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The CCR5-delta32 mutation: impact on disease outcome in individuals
with hepatitis C infection from a single source

Gut August 2005;54:1157-1161

abstracted:
The effect that this mutation may have on HCV clearance an and
severity may be not only important in relation to those solely
infected with HCV, but also of vital importance to the vast numbers
who are coinfected with HIV, particularly as anti-CCR5 directed
medications are already being investigated for the treatment of HIV.

Authors- C Goulding1, A Murphy1, G MacDonald1, S Barrett2, J Crowe2, J
Hegarty3, S McKiernan1 and D Kelleher1

1 Department of Clinical Medicine and the Dublin Molecular Medicine
Centre, Trinity Centre for Health Sciences, St. James Hospital,
Dublin, Ireland
2 Centre for Liver Diseases, Mater Misericordiae Hospital, Dublin,
Ireland
3 Liver Unit, St Vincent's University Hospital, Dublin, Ireland

ABSTRACT
Background and aims: Chemokines are small polypeptides, a major
function of which is lymphocyte recruitment and trafficking. The aim
of this study was to assess the involvement of inherited variations in
CCR2, CCR5, and the ligand RANTES in determining disease outcome in
hepatitis C virus (HCV) infected individuals.

Methods: A total of 283 women, all exposed to HCV genotype 1b from a
single donor, and including those who had spontaneously cleared the
virus and those chronically infected, were genotyped for CCR2, CCR5,
and RANTES polymorphisms. The frequencies of these polymorphisms were
then compared with disease activity and severity.

Results: CCR5, CCR2, and RANTES genotypes were compared with HCV
polymerase chain reaction (PCR) status, alanine aminotransferase
levels, and liver histology. There was no significant relationship
between CCR2 or RANTES polymorphisms and disease outcome or severity.
However, CCR5delta32 heterozygotes were more likely to have
spontaneous clearance of the virus than those without the mutation
(42% PCR negative v 28.3% negative; p = 0.044, odds ratio 1.83 (95%
confidence interval 1.1-3.6)). Among the subgroup of DRB1*03011
negative individuals, previously found to be associated with more
severe inflammation, the difference in histological inflammatory score
(CCR5WT/WT = 4.9 v CCR5delta32/WT = 3.53; p = 0.043) was significant.

Conclusion: Heterozygosity for CCR5delta32 was shown to be
significantly associated with spontaneous hepatitis C viral clearance
and with significantly lower hepatic inflammatory scores in subgroups
within this cohort. Both controls and the HCV population had similar
heterozygosity frequencies.

DISCUSSION
This study showed significantly higher spontaneous HCV viral clearance
in the CCR5delta32/WT over the CCR5WT/WT group (p = 0.044). This
association was not found in Hellier's16 or Promrat's17 studies, both
of which had several confounding issues in relation to HCV genotype,
sex, and ethnicity. Specifically, Hellier's study comprised
individuals from multiple European populations and contained a lower
percentage of viral negative patients. HCV genotype was not specified
in this study. In addition, the infection came from multiple sources,
suggesting a high degree of HCV genetic heterogeneity. Hence a number
of confounding variables are inherent in these studies that may have
hindered the ability to detect changes in viral clearance. Our study
population had a number of relatively unique features, principally
that (i) all subjects were female and Caucasians of Irish descent and
(ii) all were infected by a single inoculum of HCV genotype 1b through
anti-D immunoglobulin in 1977.2 They had no other risk factors for
liver disease and no other significant comorbid illnesses.

The CCR5delta32 mutation arises from a 32 bp deletion causing a frame
shift mutation and premature termination of the protein. The resultant
CCR5 mutant protein is likely to be functionally inert as it not only
lacks the last three of seven putative transmembrane regions but also
the domains involved in G protein coupling and signal transduction.18
Indeed, Liu et al showed that the resultant protein was not detectable
on the surface of cells that would normally express it, and therefore
cannot act as a receptor.19 However, the role of the CCR5 mutation in
HCV is not the same as for HIV as the method by which HCV gains entry
into the cell is unknown, but unlike HIV, it is generally not believed
to be related to the CCR5 receptor.

The heterozygous genotype was present in 17.6% of the HCV population
and in 17.9% of the control population, with the CCR5delta32/delta32
genotype found in 0.32% and 0.69%, respectively, one homozygote in
each group. This is one of the highest carrier rates reported for any
European population and is consistent with the results of Libert et al
who investigated gene frequency in 18 European countries and found a
North/South gradient, the highest frequencies being in Finland (16%)
and the lowest in Sardinia (4%).20 Controversially, in 2002, Woitas et
al reported that CCR5delta32 homozygosity occurred three times more
frequently in anti-HCV antibody positive HIV negative individuals.21
The fact that this group remained HIV negative, despite multiple
exposure, would suggest that they were a selected population, most
probably on the basis of the CCR5delta32 genotype. Hence the increased
CCR5delta32 homozygosity most likely reflected resistance to HIV,
rather than increased risk of HCV infection.

In explaining how the CCR5delta32 polymorphism could alter HCV
clearance, we must consider that the effect of CCR5 heterozygosity in
acute HCV may not be representative of what happens in chronic HCV
infection. In acute HCV infection, clearance is associated with a
strong T cell response to a wide range of HCV specific antigens.22
Counterintuitively, lack of CCR5 may actually lead to increased T cell
expansion. This was demonstrated using an acute lymphocytic
choriomeningitis infection model in CCR5 knockout mice where clonal
expansion of antigen specific T cells was increased, not decreased,
among CD8+ and CD4+ T cells.23 Likewise, lack of CCR5 has been
associated with increased T cell production of IFN-, leading to the
suggestion that CCR5 might be part of a negative regulatory feedback
loop on acute T cell activation.24 In CCR5 deficient mice infected
with mouse hepatic virus there was reduced T cell infiltration at day
7, but by day 12 T cell infiltration was similar to wild-type and this
study also suggested that IFN production may have been increased in
the CCR5-/- group.25 Infection with leishmania donovani showed a shift
from an initial low to an exaggerated antigen specific IFN response at
eight weeks post infection in CCR5-/- mice, suggesting that perhaps
the impact of CCR5 alters during the course of an infection.26 In
contrast with the above, a study by Belnoue et al showed that CCR5-/-
mice infected with cerebral malaria had significantly reduced T cell
cerebral infiltration.27 These contrasting results may reflect CCR5
interaction with parasitic rather than viral infection.

This study also showed a trend towards less severe hepatic
inflammatory scores in CCR5WT/delta32 versus CCR5WT/WT individuals. In
a previous study, we identified HLA DRB1*03011 positivity as being
associated with reduced hepatic inflammation in this cohort. We did
not observe an additive effect of CCR5delta32 in DRB1*03011 positive
individuals, suggesting a dominant role for this HLA allele. However,
we observed significantly lower hepatic inflammatory scores for the
CCR5delta32/WT groups who were DRB1*03011 negative (p = 0.043). In a
recent publication by Hellier et al, a significant decrease in portal
inflammation, but not in overall necroinflammatory score, was found
among CCR5delta32 heterozygotes.16 CCR5delta32 is associated with
reduced migration of circulating lymphocytes in response to ligands
such as MIP-119 and we also observed this in vitro in CCR5delta32
heterozygotes (data not shown).

In HCV there is predominantly a Th1 response in the liver. CCR5 is
expressed on Th1 cells and facilitates the migration of T cells primed
by antigen. Although the number of HCV specific cytotoxic lymphocytes
in the liver is low during infection, there are many activated/memory
T cells present, most of which express CCR5. It has been reported that
in HCV patients, liver infiltrating lymphocytes showed increased
expression of CCR5,28 which correlated with histological severity.29
Similarly, animal studies have shown a key role for CCR5 in hepatic
lymphocyte migration.30 Hence reduced expression of CCR5, associated
with heterozygosity for CCR532, could be mechanistically associated
with less hepatic inflammation, due to reduced migration of CCR5
expressing cells.

Both Hellier and Promrat found an association with the RANTES -403
promoter polymorphism and reduced hepatic inflammation in a subgroup
of patients, which was not found in this study. It is possible that
ethnic variation in the RANTES polymorphism and patient numbers may
partly explain differences between these studies.

There is clearly much work yet to be done in this very exciting area,
particularly detailed functional studies. While there is a detectable
effect on HCV clearance seen in this study, further studies are
required to determine whether such data are generalisable to the
broader HCV infected population. Such studies will require large
numbers of patients and will also require either genetic homogeneity
regarding ethnic origin or stratification given the wide diversity in
allele frequency for this polymorphism even in Caucasian European
populations. The effect that this mutation may have on HCV clearance
and severity may be not only important in relation to those solely
infected with HCV, but also of vital importance to the vast numbers
who are coinfected with HIV, particularly as anti-CCR5 directed
medications are already being investigated for the treatment of
HIV.31,32

BACKGROUND
Chronic hepatitis C virus (HCV) infection has a prevalence of
approximately 2% worldwide.1 Up to 70% of those exposed to HCV remain
chronically infected. Many aspects of response to HCV are poorly
understood, including the reasons for the wide variation in disease
severity and spontaneous clearance versus chronic infection. Viral
genotype, age at exposure, and alcohol consumption are known
determinants of disease severity but the role of genetic
predisposition to chronic infection remains unclear. The population in
this study, comprising 283 women infected by contaminated anti-D
immunoglobulin in 1977, represent a unique opportunity to assess a
large HCV population with minimal confounding issues.2 The anti-D
immunoglobulin was contaminated from a single source and therefore all
women were infected with the same genotype (that is, genotype 1b),
minimising the varying effect that genotype could have on the study.

Chemokines are small polypeptides with a significant role in leucocyte
recruitment and trafficking. Leucocyte recruitment during inflammation
requires intercellular communication between infiltrating leucocytes,
endothelial cells, and parenchymal cells, mediated by early response
chemokines. Migration of T cells is modulated in vitro and in vivo by
conditioning with chemokines.3 CCR5 is a receptor for the
proinflammatory chemokines macrophage inflammatory protein 1±
(MIP-1±), MIP-1ß, and RANTES, which have key roles in host responses
to viruses in both human and murine disease.4 A 32 base pair (bp)
deletion in CCR5 results in a protein that is not detectable at the
cell surface. Homozygosity for this deletion is found in 1% of
Caucasians and has been shown to be protective against human
immunodeficiency virus (HIV) infection, while the heterozygote state,
which is found in 10% of Caucasians, leads to slower disease
progression. Its role in other viral infections remains to be
determined.

CCR5 and other chemokines play an important role in T cell
differentiation. CD4+T cells can differentiate into Th1 or Th2 cells
depending on their exposure to chemokines. CCR5 is expressed on Th
cells and also on memory and activated T cells. Migration of antigen
primed T cells is facilitated by CCR5.5 When human T cell clones were
analysed, CCR5 appeared to be expressed at higher levels on Th1 cells,
whereas many Th2 clones had no expression of CCR5. Sallusto et al
demonstrated that CCR5 expression depends on the activation state of T
cells and that its expression is upregulated by IL-2.6 It has been
proposed that in HCV, there is predominantly a Th1 response in the
liver.7,8 Indeed, progressive liver damage in HCV is associated with
upregulation of Th1 cytokines (interferon (IFN) and interleukin
(IL)-2), as shown by Napoli et al, where increased expression of
IFN-“ and IL-2 correlated with both fibrotic and portal inflammatory
histological scores.7

CCR5 along with CCR2 are two of a cluster of six chemokine receptor
genes mapped to 3p21. CCR2 codes for a minor HIV receptor, for which a
G to A coding sequence polymorphism resulting in a valine to
isoleucine substitution, designated CCR264I, has been described. It
appears that CCR2WT is in complete linkage disequilibrium with
CCR5?32, which is 10 kb away. Thus in a study of 3000 individuals,
Smith and colleagues9 demonstrated that the delta32 mutation is never
seen on the same haplotype as the CCR264I mutation. The distribution
of CCR2 and CCR5 in cells and tissues is very similar. CCR2 signalling
also promotes Th1 development in infection models and studies using a
CCR2 knockout mouse have shown that these mice have a 46% reduction in
lymphocyte recruitment to sites of infection and inflammation and an
80% reduction in CD4+T cells locally.10 RANTES, the CCR5 ligand, is
also critical for lymphocyte recruitment, as it attracts memory and
activated T cells. An A to G mutation of RANTES has been described at
position -403, resulting in an additional GATA transcription factor
binding site, with the mutant promoter having up to an eightfold
higher constitutive transcriptional activity than the wild-type.11

In normal liver, RANTES expression is restricted to a few scattered
hepatocytes. However, in HCV infected livers its expression was
significantly elevated, especially in periportal and lobular areas
that had the most lymphocytic infiltration.12 In view of this, we
postulated that genetic variation in either CCR5 receptors or in the
chemokines binding to such receptors might have an impact on outcome
of hepatitis C infection. The impact of such variation might be
difficult to detect in populations in which there was heterogeneity in
terms of ethnicity, viral genotype, and source and dose of infection.
Hence we have undertaken a study of the genetic impact of both the
CCR532 mutation and the RANTES position -403 mutation on the outcome
of hepatitis C infection in a genetically homogenous population
infected through a single source.

PATIENTS AND METHODS
Study population
The study population of 283 women was recruited from the outpatient
hepatology units in St James, St Vincent's, and the Mater
Misericordiae Hospitals in Dublin. All women attending these units who
had been exposed to HCV via contaminated anti-D immunoglobulin in 1977
were invited to participate. The group included those who were
chronically infected, persistently HCV RNA positive as determined by
reverse transcription-polymerase chain reaction (RT-PCR), and those
who had cleared the virus (that is, remained HCV antibody positive but
RNA negative). None of the participants had any other risk factors for
acquisition of viral hepatitis (for example, blood transfusion or past
history of intravenous drug abuse). All had an alcohol consumption of
less than 14 U/week and other forms of chronic liver disease were
excluded in all cases. Of the 283 initially exposed to contaminated
anti-D immunoglobulin, 196 remained chronically infected (that is, RT-
PCR (RNA) positive) and 87 were anti-HCV antibody positive but
persistently RNA negative (that is, they had cleared the HCV
infection). RNA negative individuals had an average of six RT-PCR
reactions carried out at different time points to confirm spontaneous
viral clearance. The majority of these subjects had already been
genotyped for both class I and class II HLA polymorphisms, and a
significant association was found between viral chronicity and the
presence of DRB1*03011 and DQB1*0201.13 All subjects gave informed
consent prior to participating in the study, which received ethics
approval from the research and ethics committees at all three
institutions.

Controls
To estimate the frequency of the CCR532 allele in the Irish
population, a control group of 120 unselected unrelated healthy
volunteers were genotyped. These were health care workers and all of
Irish descent.

RESULTS
The results of the genotyping for the three different polymorphisms
were compared with HCV PCR status (table 1), histological scores, and
ALT levels.

Genotypes associated with viral clearance

Heterozygote frequency for the CCR5delta32 mutation in the general
population was similar to that of the HCV study group (17.9% and
17.6%, allele frequency 0.193 and 0.186, respectively).

There was only one CCR5delta32/delta32 individual in each group. The
presence of the CCR5delta32/WT (wild-type) genotype was significantly
associated with spontaneous viral clearance: 42.0% of those who were
CCR5delta32/WT were HCV PCR negative versus only 28.3% of CCR5WT/WT (p
= 0.044, one sided Fisher's exact test, OR 1.9 (95% confidence
interval (CI) 1.1-3.6)).

Only one patient was homozygotic CCR5delta32/delta32 and she was HCV
PCR negative. Allele frequency was in Hardy-Weinberg equilibrium for
both patient and control groups. When the association of CCR5 genotype
and viral clearance was looked at in the DRB1*03011 and DQB1*0201
negative groups, none was found (p = 0.563 and 0.68, respectively).
Analysis of the CCR264I (p = 0.327, OR 0.66 (95% CI 0.23-1.6)) and
RANTES (p = 0.441, OR 1.01 (95% CI 0.58-1.7)) genotypes failed to
reveal any relationship with HCV clearance.

Relationship between genotypes and histological severity

There was no significant difference in hepatic inflammatory scores
between heterozygotes for the delta32 mutation and those without a
copy of this mutation (HAI 3.82 v 4.53; p = 0.098) in this cohort.
Furthermore, in the DRB1*03011 positive group, previously found to be
associated with less severe inflammation, CCR5delta32 had no further
additive impact on histological severity, with a mean HAI of 4.16 for
non-CCR5WT/WT and 3.80 for CCR5delta32 heterozygotes (p = 0.78). In
contrast, within the DRB1*03011 negative group, associated with more
severe inflammation, CCR5delta32 heterozygotes had significantly lower
inflammatory scores than the CCR5WT/WT group (mean inflammatory score
3.53 v 4.91; p = 0.043).

Relationship between genotypes and ALT levels
ALT levels were slightly higher in the CCR5 and CCR2 wild-type group
compared with the heterozygotes, while the opposite was observed for
the RANTES group. However, none of these differences reached
statistical significance

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