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Lower copy numbers of the chemokine CCL3L1 gene in patients with chronic hepatitis C
Research Article
Lower copy numbers of the chemokine CCL3L1 gene in patients with chronic hepatitis C Frank Grünhage1,*, , Jacob Nattermann2, , Olav A. Gressner2,5, Hermann E. Wasmuth3, Claus Hellerbrand4, Tilman Sauerbruch2, Ulrich Spengler2, Frank Lammert1 1
Department of Medicine II, Saarland University Hospital, Saarland University, Kirrberger Str. 1, 68421 Homburg, Germany; 2Department of Internal Medicine I, University Hospital Bonn, University of Bonn, Bonn, Germany; 3Department of Medicine III, University Hospital Aachen, Aachen University (RWTH), Germany; 4Department of Medicine I, University Hospital Regensburg, University of Regensburg, Germany; 5 Institute of Clinical Chemistry and Pathobiochemistry and Central Laboratory, RWTH-University Hospital, Aachen, Germany
Background & Aims: Recently, variation of gene copy numbers was recognized as a novel type of common genetic diversity, but its impact on viral hepatitis is unknown. Here, we determine the influence of copy number variation on the susceptibility and disease severity in hepatitis C virus (HCV) infection, investigating copy number variants (CNVs) of the chemokine CCL3L1 gene, which encodes a potent CCR5 ligand. Methods: CNVs were determined in 254 patients with chronic hepatitis C, 144 HCV/HIV co-infected patients, and 210 HCV negative controls, using quality-controlled real-time fluorescent dyelabeled quantitative PCR. Liver biopsies were obtained from HCV infected patients. Results: Copy numbers of the CCL3L1 gene range from 0 to 12 (mean 2.7 ± 1.4 copies). Patients with two or less copies are over-represented in the HCV infected cohort compared to HCV negative controls (odds ratio [OR] 1.54; p = 0.02). CCL3L1 copies are shifted to lower numbers in HCV infected patients (means 2.6 vs. 2.9 in controls; p = 0.011). HCV/HIV co-infected patients carry even lower CCL3L1 copy numbers compared to controls (means 2.2 vs. 2.9; p < 0.001), with a higher proportion of patients possessing two or less copies (OR = 3.42; p < 0.001). No association was detected between CCL3L1 copy numbers and histological grades of inflammation or stages of fibrosis. Conclusions: Lower CCL3L1 gene copy number compared to the population median is associated with chronic hepatitis C. Copy number variation of host genes represents a novel class of genetic diversity associated with viral hepatitis. Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Association study; Complex disease; Gene duplication; Gene variant; Hepatitis C virus; Polymorphism; Susceptibility factor. Received 5 February 2009; received in revised form 2 August 2009; accepted 1 September 2009; available online 27 November 2009 * Corresponding author. Tel.: +49 684 11623202; fax: +49 6841 1623267. E-mail address: [email protected] (F. Grünhage). These authors contributed equally. Abbreviations: BP, base pair; CCL3L1, CC-chemokine 3-like 1; CI, confidence interval; CNV, copy number variant; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; OR, odds ratio; SNP, single nucleotide polymorphism.
Introduction Environmental and host genetic factors are suspected to modify disease susceptibility and severity in chronic hepatitis C [1,2]. However, previous genetic studies focused on the role of MHC loci or associated phenotypes with a specific type of genetic variation, namely single nucleotide polymorphisms (SNPs). Humans typically have two copies of each gene (one from each parent), but the development of new technologies to detect the extent and position of genomic alterations has demonstrated that many fragments of our genome have been duplicated or deleted. These genomic rearrangements can change the copy numbers of genes that lie within the affected regions and represent an additional common source of genetic diversity. Only recently has the widespread abundance of copy number variants (CNVs) in the human genome been recognized [3–5]. CNVs are defined as DNA segments that are 1 kb or larger in size and are present at variable copy numbers [6]; following a recent recommendation, we use the term ‘variant’, which carries no implication of frequency or phenotypic effect, to denote these alterations [7]. Albeit CNVs are far less numerous than SNPs, they contribute substantially, to genetic diversity, since 10% of the human genome might be affected by CNVs, which also tend to have higher locus-specific mutation rates than SNPs [8]. At present, studies on the relevance of CNVs for common diseases are sparse [9–13], and no studies have yet investigated the potential influence of CNVs on viral hepatitis. There is accumulating evidence that host variability in the innate immune response and T cell activation determine primary clearance of HCV [14]. Particularly, the ability to mount a strong antiviral type 1 immune response is crucial for viral clearance in acute hepatitis C [15]. CC-chemokines play a key role in recruitment, and activation and differentiation of monocytes and lymphocytes in response to viral infections via binding to G-protein coupled receptors [16,17]. In particular, the interaction between the CC-chemokine ligands 3 (CCL3), 4 and 5 and their receptor CCR5 regulate T cell chemotaxis, and the CCL3 isoform CCL3-like 1 (CCL3L1, a.k.a. macrophage inflammatory protein 1aP) is the most potent ligand for CCR5 [18–20]. Fig. 1 illustrates the location of the CCL3L1 chemokine gene on chromosome 17q12 in a hotspot for segmental duplications.
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Research Article 17p13.3
17p13.2
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17p11.1 17q11.1
17q11.2
17q12 17q21.1 17q21.2
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17q21.32 17q21.33 17q22 17q23.1 17q23.2 17q23.3 17q24.1 17q24.2 17q24.3
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17q25.1 17q25.2
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chr 17
chr 17 31500K
CCL3|NM_002983
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CCL3L1|NM_021006 CCL3L1|NM_021006_copy_2
segmental duplication (TACG) DC3280 DC3281
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CCL3L1|NM_021006
segmental duplication (TACG) DC3280 DC3281
CCL3L1 mRNA
CCL3L1 mRNA
CCL3L1
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CCR5 receptor
CCR5 receptor
CD4
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Chemotaxis, functional and proliferative activation of leukocytes, lymphocytes and macrophages Fig. 1. Organization of the CCL3 locus on chromosome (chr) 17 (top panels). Due to segmental duplications, the locus harbors different numbers of CCL3L1 genes. As example, we plotted two copies (top left panel) and one copy of the CCL3L1 gene (top right panel), respectively. The schematic panels below illustrate how CCL3L1 gene copy number variation might cause differences in T cell activation via the CCR5 receptor: The variable CCL3L1 gene copy numbers lead to differences in CCL3L1 mRNA dosage, CCL3L1 protein expression and chemokine secretion, and CCR5 receptor binding, which ultimately affects T cell functions.
Recently, Gonzalez et al. [9] provided evidence for the influence of copy number variation at the CCL3L1 gene locus on susceptibility to human immunodeficiency virus (HIV) infection. The possession of CCL3L1 gene copy numbers lower than the population median was associated with HIV/AIDS and influences immune reconstitution, i.e. CD4+ T cell recovery [9,21]. Of note, the 32-base pair (bp) deletion in the CCR5 receptor, which is protective against HIV infection, has also been associated with susceptibility to HCV infection [22], albeit clinical studies have reported conflicting results [23–25]. In this study, we tested the hypothesis that low copy numbers of the CCL3L1 gene resemble the effect of a dysfunctional CCR5 mutation and are also associated with chronic hepatitis C and/ or more advanced disease in terms of severe inflammation or fibrosis (Fig. 1). Hence, we compared the distribution of CCL3L1 154
gene copy numbers between patients with chronic hepatitis C, patients co-infected with HCV and HIV, which accelerates HCV liver disease [26], and HCV negative controls. Patients and methods Patient population Patients with chronic HCV infection as determined by consistently detectable HCV-RNA in serum, using qualitative reverse-transcriptase PCR with a sensitivity of 100 copies [50 IU]/mL (Amplicor HCV test 2.0, Roche Diagnostics, Mannheim, Germany) were included in the study. Co-infection status was determined by detection of HIV-RNA in serum [27]. Patients with hepatitis B virus (HBV) coinfection as determined by the presence of HBs-antigen and/or HBV-DNA in serum as well as patients with other liver diseases and/or hemophilia were excluded. All patients were of Caucasian ethnicity and recruited at university hos-
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JOURNAL OF HEPATOLOGY pitals (Aachen, Bonn, Homburg, Regensburg). HCV negative controls of Caucasian descent were recruited in the Department of Internal Medicine I at the University Hospital Bonn to which they were admitted for screening colonoscopy. In total, we included 254 patients with chronic hepatitis C (median age 40 years; range 16–83 years; 154 males, 100 females), 144 patients with HCV/HIV co-infection (median age 41 years; range 23–73 years; 122 males, 22 females) and 210 HCV negative controls (median age 64 years, range 23–102 years; 110 males, 100 females) (Table 1). All patients and controls gave written informed consent. The study was approved by the by the local Research Ethics Committees of the Medical Faculties and the committee for data privacy of the German Network of Excellence for Viral Hepatitis (Hep-Net). Determination of copy numbers of the CCL3L1 gene by real-time quantitative PCR assay DNA was extracted from EDTA-anti-coagulated blood using a membrane-based extraction kit (Qiagen, Hilden, Germany). DNA concentration was calibrated to 5–20 ng/lL. CCL3L1 copy numbers were determined by real-time quantitative PCR, as described and validated by Gonzalez et al. [9]. PCRs were performed in 20 ll reaction volumes, using the ABI 7300 Sequence Detection System (TaqmanÒ; Applera, Norwalk, CT), which measures emitted fluorescence from a 6-carboxyfluorescein (FAM)-labeled probe detecting CCL3L1 (forward primer: 50 -tctccacagcttcctaacca aga; reverse primer: 50 -ctggacccactcctcactgg; probe 50 -FAM-aggccgg caggtct gtgctga-tetramethyl-6-carboxyrhodamine) and a VIC-labeled probe detecting the b-hemoglobin (HBB) gene as internal standard (forward primer: 50 -ggcaaccc taaggtgaaggc; reverse primer: 50 -ggtgagccaggccatcacta; probe 50 -VIC-cat ggcaagaaagtgctcggtgcct-tetramethyl-6-carboxyrhodamine). All humans carry two copies of the HBB gene [9]. The amount of DNA added to each PCR reaction was between 2 and 20 ng. PCR conditions were 50 °C for 2 min, 95° for 1 min, and 40 cycles at 92 °C for 15 s and 60 °C for 1 min. Standard curves for CCL3L1 and HBB were generated by serial 1:2 dilutions (25–0.78 ng/20 lL reaction volume) of genomic DNA from A431 cells known to have exactly two copies of the CCL3L1 gene per diploid genome [9]. Genomic DNA from A431 cells was obtained from DSMZ (Braunschweig, Germany). DNA concentrations of samples were determined by comparison to the standard curve, and copy numbers were calculated as described, rounding the mean to the closest integer [9]. The slopes obtained in real-time PCR assays for CCL3L1 and HBB were very similar, rendering HBB a good gene for normalization to determine CCL3L1 copy numbers [9].
by conventional PCR for CCL3L1 (forward primer 50 -gatgctattcttggatatcctgag; reverse primer 50 -gtgcagagaggacctggttg). Each real-time quantitative PCR was performed in duplicate. The average intra-sample variation Vi was determined using the control chart method [28], and the variation of 17% is comparable to the quality control study by Gonzalez et al. [9]; following this study [9], all samples exceeding Vi by 3 SD were repeated in duplicate. To assess intra-assay variability of the CCL3L1 gene copy number measurements, we estimated multi-category weighted Cohen’s kappa. We observed a kappa of 0.91 with a small p-value (p < 0.07; Z = 22.22). Second, we generated a bootstrap confidence interval (CI) around the intra-class correlation coefficient (ICC) between the two copy number measurements. The estimate of the ICC is 0.92 (95% CI = 0.90–0.93). Since the variability increases with copy number, we also calculated Pearson’s correlation coefficients between pairs of log transformed replicate measurements (R2 = 0.834, p < 0.001). We assessed the inter-assay variability on a randomly chosen subset of 60 samples, which derived from each of the three patient groups and were measured on the same plates in two independent assays. The mean difference in CCL3L1 gene copy numbers is 0.1, and the multi-category weighted Cohen’s kappa is 0.84 (p < 0.001), indicating high consistency between each pair of copy numbers. Our findings confirm that the real-time quantitative PCR assay is sensitive to discriminate between CCL3L1 gene copy numbers over a wide range and has low intra- and inter-assay variability. Liver histology Liver biopsies were obtained from HCV infected patients by the percutaneous Menghini technique under ultrasound guidance. Grading and staging were performed by a pathologist blinded to the study protocol, using the scoring system by Desmet et al. [29]. Statistical analysis Statistical analysis was performed with SPSS 13.0 (SPSS, Munich, Germany). Data are given as means ± SD, unless stated otherwise. Mean CCL3L1 gene copy numbers were compared between groups using Student‘s t-test. Copy number distributions were compared using nonparametric two-sided Mann–Whitney-U tests. Contingency tables and linear regression analyses were applied for calculation of odds ratios.
Results
Quality control of real-time quantitative PCR Several procedures were followed to ensure data quality of the quantitative PCR assays. All signals obtained for test DNA samples fell on the standard curve range, which allowed the determination of CCL3L1 gene copy numbers within the range of 0–20 copies. When a result of 0 copies was obtained, the sample was checked
Variation of copy numbers of the CCL3L1 gene There is significant inter-individual variation in CCL3L1 gene copy numbers. Overall, copy numbers of the CCL3L1 gene range from 0
Table 1. Patient characteristics.
Abbreviations: f, female; HCV, hepatitis C virus; HIV, human immunodeficiency virus; m, male; n, number; n.a., not applicable; n.d., not determined; SD, standard deviation.
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Research Article
Lower CCL3L1 gene copy numbers in patients with chronic hepatitis C and HCV/HIV co-infection Fig. 2 demonstrates that the distributions of CCL3L1 gene copy numbers differ significantly among the groups. The copy number distribution is shifted to lower copy numbers in patients with chronic hepatitis C, when compared to HCV negative controls (p = 0.007). The same phenomenon is observed in HCV/HIV coinfected patients in comparison to controls (p < 0.001). The difference between HCV infected patients and HCV/HIV co-infected patients is also significant with a more pronounced shift to lower CCL3L1 gene copy numbers in HCV/HIV co-infected patients (p = 0.001; Fig. 2). The mean CCL3L1 gene copy number is lower in HCV infected patients compared to HCV negative controls (2.6 vs. 2.9; p = 0.011). HCV/HIV co-infected patients carried even lower mean CCL3L1 copy numbers compared to controls (2.2 vs. 2.9; p < 0.001), and this value differs also significantly from patients with chronic hepatitis C (p = 0.004). To better illustrate the differences in copy number distributions, we calculated and plotted the cumulative copy number frequencies in HCV infected patients, HCV/HIV co-infected patients and controls (Fig. 3). In analogy to Gonzalez et al. [9], we performed a logistic regression analysis to assess the OR for patients with lower copy numbers. Accordingly, lower copy numbers are associated with an increased OR for HCV infected patients and HCV/HIV co-infected patients in comparison to controls (OR = 1.2; 95% CI 1.0–1.4; p = 0.01 and OR = 1.5; 95% CI = 1.3– 1.9; p < 0.01, respectively).
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HCV vs. Controls
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Fig. 3. Cumulative frequencies of CCL3L1 gene copy numbers in HCV infected patients, HCV/HIV co-infected patients and controls. There is a higher proportion of patients with lower CCL3L1 copy numbers in the HCV cohort as compared to controls. The predominance of patients with lower copy number is even more pronounced in HCV/HIV co-infected patients.
As additional test for disease association [33], we dichotomized the CCL3L1 genotypes into two classes, carrying either two or less CCL3L1 gene copies or more than two copies. Accordingly, Fig. 4 illustrates that patients with two or less copies of the CCL3L1 gene are significantly over-represented both in the HCV infected cohort (OR = 1.54; 95% CI 1.1–2.6; p = 0.02) and the HCV/HIV co-infected cohort (OR = 3.42; 95% CI 2.2–5.4; p < 0.001) compared to HCV negative controls.
HCV/HIV vs. Controls
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50 Mann-Whitney-U test p = 0.007
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to 12 copies (median 2 copies; mean ± SD 2.7 ± 1.4 copies). CCL3L1 gene copy numbers are comparable to those previously observed in Caucasian populations [9,30,31], and individuals with 0 gene copies have been described before [9,32]. In line with the reported copy number distributions, the majority of individuals (64.7%) carried two or three copies of the CCL3L1 gene. No differences in CCL3L1 gene copy numbers between women and men were observed (p > 0.05 in all three cohorts).
Mann-Whitney-U test p < 0.001
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Fig. 2. Histograms displaying copy number distributions of the CCL3L1 gene. (A) Frequencies (%) of CCL3L1 gene copy numbers in HCV infected patients (n = 254; blue bars) and non-infected controls (n = 210; white bars). (B) Frequencies of CCL3L1 gene copy numbers in HCV/HIV co-infected patients (n = 144; red bars) and controls. (C) Frequencies of CCL3L1 gene copy numbers HCV/HIV co-infected patients and HCV infected patients. Copy numbers were determined by quantitative PCR assays and rounded to the closest integer; p-values were determined using nonparametric Mann–Whitney-U tests. The CCL3L1 gene copy number distributions are shifted to lower numbers both in HCV infected patients compared to controls (panel A; p = 0.007) and in HCV/HIV co-infected patients compared to controls (panel B; p < 0.001). In addition, there is a shift to lower gene copy numbers in co-infected patients in comparison to patients infected with HCV only (panel C; p = 0.001).
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JOURNAL OF HEPATOLOGY OR = 3.42; 95% CI 2.18 -5.38; p < 0.001 OR = 1.54; 95% CI 1.07-2.55; p = 0.02 100 90 80
Frequency (%)
70 60 50 40 30 20 10 0 Controls
HCV
HCV/HIV
>2 CCL3L1 copies ≤ 2 CCL3L1 copies Fig. 4. Distributions of CCL3L1 gene copy numbers > or 6 2 in controls, HCV infected and HCV/HIV co-infected patients. As determined by v2 tests (p 6 0.02), HCV infected and HCV/HIV co-infected patients carry significantly more frequently 6 2 copies of the gene.
No association of CCL3L1 gene copy numbers with grade of inflammation, stage of fibrosis or viral load Liver histology was available from 173 (68.1%) patients with chronic hepatitis C. The majority of patients show only mild to moderate grades of inflammation (grade 0: 1.2%: grade 1: 50.2%; grade 2: 32.9%; grade 3: 9.2%; grade 4: 1.7%). Most patients present with mild to moderate fibrosis (stage 0: 8.6%; stage 1: 37.0%; stage 2: 35.3%; stage 3: 12.1%; stage 4/cirrhosis: 6.9%). However, neither histological grades of inflammation nor stages of fibrosis are correlated to copy numbers of the CCL3L1 gene (all p > 0.05). We did not detect an association with HCV or HIV levels in serum either, albeit limited data on viral loads was available in our cohort (Table 1).
Discussion The activation of CD4+ and CD8+ T cell populations has been associated with spontaneous viral clearance in the acute phase of HCV infection and in responders to interferon treatment. Individual failure to create an effective immune response has been attributed to host factors [2,15,34]. However, these genetic risk factors
for chronic hepatitis C have yet to be defined systematically, and most human genetic studies have focused on the role of HLA class I and II alleles or SNPs in non-MHC genes. In contrast, it has been noted recently that CNVs of many genes contribute to inter-individual differences in the human genome and might also have an impact on disease susceptibility and progression [3]. Nonetheless, studies on the influence of variable gene copy numbers on liver diseases have not yet been performed. This study provides evidence that patients with chronic hepatitis C possess lower copy numbers of the chemokine CCL3L1 gene (Fig. 2). Since our study cohorts do not represent incident but rather prevalent cases, this association could be related with chronification of HCV infection and/or non-response to treatment. To our knowledge, this is the first report of an influence of gene copy number variation on HCV infection. CCL3L1 is one of the natural ligands of the T cell receptor CCR5, and stimulation of this receptor induces T cell activation [17]. A 32-bp deletion in the CCR5 gene leads to truncation and loss of surface expression of the receptor, and it has been shown that homozygous carriers of the CCR5D32 mutation are protected against HIV infection; furthermore, HIV progression seems to be delayed in heterozygous carriers compared to controls [35–37]. In one of our previous studies [22], homozygous CCR5D32 carriers were over-represented in patients with chronic hepatitis C, consistent with the hypothesis that reduced capability of T cell stimulation via CCR5 might be a risk factor for persistence of HCV. Since additional clinical studies yielded conflicting results [23–25,38] and pointed to a potential selection bias due to the inclusion of a large proportion of hemophilia A patients in the study by Woitas et al. [22], we have consciously not included any hemophilic patients in the present study. Our results indicate that low copy numbers of the CCL3L1 gene resemble the effect of the CCR5 loss-of-function mutation on HCV infection, possibly due to diminished CCR5 binding and receptor stimulation. CCL3L1 copy numbers vary substantially across ethnic groups with a median copy number of 3 in non-African to 6 in African populations [9]. The observed copy numbers in our cohorts fit to those reported by Gonzalez et al. [9] in European populations. Whereas a CCL3L1 gene copy number lower than the population average might enhance HIV susceptibility, presumably by influencing viral entry [9] as well as cell-mediated immunity [39], in hepatitis C lower CCL3L1 gene copy numbers may influence the fate towards chronic infection. However, our cases resemble prevalent cases of HCV infection rather than incidental cases. Hence, further studies are needed to clarify whether CCL3L1 copy number variation may also have an impact on spontaneous or therapy-induced HCV clearance. Decreased CCR5 signaling could contribute to the diminished type 1 cytokine response reported by others and our group [40,41], since CCR5 is predominantly expressed by a subset of CD8+ cytotoxic T lymphocytes and CD4+ T helper cells producing type 1 cytokines [42]. Lymphocytes expressing CCR5 are critically involved in the antiviral response [43], but show sustained dysfunction with reduced CCR5 expression in chronic HCV infection [44]. Importantly, the genetic association is supported by functional data showing correlations between CCL3L1 copy numbers and CCL3L1 steady-state mRNA levels in LPS-stimulated monocytes and CCL3L1 secretion by freshly isolated peripheral mononuclear neutrophils (Fig. 1) as well as with the proportion of T cells mounting strong cell-mediated immune responses [9,39]. Whereas effective T cell activation is a key event that determines viral clearance in the early phase of HCV infection, it
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Research Article may have deleterious effects in the chronic phase via enhanced recruitment of cytotoxic T cell populations that promote inflammatory activity and liver fibrogenesis [45,46]. Thus, the influence of the CCR5D32 mutation on disease severity and progression has been investigated in patients with chronic hepatitis C, albeit with conflicting results [23–25,38,47]. However, we could not detect any association between CCL3L1 gene copy numbers and the grade of inflammation or the stage of fibrosis. Furthermore, the considerable overlap between the groups suggests a polygenic effect, i.e. there are multiple and possibly interacting genetic as well as viral and other exogenous factors that determine the patients’ phenotypes in response to HCV infection [48]. We could demonstrate that compared to HCV negative controls, patients with HCV/HIV co-infection show also a shift to lower CCL3L1 gene copy numbers that is even more pronounced than the copy number shift observed in HCV infected patients (Fig. 2). This confirmatory observation is in line with previous reports of lower CCL3L1 copy numbers in HIV positive patients [9]. However, it remains unclear to which extent the copy number shift in HCV/HIV co-infected patients is due to susceptibility to one of these viral infections and/or positive selection. In conclusion, this study shows that CNVs of the CCL3L1 gene are associated with lower CCL3L1 gene copy numbers in patients with chronic hepatitis C as compared to controls. Our findings provide a precedent for a link between a chromosomal duplication event that leads to changes in the dose of an immune response gene and variability to HCV susceptibility. The study underscores the recent recommendation [8] that future genetic studies in complex human diseases should incorporate an evaluation of CNVs to determine whether in addition to SNPs, individual variation in gene copy numbers might contribute to the complex disease being investigated. Acknowledgements The authors who have taken part in this study declared that they do not have anything to declare regarding funding from industry or conflict of interest with respect to this manuscript. This study was presented, in part, as Poster of Distinction at the Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, October 2007, and published in abstract form (Hepatology 2007;46:619A). This work was supported by University of Bonn (BONFOR O-107.0083 to F.G.), Saarland University (HOMFOR T201000385 to F.G.), Deutsche Forschungsgemeinschaft (SFB/TRR 57 to F.L., J.N., T.S., U.S. and H.E.W.), German Network of Excellence for Viral Hepatitis (Hep-Net Genetic Epidemiology Group TP 10.1.5 to F.L.) and EU (COST Action BM0901). References [1] Bataller R, North KE, Brenner DA. Genetic polymorphisms and the progression of liver fibrosis: a critical appraisal. Hepatology 2003;37:493–503. [2] Asselah T, Bieche I, Paradis V, Bedossa P, Vidaud M, Marcellin P. Genetics, genomics, and proteomics: implications for the diagnosis and the treatment of chronic hepatitis C. Semin Liver Dis 2007;27:13–27. [3] Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global variation in copy number in the human genome. Nature 2006;444:444–454. [4] Abecasis G, Tam PK, Bustamante CD, Ostrander EA, Scherer SW, Chanock SJ, et al. Human genome variation 2006: emerging views on structural variation and large-scale SNP analysis. Nat Genet 2007;39:153–155. [5] Jakobsson M, Scholz SW, Scheet P, Gibbs JR, VanLiere JM, Fung HC, et al. Genotype, haplotype and copy-number variation in worldwide human populations. Nature 2008;451:998–1003.
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JOURNAL OF HEPATOLOGY [31] McKinney C, Merriman ME, Chapman PT, Gow PJ, Harrison AA, Highton J, et al. Evidence for an influence of chemokine ligand 3-like 1 (CCL3L1) gene copy number on susceptibility to rheumatoid arthritis. Ann Rheum Dis 2008;67:409–413. [32] Townson JR, Barcellos LF, Nibbs RJ. Gene copy number regulates the production of the human chemokine CCL3–L1. Eur J Immunol 2002;32:3016–3026. [33] McCarroll SA, Altshuler DM. Copy-number variation and association studies of human disease. Nat Genet 2007;39:S37–S42. [34] Lloyd AR, Jagger E, Post JJ, Crooks LA, Rawlinson WD, Hahn YS, et al. Host and viral factors in the immunopathogenesis of primary hepatitis C virus infection. Immunol Cell Biol 2007;85:24–32. [35] Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 1996;86:367–377. [36] Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996;382:722–725. [37] Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 1996;273:1856–1862. [38] Hellier S, Frodsham AJ, Hennig BJ, Klenerman P, Knapp S, Ramaley P, et al. Association of genetic variants of the chemokine receptor CCR5 and its ligands, RANTES and MCP-2, with outcome of HCV infection. Hepatology 2003;38:1468–1476. [39] Dolan MJ, Kulkarni H, Camargo JF, He W, Smith A, Anaya JM, et al. CCL3L1 and CCR5 influence cell-mediated immunity and affect HIV-AIDS pathogenesis via viral entry-independent mechanisms. Nat Immunol 2007;8:1324–1336.
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Lower copy numbers of the chemokine CCL3L1 gene in patients with chronic hepatitis C Frank Grünhage1,*, , Jacob Nattermann2, , Olav A. Gressner2,5, Hermann E. Wasmuth3, Claus Hellerbrand4, Tilman Sauerbruch2, Ulrich Spengler2, Frank Lammert1 1
Department of Medicine II, Saarland University Hospital, Saarland University, Kirrberger Str. 1, 68421 Homburg, Germany; 2Department of Internal Medicine I, University Hospital Bonn, University of Bonn, Bonn, Germany; 3Department of Medicine III, University Hospital Aachen, Aachen University (RWTH), Germany; 4Department of Medicine I, University Hospital Regensburg, University of Regensburg, Germany; 5 Institute of Clinical Chemistry and Pathobiochemistry and Central Laboratory, RWTH-University Hospital, Aachen, Germany
Background & Aims: Recently, variation of gene copy numbers was recognized as a novel type of common genetic diversity, but its impact on viral hepatitis is unknown. Here, we determine the influence of copy number variation on the susceptibility and disease severity in hepatitis C virus (HCV) infection, investigating copy number variants (CNVs) of the chemokine CCL3L1 gene, which encodes a potent CCR5 ligand. Methods: CNVs were determined in 254 patients with chronic hepatitis C, 144 HCV/HIV co-infected patients, and 210 HCV negative controls, using quality-controlled real-time fluorescent dyelabeled quantitative PCR. Liver biopsies were obtained from HCV infected patients. Results: Copy numbers of the CCL3L1 gene range from 0 to 12 (mean 2.7 ± 1.4 copies). Patients with two or less copies are over-represented in the HCV infected cohort compared to HCV negative controls (odds ratio [OR] 1.54; p = 0.02). CCL3L1 copies are shifted to lower numbers in HCV infected patients (means 2.6 vs. 2.9 in controls; p = 0.011). HCV/HIV co-infected patients carry even lower CCL3L1 copy numbers compared to controls (means 2.2 vs. 2.9; p < 0.001), with a higher proportion of patients possessing two or less copies (OR = 3.42; p < 0.001). No association was detected between CCL3L1 copy numbers and histological grades of inflammation or stages of fibrosis. Conclusions: Lower CCL3L1 gene copy number compared to the population median is associated with chronic hepatitis C. Copy number variation of host genes represents a novel class of genetic diversity associated with viral hepatitis. Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Association study; Complex disease; Gene duplication; Gene variant; Hepatitis C virus; Polymorphism; Susceptibility factor. Received 5 February 2009; received in revised form 2 August 2009; accepted 1 September 2009; available online 27 November 2009 * Corresponding author. Tel.: +49 684 11623202; fax: +49 6841 1623267. E-mail address: [email protected] (F. Grünhage). These authors contributed equally. Abbreviations: BP, base pair; CCL3L1, CC-chemokine 3-like 1; CI, confidence interval; CNV, copy number variant; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; OR, odds ratio; SNP, single nucleotide polymorphism.
Introduction Environmental and host genetic factors are suspected to modify disease susceptibility and severity in chronic hepatitis C [1,2]. However, previous genetic studies focused on the role of MHC loci or associated phenotypes with a specific type of genetic variation, namely single nucleotide polymorphisms (SNPs). Humans typically have two copies of each gene (one from each parent), but the development of new technologies to detect the extent and position of genomic alterations has demonstrated that many fragments of our genome have been duplicated or deleted. These genomic rearrangements can change the copy numbers of genes that lie within the affected regions and represent an additional common source of genetic diversity. Only recently has the widespread abundance of copy number variants (CNVs) in the human genome been recognized [3–5]. CNVs are defined as DNA segments that are 1 kb or larger in size and are present at variable copy numbers [6]; following a recent recommendation, we use the term ‘variant’, which carries no implication of frequency or phenotypic effect, to denote these alterations [7]. Albeit CNVs are far less numerous than SNPs, they contribute substantially, to genetic diversity, since 10% of the human genome might be affected by CNVs, which also tend to have higher locus-specific mutation rates than SNPs [8]. At present, studies on the relevance of CNVs for common diseases are sparse [9–13], and no studies have yet investigated the potential influence of CNVs on viral hepatitis. There is accumulating evidence that host variability in the innate immune response and T cell activation determine primary clearance of HCV [14]. Particularly, the ability to mount a strong antiviral type 1 immune response is crucial for viral clearance in acute hepatitis C [15]. CC-chemokines play a key role in recruitment, and activation and differentiation of monocytes and lymphocytes in response to viral infections via binding to G-protein coupled receptors [16,17]. In particular, the interaction between the CC-chemokine ligands 3 (CCL3), 4 and 5 and their receptor CCR5 regulate T cell chemotaxis, and the CCL3 isoform CCL3-like 1 (CCL3L1, a.k.a. macrophage inflammatory protein 1aP) is the most potent ligand for CCR5 [18–20]. Fig. 1 illustrates the location of the CCL3L1 chemokine gene on chromosome 17q12 in a hotspot for segmental duplications.
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Research Article 17p13.3
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Chemotaxis, functional and proliferative activation of leukocytes, lymphocytes and macrophages Fig. 1. Organization of the CCL3 locus on chromosome (chr) 17 (top panels). Due to segmental duplications, the locus harbors different numbers of CCL3L1 genes. As example, we plotted two copies (top left panel) and one copy of the CCL3L1 gene (top right panel), respectively. The schematic panels below illustrate how CCL3L1 gene copy number variation might cause differences in T cell activation via the CCR5 receptor: The variable CCL3L1 gene copy numbers lead to differences in CCL3L1 mRNA dosage, CCL3L1 protein expression and chemokine secretion, and CCR5 receptor binding, which ultimately affects T cell functions.
Recently, Gonzalez et al. [9] provided evidence for the influence of copy number variation at the CCL3L1 gene locus on susceptibility to human immunodeficiency virus (HIV) infection. The possession of CCL3L1 gene copy numbers lower than the population median was associated with HIV/AIDS and influences immune reconstitution, i.e. CD4+ T cell recovery [9,21]. Of note, the 32-base pair (bp) deletion in the CCR5 receptor, which is protective against HIV infection, has also been associated with susceptibility to HCV infection [22], albeit clinical studies have reported conflicting results [23–25]. In this study, we tested the hypothesis that low copy numbers of the CCL3L1 gene resemble the effect of a dysfunctional CCR5 mutation and are also associated with chronic hepatitis C and/ or more advanced disease in terms of severe inflammation or fibrosis (Fig. 1). Hence, we compared the distribution of CCL3L1 154
gene copy numbers between patients with chronic hepatitis C, patients co-infected with HCV and HIV, which accelerates HCV liver disease [26], and HCV negative controls. Patients and methods Patient population Patients with chronic HCV infection as determined by consistently detectable HCV-RNA in serum, using qualitative reverse-transcriptase PCR with a sensitivity of 100 copies [50 IU]/mL (Amplicor HCV test 2.0, Roche Diagnostics, Mannheim, Germany) were included in the study. Co-infection status was determined by detection of HIV-RNA in serum [27]. Patients with hepatitis B virus (HBV) coinfection as determined by the presence of HBs-antigen and/or HBV-DNA in serum as well as patients with other liver diseases and/or hemophilia were excluded. All patients were of Caucasian ethnicity and recruited at university hos-
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JOURNAL OF HEPATOLOGY pitals (Aachen, Bonn, Homburg, Regensburg). HCV negative controls of Caucasian descent were recruited in the Department of Internal Medicine I at the University Hospital Bonn to which they were admitted for screening colonoscopy. In total, we included 254 patients with chronic hepatitis C (median age 40 years; range 16–83 years; 154 males, 100 females), 144 patients with HCV/HIV co-infection (median age 41 years; range 23–73 years; 122 males, 22 females) and 210 HCV negative controls (median age 64 years, range 23–102 years; 110 males, 100 females) (Table 1). All patients and controls gave written informed consent. The study was approved by the by the local Research Ethics Committees of the Medical Faculties and the committee for data privacy of the German Network of Excellence for Viral Hepatitis (Hep-Net). Determination of copy numbers of the CCL3L1 gene by real-time quantitative PCR assay DNA was extracted from EDTA-anti-coagulated blood using a membrane-based extraction kit (Qiagen, Hilden, Germany). DNA concentration was calibrated to 5–20 ng/lL. CCL3L1 copy numbers were determined by real-time quantitative PCR, as described and validated by Gonzalez et al. [9]. PCRs were performed in 20 ll reaction volumes, using the ABI 7300 Sequence Detection System (TaqmanÒ; Applera, Norwalk, CT), which measures emitted fluorescence from a 6-carboxyfluorescein (FAM)-labeled probe detecting CCL3L1 (forward primer: 50 -tctccacagcttcctaacca aga; reverse primer: 50 -ctggacccactcctcactgg; probe 50 -FAM-aggccgg caggtct gtgctga-tetramethyl-6-carboxyrhodamine) and a VIC-labeled probe detecting the b-hemoglobin (HBB) gene as internal standard (forward primer: 50 -ggcaaccc taaggtgaaggc; reverse primer: 50 -ggtgagccaggccatcacta; probe 50 -VIC-cat ggcaagaaagtgctcggtgcct-tetramethyl-6-carboxyrhodamine). All humans carry two copies of the HBB gene [9]. The amount of DNA added to each PCR reaction was between 2 and 20 ng. PCR conditions were 50 °C for 2 min, 95° for 1 min, and 40 cycles at 92 °C for 15 s and 60 °C for 1 min. Standard curves for CCL3L1 and HBB were generated by serial 1:2 dilutions (25–0.78 ng/20 lL reaction volume) of genomic DNA from A431 cells known to have exactly two copies of the CCL3L1 gene per diploid genome [9]. Genomic DNA from A431 cells was obtained from DSMZ (Braunschweig, Germany). DNA concentrations of samples were determined by comparison to the standard curve, and copy numbers were calculated as described, rounding the mean to the closest integer [9]. The slopes obtained in real-time PCR assays for CCL3L1 and HBB were very similar, rendering HBB a good gene for normalization to determine CCL3L1 copy numbers [9].
by conventional PCR for CCL3L1 (forward primer 50 -gatgctattcttggatatcctgag; reverse primer 50 -gtgcagagaggacctggttg). Each real-time quantitative PCR was performed in duplicate. The average intra-sample variation Vi was determined using the control chart method [28], and the variation of 17% is comparable to the quality control study by Gonzalez et al. [9]; following this study [9], all samples exceeding Vi by 3 SD were repeated in duplicate. To assess intra-assay variability of the CCL3L1 gene copy number measurements, we estimated multi-category weighted Cohen’s kappa. We observed a kappa of 0.91 with a small p-value (p < 0.07; Z = 22.22). Second, we generated a bootstrap confidence interval (CI) around the intra-class correlation coefficient (ICC) between the two copy number measurements. The estimate of the ICC is 0.92 (95% CI = 0.90–0.93). Since the variability increases with copy number, we also calculated Pearson’s correlation coefficients between pairs of log transformed replicate measurements (R2 = 0.834, p < 0.001). We assessed the inter-assay variability on a randomly chosen subset of 60 samples, which derived from each of the three patient groups and were measured on the same plates in two independent assays. The mean difference in CCL3L1 gene copy numbers is 0.1, and the multi-category weighted Cohen’s kappa is 0.84 (p < 0.001), indicating high consistency between each pair of copy numbers. Our findings confirm that the real-time quantitative PCR assay is sensitive to discriminate between CCL3L1 gene copy numbers over a wide range and has low intra- and inter-assay variability. Liver histology Liver biopsies were obtained from HCV infected patients by the percutaneous Menghini technique under ultrasound guidance. Grading and staging were performed by a pathologist blinded to the study protocol, using the scoring system by Desmet et al. [29]. Statistical analysis Statistical analysis was performed with SPSS 13.0 (SPSS, Munich, Germany). Data are given as means ± SD, unless stated otherwise. Mean CCL3L1 gene copy numbers were compared between groups using Student‘s t-test. Copy number distributions were compared using nonparametric two-sided Mann–Whitney-U tests. Contingency tables and linear regression analyses were applied for calculation of odds ratios.
Results
Quality control of real-time quantitative PCR Several procedures were followed to ensure data quality of the quantitative PCR assays. All signals obtained for test DNA samples fell on the standard curve range, which allowed the determination of CCL3L1 gene copy numbers within the range of 0–20 copies. When a result of 0 copies was obtained, the sample was checked
Variation of copy numbers of the CCL3L1 gene There is significant inter-individual variation in CCL3L1 gene copy numbers. Overall, copy numbers of the CCL3L1 gene range from 0
Table 1. Patient characteristics.
Abbreviations: f, female; HCV, hepatitis C virus; HIV, human immunodeficiency virus; m, male; n, number; n.a., not applicable; n.d., not determined; SD, standard deviation.
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Lower CCL3L1 gene copy numbers in patients with chronic hepatitis C and HCV/HIV co-infection Fig. 2 demonstrates that the distributions of CCL3L1 gene copy numbers differ significantly among the groups. The copy number distribution is shifted to lower copy numbers in patients with chronic hepatitis C, when compared to HCV negative controls (p = 0.007). The same phenomenon is observed in HCV/HIV coinfected patients in comparison to controls (p < 0.001). The difference between HCV infected patients and HCV/HIV co-infected patients is also significant with a more pronounced shift to lower CCL3L1 gene copy numbers in HCV/HIV co-infected patients (p = 0.001; Fig. 2). The mean CCL3L1 gene copy number is lower in HCV infected patients compared to HCV negative controls (2.6 vs. 2.9; p = 0.011). HCV/HIV co-infected patients carried even lower mean CCL3L1 copy numbers compared to controls (2.2 vs. 2.9; p < 0.001), and this value differs also significantly from patients with chronic hepatitis C (p = 0.004). To better illustrate the differences in copy number distributions, we calculated and plotted the cumulative copy number frequencies in HCV infected patients, HCV/HIV co-infected patients and controls (Fig. 3). In analogy to Gonzalez et al. [9], we performed a logistic regression analysis to assess the OR for patients with lower copy numbers. Accordingly, lower copy numbers are associated with an increased OR for HCV infected patients and HCV/HIV co-infected patients in comparison to controls (OR = 1.2; 95% CI 1.0–1.4; p = 0.01 and OR = 1.5; 95% CI = 1.3– 1.9; p < 0.01, respectively).
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Fig. 3. Cumulative frequencies of CCL3L1 gene copy numbers in HCV infected patients, HCV/HIV co-infected patients and controls. There is a higher proportion of patients with lower CCL3L1 copy numbers in the HCV cohort as compared to controls. The predominance of patients with lower copy number is even more pronounced in HCV/HIV co-infected patients.
As additional test for disease association [33], we dichotomized the CCL3L1 genotypes into two classes, carrying either two or less CCL3L1 gene copies or more than two copies. Accordingly, Fig. 4 illustrates that patients with two or less copies of the CCL3L1 gene are significantly over-represented both in the HCV infected cohort (OR = 1.54; 95% CI 1.1–2.6; p = 0.02) and the HCV/HIV co-infected cohort (OR = 3.42; 95% CI 2.2–5.4; p < 0.001) compared to HCV negative controls.
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to 12 copies (median 2 copies; mean ± SD 2.7 ± 1.4 copies). CCL3L1 gene copy numbers are comparable to those previously observed in Caucasian populations [9,30,31], and individuals with 0 gene copies have been described before [9,32]. In line with the reported copy number distributions, the majority of individuals (64.7%) carried two or three copies of the CCL3L1 gene. No differences in CCL3L1 gene copy numbers between women and men were observed (p > 0.05 in all three cohorts).
Mann-Whitney-U test p < 0.001
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0 1 2 3 4 5 6 7 8 9 10
CCL3L1 copy number
Fig. 2. Histograms displaying copy number distributions of the CCL3L1 gene. (A) Frequencies (%) of CCL3L1 gene copy numbers in HCV infected patients (n = 254; blue bars) and non-infected controls (n = 210; white bars). (B) Frequencies of CCL3L1 gene copy numbers in HCV/HIV co-infected patients (n = 144; red bars) and controls. (C) Frequencies of CCL3L1 gene copy numbers HCV/HIV co-infected patients and HCV infected patients. Copy numbers were determined by quantitative PCR assays and rounded to the closest integer; p-values were determined using nonparametric Mann–Whitney-U tests. The CCL3L1 gene copy number distributions are shifted to lower numbers both in HCV infected patients compared to controls (panel A; p = 0.007) and in HCV/HIV co-infected patients compared to controls (panel B; p < 0.001). In addition, there is a shift to lower gene copy numbers in co-infected patients in comparison to patients infected with HCV only (panel C; p = 0.001).
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JOURNAL OF HEPATOLOGY OR = 3.42; 95% CI 2.18 -5.38; p < 0.001 OR = 1.54; 95% CI 1.07-2.55; p = 0.02 100 90 80
Frequency (%)
70 60 50 40 30 20 10 0 Controls
HCV
HCV/HIV
>2 CCL3L1 copies ≤ 2 CCL3L1 copies Fig. 4. Distributions of CCL3L1 gene copy numbers > or 6 2 in controls, HCV infected and HCV/HIV co-infected patients. As determined by v2 tests (p 6 0.02), HCV infected and HCV/HIV co-infected patients carry significantly more frequently 6 2 copies of the gene.
No association of CCL3L1 gene copy numbers with grade of inflammation, stage of fibrosis or viral load Liver histology was available from 173 (68.1%) patients with chronic hepatitis C. The majority of patients show only mild to moderate grades of inflammation (grade 0: 1.2%: grade 1: 50.2%; grade 2: 32.9%; grade 3: 9.2%; grade 4: 1.7%). Most patients present with mild to moderate fibrosis (stage 0: 8.6%; stage 1: 37.0%; stage 2: 35.3%; stage 3: 12.1%; stage 4/cirrhosis: 6.9%). However, neither histological grades of inflammation nor stages of fibrosis are correlated to copy numbers of the CCL3L1 gene (all p > 0.05). We did not detect an association with HCV or HIV levels in serum either, albeit limited data on viral loads was available in our cohort (Table 1).
Discussion The activation of CD4+ and CD8+ T cell populations has been associated with spontaneous viral clearance in the acute phase of HCV infection and in responders to interferon treatment. Individual failure to create an effective immune response has been attributed to host factors [2,15,34]. However, these genetic risk factors
for chronic hepatitis C have yet to be defined systematically, and most human genetic studies have focused on the role of HLA class I and II alleles or SNPs in non-MHC genes. In contrast, it has been noted recently that CNVs of many genes contribute to inter-individual differences in the human genome and might also have an impact on disease susceptibility and progression [3]. Nonetheless, studies on the influence of variable gene copy numbers on liver diseases have not yet been performed. This study provides evidence that patients with chronic hepatitis C possess lower copy numbers of the chemokine CCL3L1 gene (Fig. 2). Since our study cohorts do not represent incident but rather prevalent cases, this association could be related with chronification of HCV infection and/or non-response to treatment. To our knowledge, this is the first report of an influence of gene copy number variation on HCV infection. CCL3L1 is one of the natural ligands of the T cell receptor CCR5, and stimulation of this receptor induces T cell activation [17]. A 32-bp deletion in the CCR5 gene leads to truncation and loss of surface expression of the receptor, and it has been shown that homozygous carriers of the CCR5D32 mutation are protected against HIV infection; furthermore, HIV progression seems to be delayed in heterozygous carriers compared to controls [35–37]. In one of our previous studies [22], homozygous CCR5D32 carriers were over-represented in patients with chronic hepatitis C, consistent with the hypothesis that reduced capability of T cell stimulation via CCR5 might be a risk factor for persistence of HCV. Since additional clinical studies yielded conflicting results [23–25,38] and pointed to a potential selection bias due to the inclusion of a large proportion of hemophilia A patients in the study by Woitas et al. [22], we have consciously not included any hemophilic patients in the present study. Our results indicate that low copy numbers of the CCL3L1 gene resemble the effect of the CCR5 loss-of-function mutation on HCV infection, possibly due to diminished CCR5 binding and receptor stimulation. CCL3L1 copy numbers vary substantially across ethnic groups with a median copy number of 3 in non-African to 6 in African populations [9]. The observed copy numbers in our cohorts fit to those reported by Gonzalez et al. [9] in European populations. Whereas a CCL3L1 gene copy number lower than the population average might enhance HIV susceptibility, presumably by influencing viral entry [9] as well as cell-mediated immunity [39], in hepatitis C lower CCL3L1 gene copy numbers may influence the fate towards chronic infection. However, our cases resemble prevalent cases of HCV infection rather than incidental cases. Hence, further studies are needed to clarify whether CCL3L1 copy number variation may also have an impact on spontaneous or therapy-induced HCV clearance. Decreased CCR5 signaling could contribute to the diminished type 1 cytokine response reported by others and our group [40,41], since CCR5 is predominantly expressed by a subset of CD8+ cytotoxic T lymphocytes and CD4+ T helper cells producing type 1 cytokines [42]. Lymphocytes expressing CCR5 are critically involved in the antiviral response [43], but show sustained dysfunction with reduced CCR5 expression in chronic HCV infection [44]. Importantly, the genetic association is supported by functional data showing correlations between CCL3L1 copy numbers and CCL3L1 steady-state mRNA levels in LPS-stimulated monocytes and CCL3L1 secretion by freshly isolated peripheral mononuclear neutrophils (Fig. 1) as well as with the proportion of T cells mounting strong cell-mediated immune responses [9,39]. Whereas effective T cell activation is a key event that determines viral clearance in the early phase of HCV infection, it
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Research Article may have deleterious effects in the chronic phase via enhanced recruitment of cytotoxic T cell populations that promote inflammatory activity and liver fibrogenesis [45,46]. Thus, the influence of the CCR5D32 mutation on disease severity and progression has been investigated in patients with chronic hepatitis C, albeit with conflicting results [23–25,38,47]. However, we could not detect any association between CCL3L1 gene copy numbers and the grade of inflammation or the stage of fibrosis. Furthermore, the considerable overlap between the groups suggests a polygenic effect, i.e. there are multiple and possibly interacting genetic as well as viral and other exogenous factors that determine the patients’ phenotypes in response to HCV infection [48]. We could demonstrate that compared to HCV negative controls, patients with HCV/HIV co-infection show also a shift to lower CCL3L1 gene copy numbers that is even more pronounced than the copy number shift observed in HCV infected patients (Fig. 2). This confirmatory observation is in line with previous reports of lower CCL3L1 copy numbers in HIV positive patients [9]. However, it remains unclear to which extent the copy number shift in HCV/HIV co-infected patients is due to susceptibility to one of these viral infections and/or positive selection. In conclusion, this study shows that CNVs of the CCL3L1 gene are associated with lower CCL3L1 gene copy numbers in patients with chronic hepatitis C as compared to controls. Our findings provide a precedent for a link between a chromosomal duplication event that leads to changes in the dose of an immune response gene and variability to HCV susceptibility. The study underscores the recent recommendation [8] that future genetic studies in complex human diseases should incorporate an evaluation of CNVs to determine whether in addition to SNPs, individual variation in gene copy numbers might contribute to the complex disease being investigated. Acknowledgements The authors who have taken part in this study declared that they do not have anything to declare regarding funding from industry or conflict of interest with respect to this manuscript. This study was presented, in part, as Poster of Distinction at the Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, October 2007, and published in abstract form (Hepatology 2007;46:619A). This work was supported by University of Bonn (BONFOR O-107.0083 to F.G.), Saarland University (HOMFOR T201000385 to F.G.), Deutsche Forschungsgemeinschaft (SFB/TRR 57 to F.L., J.N., T.S., U.S. and H.E.W.), German Network of Excellence for Viral Hepatitis (Hep-Net Genetic Epidemiology Group TP 10.1.5 to F.L.) and EU (COST Action BM0901). References [1] Bataller R, North KE, Brenner DA. Genetic polymorphisms and the progression of liver fibrosis: a critical appraisal. Hepatology 2003;37:493–503. [2] Asselah T, Bieche I, Paradis V, Bedossa P, Vidaud M, Marcellin P. Genetics, genomics, and proteomics: implications for the diagnosis and the treatment of chronic hepatitis C. Semin Liver Dis 2007;27:13–27. [3] Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global variation in copy number in the human genome. Nature 2006;444:444–454. [4] Abecasis G, Tam PK, Bustamante CD, Ostrander EA, Scherer SW, Chanock SJ, et al. Human genome variation 2006: emerging views on structural variation and large-scale SNP analysis. Nat Genet 2007;39:153–155. [5] Jakobsson M, Scholz SW, Scheet P, Gibbs JR, VanLiere JM, Fung HC, et al. Genotype, haplotype and copy-number variation in worldwide human populations. Nature 2008;451:998–1003.
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