+
MODEL
Microbes and Infection xx (2014) 1e5 www.elsevier.com/locate/micinf
Short communication
Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population Andrey V. Barkhash a,*, Galina V. Kochneva b, Elena V. Chub b, Svetlana V. Mikhailova a, Aida G. Romaschenko a a
Laboratory of Human Molecular Genetics, Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Branch, 10 Lavrentyeva Ave., Novosibirsk 630090, Russia b Laboratory of Viral Hepatitis, State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region1 630559, Russia Received 28 September 2013; accepted 14 February 2014
Abstract Chronic hepatitis C is a severe liver disease caused by positive-strand RNA virus. Previously, we reported an association between seven single nucleotide polymorphisms (SNPs) in four innate immunity genes (OAS2, OAS3, CD209, and TLR3) and human predisposition to tick-borne encephalitis, caused by a virus from the same Flaviviridae family, in a Russian population. Currently, genotype and allele frequencies for these SNPs were analyzed in 75 chronic hepatitis C patients and compared with the population control (269 Novosibirsk citizens). Data obtained suggest that the OAS2 rs1293762 and CD209 rs2287886 SNPs are associated with predisposition to chronic hepatitis C in Russian population. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Chronic hepatitis C; Genetic predisposition to disease; Single nucleotide polymorphism
1. Introduction Chronic hepatitis C (HC) is a severe liver disease which is found worldwide. HC is caused by an enveloped, positivestrand RNA virus (HC virus, HCV) from the Hepacivirus genus (the Flaviviridae family). About 75e85% of newly HCV-infected individuals develop chronic disease that can progress to liver cirrhosis and hepatocellular carcinoma. Approximately 150 million individuals in the world are chronically infected with HCV, and more than 350,000 individuals annually die from HC-related liver diseases. The development of efficient vaccine against HCV is currently only in progress [1e4]. It was established that human genetic factors play an important role in determining the course and outcome of * Corresponding author. Tel.: þ7 383 3634974; fax: þ7 383 3331278. E-mail address: [email protected] (A.V. Barkhash). 1 Koltsovo is located not within Novosibirsk city, but in Novosibirsk region.
different infectious diseases (including chronic HC) [5]. Polymorphisms in several genes involved in both innate and acquired immunity pathways were associated with different aspects of HC pathogenesis (viral clearance, chronic HC and fibrosis progression, response to combined antiviral therapy) in different human populations. Among them, several single nucleotide polymorphisms (SNPs) located within interleukin 28B (IL28B) gene are considered to be the most strong predictors, since their involvement in the control of different aspects of HCV infection was confirmed in replication studies and meta-analyses [6e8]. Since human genetic control of susceptibility to infectious disease is likely to be complex, other genetic loci can also influence on predisposition to HC. Therefore, the search on these loci should be continued in different human populations. Previously, seven SNPs located in four human genes, including interferon-induced oligoadenylate synthetase 2 (OAS2) and oligoadenylate synthetase 3 (OAS3) genes, tolllike receptor 3 (TLR3) gene, and CD209 gene encoding
http://dx.doi.org/10.1016/j.micinf.2014.02.004 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
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A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5
Table 1 Characteristic of single nucleotide polymorphisms (SNPs). Gene, SNP
SNP localization
Nucleotide change
Amino acid change
OAS2, rs1293762 OAS2, rs15895 OAS2, rs1732778 OAS3, rs2285932 OAS3, rs2072136 CD209, rs2287886 TLR3, rs3775291
Intron 2 0 3 UTRa/exon 11b 0 3 flanking region Exon 6 Exon 8 Promoter region Exon 4
G/T G/A G/A C/T G/A G/A G/A
e Trpb720Ter e Ile438Ile Ser567Ser e Leu412Phe
UTR, untranslated region. a According to GenBank NM_002535.2 OAS2 mRNA isoform (p. 69). b According to GenBank NM_016817.2 OAS2 mRNA isoform (p. 71).
dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), were reported to be associated with predisposition to tick-borne encephalitis (TBE) in a Russian population [9e11]. Since all these genes are involved in the antiviral defense, they can be also considered as candidate genes for the study of genetic predisposition to chronic HC. Moreover, although HC and TBE differ in the routes of infection (parenteral and transmissive, respectively), in tropism of the infectious agents (hepato- and neurotropism, respectively), and in prevalent course of the diseases (chronic and acute, respectively), they are caused by viruses that belong to the same Flaviviridae family and have similar genome organization (single-stranded positive sense RNA viruses). However, it is still not clear whether molecular protective mechanisms against these two viruses are similar or different in the same human population. Thus, the aim of this study was to estimate a possible association between predisposition to chronic HC and those seven SNPs, including OAS2 gene rs1293762, rs15895, and rs1732778 SNPs, OAS3 gene rs2285932 and rs2072136 SNPs, CD209 gene rs2287886 SNP, and TLR3 gene rs3775291 SNP (SNP characteristics are given in Table 1), in the Russian population. 2. Materials and methods 2.1. Subjects Blood samples were collected from unrelated HC patients from hospitals in Novosibirsk between 2008 and 2011. Only patients with PCR-positive results of HCV detection were
included in the studied sample; patients with PCR-negative results, as well as patients with hepatitis B (that were initially considered as HC patients), were finally excluded from the sample. All patients had prolonged (no less than 3 years) chronic HCV infection. This research was approved by the Bioethics Committee of the Institute of Cytology and Genetics (Russian Academy of Sciences, Siberian Branch). Written informed consent was obtained from each participant. All studied individuals were white (mainly Russians). In total, 75 samples from patients with the confirmed diagnosis “chronic HC” were studied (age 18e62; males/females approximately 1/1). HCV genotype was determined for 60 out of 75 studied samples; among them, 1b and 3a genotypes prevailed (in samples of 36 and 15 patients, respectively). The population control was used in this study. Control samples (Russian population) were obtained from 269 Novosibirsk citizens (age 25e64; males/females approximately 1/1) from The World Health Organization MONICA project (Russia). The control group was developed by random selection of individuals from the voting list of the population of one of the Novosibirsk district. No information about whether or not they have been exposed to HCV was available. Since the current study is conducted on the same population as our previous study devoted to predisposition to TBE, data on genotype and allele frequencies for the studied SNPs for the control sample were taken from our previously published articles [9e11] (see Results section). 2.2. Genotyping PCR and restriction fragment length polymorphism analysis was used to genotype the studied SNPs in DNA samples of HC patients and the control group. Genotyping assays for the OAS2 gene rs15895 and rs1732778 SNPs, OAS3 gene rs2285932 and rs2072136 SNPs, and TLR3 gene rs3775291 SNP were the same as used by Barkhash et al. [9,11]. The details of the genotyping assays for the OAS2 gene rs1293762 and CD209 gene rs2287886 SNPs are summarized in Table 2. To facilitate these SNP assays, new restriction sites were generated in the PCR products by substituting one or two nucleotides in one of the primers [12]. The PCR and restriction products were separated on 5% polyacrylamide gels and visualized by staining with ethidium bromide.
Table 2 Genotyping assays for OAS2 gene rs1293762 and CD209 gene rs2287886 single nucleotide polymorphisms (SNPs). Gene, SNP
Primer sequences 0
0
OAS2, rs1293762
5 -gaagctgccactagatggCCg-3 0 0 5 -caagttaccaattcccctgtag-3
CD209, rs2287886
5 -atgctctgatgctttccactGg-3 a 0 0 5 -cactcatgtcaccccactctcc-3
0
0
a
Tm ( C)
Product length (bp)
Restriction enzyme
Restriction fragment lengths (bp)
60
120
HpaII
65
168
HaeIII
G/G: 100, 20 G/T: 120, 100, 20 T/T: 120 G/G: 100, 46,b 22 G/A: 122, 100, 46,b 22 A/A: 122, 46b
Tm, annealing temperature; bp, base pair. a Nucleotides designated by capitalization were changed in the primer sequence in order to generate a new restriction site, as described by Neff et al. [12]. b A 46 bp fragment is produced for any genotype due to the presence of second HaeIII recognition site within the amplified 168 bp fragment. Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5 Table 3 Genotypic and allelic frequencies for the OAS3, OAS2, CD209, and TLR3 gene single nucleotide polymorphisms (SNPs) in hepatitis C (HC) patients and in the control group.b Genes, SNPs; Genotype (allele) frequency: % (numbera) genotypes or alleles HC patients Control groupb
Pd values
OAS3, rs2285932 C/C C/T T/T C T Nc c2 HWe
61.3 (46) 32.0 (24) 6.7 (5) 77.3 22.7 75 0.57
57.0 (151) 35.8 (95) 7.2 (19) 74.9 25.1 265 0.57
>0.05 >0.05 >0.05 >0.05 >0.05
OAS3, rs2072136 G/G G/A A/A G A Nc c2 HWe
48.0 (35) 39.7 (29) 12.3 (9) 67.8 32.2 73 0.59
50.0 (130) 41.2 (107) 8.8 (23) 70.6 29.4 260 0.02
>0.05 >0.05 >0.05 >0.05 >0.05
OAS2, rs1293762 G/G G/T T/T G T Nc c2 HWe
47.3 (35) 43.2 (32) 9.5 (7) 68.9 31.1 74 0.01
33.2 (62) 49.7 (93) 17.1 (32) 58.0 42.0 187 0.08
0.033 >0.05 >0.05 0.021 0.021
OAS2, rs15895 G/G G/A A/A G A Nc c2 HWe
54.3 (38) 41.4 (29) 4.3 (3) 75.0 25.0 70 0.77
54.2 (130) 35.4 (85) 10.4 (25) 71.9 28.1 240 3.69
>0.05 >0.05 >0.05 >0.05 >0.05
OAS2, rs1732778 G/G G/A A/A G A Nc c2 HWe
57.1 (40) 34.3 (24) 8.6 (6) 74.3 25.7 70 0.74
53.7 (116) 38.0 (82) 8.3 (18) 72.7 27.3 216 0.42
>0.05 >0.05 >0.05 >0.05 >0.05
CD209, rs2287886 G/G G/A A/A G A Nc c2 HWe
24.3 (18) 59.5 (44) 16.2 (12) 54.1 45.9 74 2.87
49.0 (122) 39.0 (97) 12.0 (30) 68.5 31.5 249 2.38
0.05 0.001 0.001
TLR3, rs3775291 G/G G/A A/A G A Nc c2 HWe
44.6 (33) 41.9 (31) 13.5 (10) 65.5 34.5 74 0.39
42.4 (114) 46.1 (124) 11.5 (31) 65.4 34.6 269 0.10
>0.05 >0.05 >0.05 >0.05 >0.05
a
Number of subjects with a given genotype.
3
b Genotypic and allelic frequencies for the control group are given according to our published data; particularly [9] (for SNPs rs2285932, rs2072136, rs1293762, rs15895, rs1732778), [10] (for SNP rs2287886), [11] (for SNP rs3775291). c N e number of individuals. d P values were calculated for comparisons between HC patients and the control group. e Values of c2 test for correspondence of genotype frequency distribution to the HardyeWeinberg equilibrium.
2.3. Statistical analysis The correspondence of genotype frequencies to HardyeWeinberg equilibrium was assessed by both the c2 test and CHIHW Software [13]. Genotypic and allelic frequencies were compared between groups by c2 test using SPSS Software (version 11.0). The differences were considered significant when the P-values were less than 0.05. 3. Results Genotypic and allelic frequencies for the studied seven SNPs in chronic HC patients are presented in Table 3. For all of the studied SNPs, the genotype frequency distribution was consistent with HardyeWeinberg equilibrium. Genotypic and allelic frequencies for the control group (that were taken from previously published data [9e11]) are also presented in Table 3. No significant differences between chronic HC patients and controls were found in genotype or allele frequencies for both OAS3 SNPs, OAS2 rs15895 and rs1732778 SNPs, and TLR3 rs3775291 SNP. For the OAS2 rs1293762 SNP, significant differences in the frequencies of G/G homozygotes were detected between chronic HC patients (47.3%) and the control group (33.2%) (P ¼ 0.033); in addition, the frequency of the G allele for this SNP was greater in HC patients (68.9%) than in the control group (58.0%) (P ¼ 0.021). A significant decrease in the G/G homozygote and G allele frequencies for the CD209 rs2287886 SNP was found among HC patients (24.3% and 54.1%, respectively) compared with the control group (49.0% and 68.5%, respectively) (P < 0.001 and P ¼ 0.001, respectively); an increase in the frequency of G/A genotype for the same SNP was also observed among HC patients (59.5%) compared with the controls (39.0%) (P ¼ 0.002). 4. Discussion Possible involvement of seven SNPs within four innate immunity genes (OAS2, OAS3, CD209, and TLR3) in predisposition to chronic HC was for the first time studied in a Russian population. Previously, association between these SNPs and predisposition to severe forms of TBE (caused by a virus from the same family) was demonstrated [9e11]. In the current study, two of them (OAS2 gene rs1293762 and CD209 gene rs2287886 SNPs) also demonstrated an association with predisposition to chronic HC. The OAS2 gene is located in the human chromosome 12 0 within a cluster of three interferon-induced OAS-genes (5 -
Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
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A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5 0
0
0
OAS1-OAS3-OAS2-3 ) encoding different types of 2 -5 -oligoadenylate synthetases; after binding to viral double-stranded 0 0 RNA, these enzymes catalyze the generation of 2 -5 -linked oligoadenylates that activate latent RNase L to cleave both cellular and viral RNA molecules and thus prevent the protein synthesis [14]. It is noteworthy that several associations between the OAS1 gene polymorphisms and some aspects of HC pathogenesis were previously reported for different human populations [15e17]. Moreover, the involvement of the OAS1 p46 and OAS3 p100 isoforms in mediation of the RNase Ldependent activity against HCV was recently demonstrated [18]. It is known that three types of enzymes encoded by OASgenes differ in their cellular compartmentalization, conformation, and the amount of double-stranded RNA required for their activation [14]. Data obtained in the present study are the first evidence of a possible involvement of the OAS2 gene variant (rs1293762 SNP located in the intron 2 (Table 1)) in human predisposition to chronic HC. This result requires further studies aimed to finding of yet unidentified OAS2 gene activity relative to HCV. The CD209 gene is located in the chromosome 19 and encodes dendritic cell-specific intercellular adhesion molecule 3 (ICAM3)-grabbing non-integrin (DC-SIGN), a C-type lectin that is expressed on the surface of dendritic cells and certain macrophages. DC-SIGN has several functions both as pathogen-recognition receptor (detection of specific pathogenassociated molecular patterns and subsequent processing of pathogen antigens and their presentation to the effectors of the adaptive immune response) and as cell adhesion receptor (involvement in dendritic cell migration and adhesion and in T-lymphocyte activation) [19,20]. The CD209 gene rs2287886 SNP is located in the promoter region (Table 1) at the position 139. To date, it is known that this SNP is located not far (7 bps) from the predicted binding site for the transcription factor AP-1 [21]. Such substitution can affect the expression level of the CD209 [22]. Previously, another CD209 gene promoter SNP located at the position 336 (rs4804803) was associated with more advanced liver disease, higher liver fibrosis scores and higher levels of alanine transaminase in Irish chronic HC patients [23]. However, our previous studies found that rs2287886 (but not rs4804803) is associated with predisposition to TBE (although these two SNPs are in significant (but not complete) linkage disequilibrium in a Russian population) [10]. The frequencies of the OAS2 rs1293762 G/G genotype and G allele, the CD209 rs2287886 G/A genotype and A allele are significantly higher in chronic HC patients compared to the control group (Table 3). Previously, another OAS2 rs1293762 SNP genotypes and alleles were reported to be associated with predisposition to severe forms of TBE (T/T genotype and T allele) [9]. Highly significant differences in genotype or allele frequencies for the CD209 rs2287886 SNP (P ¼ 0.002, 0.001) were found between chronic HC patients and the control group (Table 3). The genotype distribution in patient and control groups for the CD209 rs2287886 SNP is different in case of HC and TBE; however, an increase in the A allele frequency is observed in patients with both diseases (Table 3, [10]). This
fact allowed us to speculate that the CD209 gene rs2287886 SNP can be a common genetic marker of predisposition to chronic HC and severe TBE at least in the Russian population. Thus, we demonstrated that the rs1293762 SNP in the OAS2 gene and rs2287886 SNP in the CD209 gene can be genetic markers of human predisposition to chronic HC in the Russian population. To our best knowledge, this is the first study on a possible involvement of polymorphisms in these genes in predisposition to HC. Additional studies with independent HC patient samples are needed to further validate these results. However, it is noteworthy that these SNPs can be involved in predisposition to two infections caused by viruses from the Flaviviridae family (HC and TBE viruses) with common structural and functional features. Further studies on larger chronic HC patient samples, analysis of other functional SNPs from the OAS gene cluster and CD209 gene, additional selection of appropriate candidate genes, as well as genome wide association studies can be also useful in determining the involvement of these and other genes in the development of chronic HC in a Russian population. Acknowledgments This work was supported by the Russian Foundation for Basic Research (Grant Nos. 11-04-01206a and 14-04-00641a) and by Russian State Program for the Support of Leading Scientific Schools (NSh-4713.2014.4). References [1] http://www.who.int/mediacentre/factsheets/fs164/en/ [accessed 27.12.13]. [2] Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect 2011;17:107e15. [3] Thomas DL, Seeff LB. Natural history of hepatitis C. Clin Liver Dis 2005;9:383e98. [4] Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 2005;5:558e67. [5] Chapman SJ, Hill AV. Human genetic susceptibility to infectious disease. Nat Rev Genet 2012;13:175e88. [6] Rau M, Baur K, Geier A. Host genetic variants in the pathogenesis of hepatitis C. Viruses 2012;4:3281e302. [7] Romero-Gomez M, Eslam M, Ruiz A, Maraver M. Genes and hepatitis C: susceptibility, fibrosis progression and response to treatment. Liver Int 2011;31:443e60. [8] Schaefer EA, Chung RT. The impact of human gene polymorphisms on HCV infection and disease outcome. Semin Liver Dis 2011;31:375e86. [9] Barkhash AV, Perelygin AA, Babenko VN, Myasnikova NG, 0 0 Pilipenko PI, Romaschenko AG, et al. Variability in the 2 -5 -oligoadenylate synthetase gene cluster is associated with human predisposition to tick-borne encephalitis virus-induced disease. J Infect Dis 2010;202:1813e8. [10] Barkhash AV, Perelygin AA, Babenko VN, Brinton MA, Voevoda MI. Single nucleotide polymorphism in the promoter region of the CD209 gene is associated with human predisposition to severe forms of tickborne encephalitis. Antiviral Res 2012;93:64e8. [11] Barkhash AV, Voevoda MI, Romaschenko AG. Association of single nucleotide polymorphism rs3775291 in the coding region of the TLR3 gene with predisposition to tick-borne encephalitis in a Russian population. Antiviral Res 2013;99:136e8. [12] Neff MM, Turk E, Kalishman M. Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 2002;18:613e5.
Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5 [13] Zaykin DV, Pudovkin AI. Two programs to estimate significance of c2 values using pseudo-probability tests. J Hered 1993;84:152. 0 0 [14] Hovanessian AG, Justesen J. The human 2 -5 oligoadenylate synthetase 0 0 family: unique interferon-inducible enzymes catalyzing 2 -5 instead of 0 0 3 -5 phosphodiester bond formation. Biochimie 2007;89:779e88. [15] Knapp S, Yee LJ, Frodsham AJ, Hennig BJ, Hellier S, Zhang L, et al. Polymorphisms in interferon-induced genes and the outcome of hepatitis C virus infection: roles of MxA, OAS-1 and PKR. Genes Immun 2003;4:411e9. [16] Li CZ, Kato N, Chang JH, Muroyama R, Shao RX, Dharel N, et al. Polymorphism of OAS-1 determines liver fibrosis progression in hepatitis C by reduced ability to inhibit viral replication. Liver Int 2009;29:1413e21. [17] El Awady MK, Anany MA, Esmat G, Zayed N, Tabll AA, Helmy A, et al. Single nucleotide polymorphism at exon 7 splice acceptor site of OAS1 gene determines response of hepatitis C virus patients to interferon therapy. J Gastroenterol Hepatol 2011;26:843e50.
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[18] Kwon YC, Kang JI, Hwang SB, Ahn BY. The ribonuclease L-dependent 0 0 antiviral roles of human 2 ,5 -oligoadenylate synthetase family members against hepatitis C virus. FEBS Lett 2013;587:156e64. [19] Khoo US, Chan KY, Chan VS, Lin CL. DC-SIGN and L-SIGN: the SIGNs for infection. J Mol Med 2008;86:861e74. [20] Zhou T, Chen Y, Hao L, Zhang Y. DC-SIGN and immunoregulation. Cell Mol Immunol 2006;3:279e83. [21] Liu H, Yu W, Liou LY, Rice AP. Isolation and characterization of the human DC-SIGN and DC-SIGNR promoters. Gene 2003;313:149e59. [22] Koizumi Y, Kageyama S, Fujiyama Y, Miyashita M, Lwembe R, Ogino K, et al. RANTES e 28G delays and DC-SIGN e 139C enhances AIDS progression in HIV type 1-infected Japanese hemophiliacs. AIDS Res Hum Retroviruses 2007;23:713e9. [23] Ryan EJ, Dring M, Ryan CM, McNulty C, Stevenson NJ, Lawless MW, et al. Variant in CD209 promoter is associated with severity of liver disease in chronic hepatitis C virus infection. Hum Immunol 2010;71:829e32.
Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
MODEL
Microbes and Infection xx (2014) 1e5 www.elsevier.com/locate/micinf
Short communication
Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population Andrey V. Barkhash a,*, Galina V. Kochneva b, Elena V. Chub b, Svetlana V. Mikhailova a, Aida G. Romaschenko a a
Laboratory of Human Molecular Genetics, Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Branch, 10 Lavrentyeva Ave., Novosibirsk 630090, Russia b Laboratory of Viral Hepatitis, State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region1 630559, Russia Received 28 September 2013; accepted 14 February 2014
Abstract Chronic hepatitis C is a severe liver disease caused by positive-strand RNA virus. Previously, we reported an association between seven single nucleotide polymorphisms (SNPs) in four innate immunity genes (OAS2, OAS3, CD209, and TLR3) and human predisposition to tick-borne encephalitis, caused by a virus from the same Flaviviridae family, in a Russian population. Currently, genotype and allele frequencies for these SNPs were analyzed in 75 chronic hepatitis C patients and compared with the population control (269 Novosibirsk citizens). Data obtained suggest that the OAS2 rs1293762 and CD209 rs2287886 SNPs are associated with predisposition to chronic hepatitis C in Russian population. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Chronic hepatitis C; Genetic predisposition to disease; Single nucleotide polymorphism
1. Introduction Chronic hepatitis C (HC) is a severe liver disease which is found worldwide. HC is caused by an enveloped, positivestrand RNA virus (HC virus, HCV) from the Hepacivirus genus (the Flaviviridae family). About 75e85% of newly HCV-infected individuals develop chronic disease that can progress to liver cirrhosis and hepatocellular carcinoma. Approximately 150 million individuals in the world are chronically infected with HCV, and more than 350,000 individuals annually die from HC-related liver diseases. The development of efficient vaccine against HCV is currently only in progress [1e4]. It was established that human genetic factors play an important role in determining the course and outcome of * Corresponding author. Tel.: þ7 383 3634974; fax: þ7 383 3331278. E-mail address: [email protected] (A.V. Barkhash). 1 Koltsovo is located not within Novosibirsk city, but in Novosibirsk region.
different infectious diseases (including chronic HC) [5]. Polymorphisms in several genes involved in both innate and acquired immunity pathways were associated with different aspects of HC pathogenesis (viral clearance, chronic HC and fibrosis progression, response to combined antiviral therapy) in different human populations. Among them, several single nucleotide polymorphisms (SNPs) located within interleukin 28B (IL28B) gene are considered to be the most strong predictors, since their involvement in the control of different aspects of HCV infection was confirmed in replication studies and meta-analyses [6e8]. Since human genetic control of susceptibility to infectious disease is likely to be complex, other genetic loci can also influence on predisposition to HC. Therefore, the search on these loci should be continued in different human populations. Previously, seven SNPs located in four human genes, including interferon-induced oligoadenylate synthetase 2 (OAS2) and oligoadenylate synthetase 3 (OAS3) genes, tolllike receptor 3 (TLR3) gene, and CD209 gene encoding
http://dx.doi.org/10.1016/j.micinf.2014.02.004 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
2
A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5
Table 1 Characteristic of single nucleotide polymorphisms (SNPs). Gene, SNP
SNP localization
Nucleotide change
Amino acid change
OAS2, rs1293762 OAS2, rs15895 OAS2, rs1732778 OAS3, rs2285932 OAS3, rs2072136 CD209, rs2287886 TLR3, rs3775291
Intron 2 0 3 UTRa/exon 11b 0 3 flanking region Exon 6 Exon 8 Promoter region Exon 4
G/T G/A G/A C/T G/A G/A G/A
e Trpb720Ter e Ile438Ile Ser567Ser e Leu412Phe
UTR, untranslated region. a According to GenBank NM_002535.2 OAS2 mRNA isoform (p. 69). b According to GenBank NM_016817.2 OAS2 mRNA isoform (p. 71).
dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), were reported to be associated with predisposition to tick-borne encephalitis (TBE) in a Russian population [9e11]. Since all these genes are involved in the antiviral defense, they can be also considered as candidate genes for the study of genetic predisposition to chronic HC. Moreover, although HC and TBE differ in the routes of infection (parenteral and transmissive, respectively), in tropism of the infectious agents (hepato- and neurotropism, respectively), and in prevalent course of the diseases (chronic and acute, respectively), they are caused by viruses that belong to the same Flaviviridae family and have similar genome organization (single-stranded positive sense RNA viruses). However, it is still not clear whether molecular protective mechanisms against these two viruses are similar or different in the same human population. Thus, the aim of this study was to estimate a possible association between predisposition to chronic HC and those seven SNPs, including OAS2 gene rs1293762, rs15895, and rs1732778 SNPs, OAS3 gene rs2285932 and rs2072136 SNPs, CD209 gene rs2287886 SNP, and TLR3 gene rs3775291 SNP (SNP characteristics are given in Table 1), in the Russian population. 2. Materials and methods 2.1. Subjects Blood samples were collected from unrelated HC patients from hospitals in Novosibirsk between 2008 and 2011. Only patients with PCR-positive results of HCV detection were
included in the studied sample; patients with PCR-negative results, as well as patients with hepatitis B (that were initially considered as HC patients), were finally excluded from the sample. All patients had prolonged (no less than 3 years) chronic HCV infection. This research was approved by the Bioethics Committee of the Institute of Cytology and Genetics (Russian Academy of Sciences, Siberian Branch). Written informed consent was obtained from each participant. All studied individuals were white (mainly Russians). In total, 75 samples from patients with the confirmed diagnosis “chronic HC” were studied (age 18e62; males/females approximately 1/1). HCV genotype was determined for 60 out of 75 studied samples; among them, 1b and 3a genotypes prevailed (in samples of 36 and 15 patients, respectively). The population control was used in this study. Control samples (Russian population) were obtained from 269 Novosibirsk citizens (age 25e64; males/females approximately 1/1) from The World Health Organization MONICA project (Russia). The control group was developed by random selection of individuals from the voting list of the population of one of the Novosibirsk district. No information about whether or not they have been exposed to HCV was available. Since the current study is conducted on the same population as our previous study devoted to predisposition to TBE, data on genotype and allele frequencies for the studied SNPs for the control sample were taken from our previously published articles [9e11] (see Results section). 2.2. Genotyping PCR and restriction fragment length polymorphism analysis was used to genotype the studied SNPs in DNA samples of HC patients and the control group. Genotyping assays for the OAS2 gene rs15895 and rs1732778 SNPs, OAS3 gene rs2285932 and rs2072136 SNPs, and TLR3 gene rs3775291 SNP were the same as used by Barkhash et al. [9,11]. The details of the genotyping assays for the OAS2 gene rs1293762 and CD209 gene rs2287886 SNPs are summarized in Table 2. To facilitate these SNP assays, new restriction sites were generated in the PCR products by substituting one or two nucleotides in one of the primers [12]. The PCR and restriction products were separated on 5% polyacrylamide gels and visualized by staining with ethidium bromide.
Table 2 Genotyping assays for OAS2 gene rs1293762 and CD209 gene rs2287886 single nucleotide polymorphisms (SNPs). Gene, SNP
Primer sequences 0
0
OAS2, rs1293762
5 -gaagctgccactagatggCCg-3 0 0 5 -caagttaccaattcccctgtag-3
CD209, rs2287886
5 -atgctctgatgctttccactGg-3 a 0 0 5 -cactcatgtcaccccactctcc-3
0
0
a
Tm ( C)
Product length (bp)
Restriction enzyme
Restriction fragment lengths (bp)
60
120
HpaII
65
168
HaeIII
G/G: 100, 20 G/T: 120, 100, 20 T/T: 120 G/G: 100, 46,b 22 G/A: 122, 100, 46,b 22 A/A: 122, 46b
Tm, annealing temperature; bp, base pair. a Nucleotides designated by capitalization were changed in the primer sequence in order to generate a new restriction site, as described by Neff et al. [12]. b A 46 bp fragment is produced for any genotype due to the presence of second HaeIII recognition site within the amplified 168 bp fragment. Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5 Table 3 Genotypic and allelic frequencies for the OAS3, OAS2, CD209, and TLR3 gene single nucleotide polymorphisms (SNPs) in hepatitis C (HC) patients and in the control group.b Genes, SNPs; Genotype (allele) frequency: % (numbera) genotypes or alleles HC patients Control groupb
Pd values
OAS3, rs2285932 C/C C/T T/T C T Nc c2 HWe
61.3 (46) 32.0 (24) 6.7 (5) 77.3 22.7 75 0.57
57.0 (151) 35.8 (95) 7.2 (19) 74.9 25.1 265 0.57
>0.05 >0.05 >0.05 >0.05 >0.05
OAS3, rs2072136 G/G G/A A/A G A Nc c2 HWe
48.0 (35) 39.7 (29) 12.3 (9) 67.8 32.2 73 0.59
50.0 (130) 41.2 (107) 8.8 (23) 70.6 29.4 260 0.02
>0.05 >0.05 >0.05 >0.05 >0.05
OAS2, rs1293762 G/G G/T T/T G T Nc c2 HWe
47.3 (35) 43.2 (32) 9.5 (7) 68.9 31.1 74 0.01
33.2 (62) 49.7 (93) 17.1 (32) 58.0 42.0 187 0.08
0.033 >0.05 >0.05 0.021 0.021
OAS2, rs15895 G/G G/A A/A G A Nc c2 HWe
54.3 (38) 41.4 (29) 4.3 (3) 75.0 25.0 70 0.77
54.2 (130) 35.4 (85) 10.4 (25) 71.9 28.1 240 3.69
>0.05 >0.05 >0.05 >0.05 >0.05
OAS2, rs1732778 G/G G/A A/A G A Nc c2 HWe
57.1 (40) 34.3 (24) 8.6 (6) 74.3 25.7 70 0.74
53.7 (116) 38.0 (82) 8.3 (18) 72.7 27.3 216 0.42
>0.05 >0.05 >0.05 >0.05 >0.05
CD209, rs2287886 G/G G/A A/A G A Nc c2 HWe
24.3 (18) 59.5 (44) 16.2 (12) 54.1 45.9 74 2.87
49.0 (122) 39.0 (97) 12.0 (30) 68.5 31.5 249 2.38
0.05 0.001 0.001
TLR3, rs3775291 G/G G/A A/A G A Nc c2 HWe
44.6 (33) 41.9 (31) 13.5 (10) 65.5 34.5 74 0.39
42.4 (114) 46.1 (124) 11.5 (31) 65.4 34.6 269 0.10
>0.05 >0.05 >0.05 >0.05 >0.05
a
Number of subjects with a given genotype.
3
b Genotypic and allelic frequencies for the control group are given according to our published data; particularly [9] (for SNPs rs2285932, rs2072136, rs1293762, rs15895, rs1732778), [10] (for SNP rs2287886), [11] (for SNP rs3775291). c N e number of individuals. d P values were calculated for comparisons between HC patients and the control group. e Values of c2 test for correspondence of genotype frequency distribution to the HardyeWeinberg equilibrium.
2.3. Statistical analysis The correspondence of genotype frequencies to HardyeWeinberg equilibrium was assessed by both the c2 test and CHIHW Software [13]. Genotypic and allelic frequencies were compared between groups by c2 test using SPSS Software (version 11.0). The differences were considered significant when the P-values were less than 0.05. 3. Results Genotypic and allelic frequencies for the studied seven SNPs in chronic HC patients are presented in Table 3. For all of the studied SNPs, the genotype frequency distribution was consistent with HardyeWeinberg equilibrium. Genotypic and allelic frequencies for the control group (that were taken from previously published data [9e11]) are also presented in Table 3. No significant differences between chronic HC patients and controls were found in genotype or allele frequencies for both OAS3 SNPs, OAS2 rs15895 and rs1732778 SNPs, and TLR3 rs3775291 SNP. For the OAS2 rs1293762 SNP, significant differences in the frequencies of G/G homozygotes were detected between chronic HC patients (47.3%) and the control group (33.2%) (P ¼ 0.033); in addition, the frequency of the G allele for this SNP was greater in HC patients (68.9%) than in the control group (58.0%) (P ¼ 0.021). A significant decrease in the G/G homozygote and G allele frequencies for the CD209 rs2287886 SNP was found among HC patients (24.3% and 54.1%, respectively) compared with the control group (49.0% and 68.5%, respectively) (P < 0.001 and P ¼ 0.001, respectively); an increase in the frequency of G/A genotype for the same SNP was also observed among HC patients (59.5%) compared with the controls (39.0%) (P ¼ 0.002). 4. Discussion Possible involvement of seven SNPs within four innate immunity genes (OAS2, OAS3, CD209, and TLR3) in predisposition to chronic HC was for the first time studied in a Russian population. Previously, association between these SNPs and predisposition to severe forms of TBE (caused by a virus from the same family) was demonstrated [9e11]. In the current study, two of them (OAS2 gene rs1293762 and CD209 gene rs2287886 SNPs) also demonstrated an association with predisposition to chronic HC. The OAS2 gene is located in the human chromosome 12 0 within a cluster of three interferon-induced OAS-genes (5 -
Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
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A.V. Barkhash et al. / Microbes and Infection xx (2014) 1e5 0
0
0
OAS1-OAS3-OAS2-3 ) encoding different types of 2 -5 -oligoadenylate synthetases; after binding to viral double-stranded 0 0 RNA, these enzymes catalyze the generation of 2 -5 -linked oligoadenylates that activate latent RNase L to cleave both cellular and viral RNA molecules and thus prevent the protein synthesis [14]. It is noteworthy that several associations between the OAS1 gene polymorphisms and some aspects of HC pathogenesis were previously reported for different human populations [15e17]. Moreover, the involvement of the OAS1 p46 and OAS3 p100 isoforms in mediation of the RNase Ldependent activity against HCV was recently demonstrated [18]. It is known that three types of enzymes encoded by OASgenes differ in their cellular compartmentalization, conformation, and the amount of double-stranded RNA required for their activation [14]. Data obtained in the present study are the first evidence of a possible involvement of the OAS2 gene variant (rs1293762 SNP located in the intron 2 (Table 1)) in human predisposition to chronic HC. This result requires further studies aimed to finding of yet unidentified OAS2 gene activity relative to HCV. The CD209 gene is located in the chromosome 19 and encodes dendritic cell-specific intercellular adhesion molecule 3 (ICAM3)-grabbing non-integrin (DC-SIGN), a C-type lectin that is expressed on the surface of dendritic cells and certain macrophages. DC-SIGN has several functions both as pathogen-recognition receptor (detection of specific pathogenassociated molecular patterns and subsequent processing of pathogen antigens and their presentation to the effectors of the adaptive immune response) and as cell adhesion receptor (involvement in dendritic cell migration and adhesion and in T-lymphocyte activation) [19,20]. The CD209 gene rs2287886 SNP is located in the promoter region (Table 1) at the position 139. To date, it is known that this SNP is located not far (7 bps) from the predicted binding site for the transcription factor AP-1 [21]. Such substitution can affect the expression level of the CD209 [22]. Previously, another CD209 gene promoter SNP located at the position 336 (rs4804803) was associated with more advanced liver disease, higher liver fibrosis scores and higher levels of alanine transaminase in Irish chronic HC patients [23]. However, our previous studies found that rs2287886 (but not rs4804803) is associated with predisposition to TBE (although these two SNPs are in significant (but not complete) linkage disequilibrium in a Russian population) [10]. The frequencies of the OAS2 rs1293762 G/G genotype and G allele, the CD209 rs2287886 G/A genotype and A allele are significantly higher in chronic HC patients compared to the control group (Table 3). Previously, another OAS2 rs1293762 SNP genotypes and alleles were reported to be associated with predisposition to severe forms of TBE (T/T genotype and T allele) [9]. Highly significant differences in genotype or allele frequencies for the CD209 rs2287886 SNP (P ¼ 0.002, 0.001) were found between chronic HC patients and the control group (Table 3). The genotype distribution in patient and control groups for the CD209 rs2287886 SNP is different in case of HC and TBE; however, an increase in the A allele frequency is observed in patients with both diseases (Table 3, [10]). This
fact allowed us to speculate that the CD209 gene rs2287886 SNP can be a common genetic marker of predisposition to chronic HC and severe TBE at least in the Russian population. Thus, we demonstrated that the rs1293762 SNP in the OAS2 gene and rs2287886 SNP in the CD209 gene can be genetic markers of human predisposition to chronic HC in the Russian population. To our best knowledge, this is the first study on a possible involvement of polymorphisms in these genes in predisposition to HC. Additional studies with independent HC patient samples are needed to further validate these results. However, it is noteworthy that these SNPs can be involved in predisposition to two infections caused by viruses from the Flaviviridae family (HC and TBE viruses) with common structural and functional features. Further studies on larger chronic HC patient samples, analysis of other functional SNPs from the OAS gene cluster and CD209 gene, additional selection of appropriate candidate genes, as well as genome wide association studies can be also useful in determining the involvement of these and other genes in the development of chronic HC in a Russian population. Acknowledgments This work was supported by the Russian Foundation for Basic Research (Grant Nos. 11-04-01206a and 14-04-00641a) and by Russian State Program for the Support of Leading Scientific Schools (NSh-4713.2014.4). References [1] http://www.who.int/mediacentre/factsheets/fs164/en/ [accessed 27.12.13]. [2] Lavanchy D. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect 2011;17:107e15. [3] Thomas DL, Seeff LB. Natural history of hepatitis C. Clin Liver Dis 2005;9:383e98. [4] Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 2005;5:558e67. [5] Chapman SJ, Hill AV. Human genetic susceptibility to infectious disease. Nat Rev Genet 2012;13:175e88. [6] Rau M, Baur K, Geier A. Host genetic variants in the pathogenesis of hepatitis C. Viruses 2012;4:3281e302. [7] Romero-Gomez M, Eslam M, Ruiz A, Maraver M. Genes and hepatitis C: susceptibility, fibrosis progression and response to treatment. Liver Int 2011;31:443e60. [8] Schaefer EA, Chung RT. The impact of human gene polymorphisms on HCV infection and disease outcome. Semin Liver Dis 2011;31:375e86. [9] Barkhash AV, Perelygin AA, Babenko VN, Myasnikova NG, 0 0 Pilipenko PI, Romaschenko AG, et al. Variability in the 2 -5 -oligoadenylate synthetase gene cluster is associated with human predisposition to tick-borne encephalitis virus-induced disease. J Infect Dis 2010;202:1813e8. [10] Barkhash AV, Perelygin AA, Babenko VN, Brinton MA, Voevoda MI. Single nucleotide polymorphism in the promoter region of the CD209 gene is associated with human predisposition to severe forms of tickborne encephalitis. Antiviral Res 2012;93:64e8. [11] Barkhash AV, Voevoda MI, Romaschenko AG. Association of single nucleotide polymorphism rs3775291 in the coding region of the TLR3 gene with predisposition to tick-borne encephalitis in a Russian population. Antiviral Res 2013;99:136e8. [12] Neff MM, Turk E, Kalishman M. Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 2002;18:613e5.
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[18] Kwon YC, Kang JI, Hwang SB, Ahn BY. The ribonuclease L-dependent 0 0 antiviral roles of human 2 ,5 -oligoadenylate synthetase family members against hepatitis C virus. FEBS Lett 2013;587:156e64. [19] Khoo US, Chan KY, Chan VS, Lin CL. DC-SIGN and L-SIGN: the SIGNs for infection. J Mol Med 2008;86:861e74. [20] Zhou T, Chen Y, Hao L, Zhang Y. DC-SIGN and immunoregulation. Cell Mol Immunol 2006;3:279e83. [21] Liu H, Yu W, Liou LY, Rice AP. Isolation and characterization of the human DC-SIGN and DC-SIGNR promoters. Gene 2003;313:149e59. [22] Koizumi Y, Kageyama S, Fujiyama Y, Miyashita M, Lwembe R, Ogino K, et al. RANTES e 28G delays and DC-SIGN e 139C enhances AIDS progression in HIV type 1-infected Japanese hemophiliacs. AIDS Res Hum Retroviruses 2007;23:713e9. [23] Ryan EJ, Dring M, Ryan CM, McNulty C, Stevenson NJ, Lawless MW, et al. Variant in CD209 promoter is associated with severity of liver disease in chronic hepatitis C virus infection. Hum Immunol 2010;71:829e32.
Please cite this article in press as: Barkhash AV, et al., Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.02.004
