis (F0-F3 stage, n=107). As duration of
HCV infection is directly linked to liver damage and unfavorable prognosis, 90 informative patients
were divided into two groups according to the duration of infection by calculating the median for
the parameter (whether less than 20 years of infection, n=46; or more than 20 years of infection,
n=44), and then compared according to the different degrees of fibrosis in each group (F0-F2 versus
The DRB1*11 group of alleles was less frequent in the group with advanced fibrosis or
cirrhosis when compared to the group with no fibrosis or mild to moderate fibrosis (0.050 vs. 0.182,
P=0.0016, Pc=0.0208; OR=0.23, CI 95%=0.09-0.58, respectively) (Table 2). This group of alleles
was also less frequent in patients with cirrhosis than in those without cirrhosis but without statistic
significance (0.07 vs. 0.15, P=0.07). In patients with over 20 years of infection, the frequency of
HLA-DRB1*11 was absent in the F3-F4 patients when compared to F0-F2 ones (0 vs. 0.25,
P=0.009, Pc=0.117; OR=0.08, CI 95%=0.01-0.7), without statistics significance after Bonferroni
correction (Table 3). Moreover, DQA1*05 was also less frequent in the group with advanced
fibrosis or cirrhosis (0.191 vs. 0.329, P=0.013; OR=0.48, CI 95%=0.27-0.84); but the significance
was lost after Bonferroni correction, the P-value lost significance (Pc=0.065) (Table 4). No
significant differences were found when the groups were compared for HLA-DQB1* specificities.
HLA Class I Group of alleles
The frequencies of HLA Class I group of alleles are shown in Table 5. In the analysis by
duration of infection (Table 3), HLA-C*04 was associated with predisposition for liver damage (F3-
F4) in individuals infected for less than 20 years (0.384 F3-F4 vs. 0.09 F0-F2, P=0.002; OR=6.25,
CI 95%=1.97-19.7) and remained significant even after Bonferroni correction (Pc=0.0238).
Although not statistically significant, it was observed in patients infected for less than 20
years a higher frequency of HLA-B*35 in F3-F4 group when compared to F0-F2 group (0.27 vs.
0.075, P=0.03, Pc=0.75; OR=4.49, CI 95%=1.27-15.8) and in patients over 20 years was observed a
higher frequency of HLA-B*07 in F3-F4 group when compared to F0-F2 (0.146 vs. 0.025, P=0.06).
Moreover, the HLA-A*02 was more frequent in the group with advanced fibrosis when compared
to the group with no fibrosis or mild to moderate fibrosis (0.350 vs. 0.206, P=0.009; OR=2.07, CI
95%=1.24-3.52, respectively), and was also more frequent in the group with cirrhosis than in the
one without cirrhosis (0.368 vs. 0.228, P=0.02; OR=1.96, CI 95%=1.11-3.4, respectively).
However, after P correction (Bonferroni correction) the HLA-A*02 specificity lost its significance
(Pc=0.153 and Pc=0.416, respectively). Conversely, HLA-A*23 was less frequent in the F3-F4
group when compared to the F0-F2 group (0.008 vs. 0.06- P=0.029, OR=0.13, CI 95%=0.01-1.06),
and the significance was also lost after Bonferroni correction (Pc=0.493).
With regard to patients with cirrhosis (F4), although not significant, only the HLA-B*44
specificity showed higher frequency when compared to non-cirrhotic patients (0.197 vs. 0.10,
P=0.05; Pc=1.35; OR=2.14, CI 95%=1.04-4.39).
Twenty-two haplotypes were found in group F3-F4 and twenty-seven in the F0-F2. Only the
HLA-DRB1*11-DQA1*05-DQB1*03 haplotype was statistically less frequent in F3-F4 patients
than in F0-F2 patients (0.042 vs. 0.153, P=0.0032, OR=0.24, CI 95%=0.08-0.64) (Data not shown).
A high diversity of HLA specificities among the subjects with the different status of HCV
infection was showed in this study with Brazilian patients. The patients were carefully selected to
form a homogeneous group in terms of HCV genotype and absence of viral co-infections, and the
HLA-DRB1*11 specificity and the haplotype HLA-DRB1*11-DQA1*05-DQB1*03 were
associated with protection against the development of advanced fibrosis or cirrhosis, while the
HLA-C*04 was associated with predisposition for liver damage.
The HLA-DRB1*11 protection to severe liver damage is in accordance with other studies
which also found an association between DRB1*11 and protection against progression to chronic
disease or severity of hepatitis C in different populations. Asti et al. , in Italy, found the
DRB1*11:04 allele less frequently in patients with chronic liver disease of various severities, as
compared to asymptomatic individuals hosting HCV. In a study conducted in France, Renou et al.
 related the severity of hepatitis C to levels of alanine aminotransferase (ALT), by comparing
patients with chronic hepatitis C who had normal levels of ALT and patients with high ALT levels:
DRB1*11 was more frequent in the group with normal ALT levels. In another study in France, Hue
et al.  observed a lower frequency of DRB1*11 in patients with cirrhosis (level F4 in the
Knodell score) when compared to patients without cirrhosis (F0-F3 Knodell score). Tilmann et al.
 in Europe, observed that DRB1*11 was less frequent in individuals who had received
transplants for HCV-induced end-stage liver disease compared to blood donors. Although these
studies evaluate the progression of chronic hepatitis C in different ways, in all of them the
DRB1*11 specificity appeared less frequently in individuals with more advanced stages of the
disease. These data confirm that the DRB1*11 specificity as a favorable prognosis factor,
demonstrating that it plays a role not only in facilitating spontaneous HCV clearance as described in
others studies [14, 22-25], but also in avoiding further liver damage in patients chronically infected
by the virus. More important, the protection conferred by DRB1*11 in our study becomes evident
in the analysis by duration of infection, in which DRB1*11 was completely absent in patients with
more advanced stages of the disease and over 20 years of infection.
One possible explanation for the influence of HLA-DRB1*11 in HCV infection was
suggested by Godkin et al. , who identified a group of HCV epitopes restricted to HLA-DR11
that lead to a different T CD4+ lymphocyte response against HCV in infected patients. These
epitopes lead to an increase in the number of interferon gamma (INF-γ )-producing T cells, which
can be important in viral clearance and maintenance of its status as a self-limited infection, and may
also explain the association of HLA-DR11 with low liver injury in chronically infected HCV
With respect to the HLA-DQ specificities, the DQA1*05 also appeared less frequently in F3-
F4 patients, suggesting a protective role of this allele against liver damage; however, after
Bonferroni correction, its P value lost significance. Furthermore, this allele is in linkage
disequilibrium with DRB1*11(?’=0.46), indicating that this association is secondary to the
association with DRB1*11.
DRB1*11 has also been described in conjunction with DQB1*03 as a protective factor
against the development of chronic HCV infection [14, 27, 28]. However, our data did not confirm
the association between DQB1*03 and progression and/or severity of chronic hepatitis C. On other
hand, the DQB1*03 specificity appears in conjunction with DRB1*11 in haplotype analysis, in
which the HLA-DRB1*11-DQA1*05-DQB1*03 haplotype was significantly less frequent in F3-F4
patients than in F0-F2 patients. In a study conducted in Italy, this same haplotype was also
associated with protection against the development of hepatitis C. The author suggests that the
association between HCV infection and this haplotype is due primarily to the allele DRB1*11:04
. In the present study, the protective effect of the HLA-DRB1*11-DQA1*05-DQB1*03
haplotype seems to be due primarily to DRB1*11, as DQB1*03 was not associated with mild or
moderate levels of fibrosis.
DRB1*07 has been associated with higher susceptibility to persistent HCV infection [30, 31].
Although our data did not characterize an association of this specificity with severe liver damage in
chronic HCV patients, it is interesting to observe that in the F0-F2 group, only 2 of 31 DRB1*11
patients (0.06) concomitantly expressed the DRB1*07 specificity. On the other hand, in the F3-F4
group, 3 of the 6 DRB1*11 patients (0.5) featured DRB1*07 as well. Thus, these 3 patients would
have one protective and one susceptibility factor for the disease, and perhaps the DRB1*07 in these
individuals counteracts the protection afforded by the DRB1*11 specificity.
Virus-specific cytotoxic CD8+ T lymphocytes also play a role in viral control and liver
damage, and the activation of these cells requires interaction with HLA class I molecules . In
the present work, an association was observed between HLA-C*04 and the development of liver
damage in individuals with less than 20 years of infection. Although the stratification of the sample
by duration of infection significantly decreases sample size, this result is similar to other studies that
found an association of C*04 with persistence of HCV infection . Also in individuals with less
than 20 years of infection, the study found a trend of an association between B*35 and the
development of liver damage. This result is in accordance with a study in Korean patients, in which
the HLA-B35 antigen was found more frequently in HCV carriers when compared to healthy
individuals . It is worth observing that in HLA-C*04 or B*35 patients, the infection seems to
progress rapidly into more advanced forms of the disease, as these specificities were found
associated with the development of severe liver damage only in patients with less than 20 years of
infection. Nevertheless, the association of HLA-B*35 with advanced degree of fibrosis may be the
consequence of the strong linkage disequilibrium of this specificity with HLA-C*04, as all F3-F4
HLA-B*35 patients (11/11) also presented the C*04 specificity. On the other hand, two of the seven
F0-F2 HLA-B*35 patients were C*04-negative (data not shown). Furthermore, it was observed that
when HLA-B*35 individuals are excluded from the analysis, the association of C*04 with the
development of advanced fibrosis or cirrhosis (F3-F4) remained significant (0.20 vs. 0.017, P=0.01,
As with HLA-C*04 and HLA-B*35, the HLA-B*18 specificity was also more frequent in F3-
F4 individuals with less than 20 years of infection, but without statistical significance. Although our
data does not have the statistical power to confirm this association, this result is in accordance with
a study performed in England, in which a higher ratio of F2-F4 patients was observed expressing
HLA-B*18 when compared to controls . This specificity was also more frequent in patients
with hepatocellular carcinoma in a study carried out in Spain, suggesting that it may be involved in
the progression into more severe forms of the disease and more unfavorable prognosis .
Regarding HLA-A specificities, the HLA-A*23 was less frequent in the F3-F4 group,
suggesting protection against the development of liver damage; however, after Bonferroni
correction the P value lost significance. Contrary to this result, Thio et al.  found a higher
susceptibility to the development of chronic HCV infection in African-American patients who
showed HLA-A*23. On the other hand, the HLA-A*02 was more frequent in the F3-F4 group, but
the P value lost significance after Bonferroni correction. These associations must be confirmed in
separate studies. It is important to highlight that – unlike the present study – many HLA-hepatitis C
association studies included several viral genotypes in a single patient group, or the patients
featured co-infection by other viruses such as HBV and HCV, which can cause spurious
associations or mask genuine ones.
The associations found between HLA class I alleles and the development or protection against
liver damage in patients chronically infected by HCV may be related to the ability of these
molecules to feature HCV peptides with different affinities to cytolytic T lymphocytes. On the other
hand, some of these alleles, including C*04, interact with killer cell immunoglobulin-like receptors
(KIR) present in natural killer cells, which can influence the natural history of HCV infection [38-
40]. In fact, the involvement of diverse immune response genes, isolated or in combination can be
related to HCV clearance and disease progression. Suppiah et al.  in a large study investigated
the HLA-C alleles, KIR genes and the IL28B polymorphisms in patients with chronic hepatitis C
and found that these factors additively can predict response to therapy in chronic hepatitis C.
Finally, our findings suggest that HLA-DRB1*11 specificity may constitute an important
genetic factor not only in protecting against chronic infection as characterized in literature, but also
in protecting against severe liver damage in patients chronically infected by HCV. On the other
hand, HLA-C*04 may be a factor for rapid progression into more severe forms of the disease. In
this study, patients were carefully characterized regarding the “status” of chronic infection, duration
of infection, and other possible non-genetic interfering factors, forming a group with quite
homogenous characteristics. This effort may have more clearly characterized the host’s genetic
interfering factors leading to liver damage of chronically HCV-1 infected patients. We conclude
that the development of tissue liver injure in these patients seems to be under the influence of HLA
specificities, either positively or negatively, opening a role for HLA typing of chronically infected
HCV-1 patients as a way to evaluate both disease prognosis and the choice of a more aggressive
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