Regulatory Polymorphisms in the Promoter of CXCL10 Gene and Disease Progression in Male Hepatitis B Virus Carriers
2008; Elsevier BV; Volume: 134; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.12.044
ISSN1528-0012
AutoresGuohong Deng, Gangqiao Zhou, Rong Zhang, Yun Zhai, Wenli Zhao, Zehui Yan, Chunqing Deng, Xiaoyan Yuan, Baoyan Xu, Xiaojia Dong, Xiumei Zhang, Xuqing Zhang, Zhijian Yao, Yan Shen, Boqing Qiang, Yuming Wang, Fuchu He,
Tópico(s)Animal Virus Infections Studies
ResumoBackground & Aims: The importance of expression of interferon gamma–inducible protein of 10 kilodaltons (IP-10, CXCL10) during chronic hepatitis B virus (HBV) infection has been recently emphasized. In this report, we investigated whether the naturally occurred sequence variations in the CXCL10 gene impact liver damage and disease progression of chronic HBV infection. Methods: A hospital-based case-control study was conducted, and a total of 613 and 1787 unrelated Han Chinese HBV carriers were recruited from Beijing and Chongqing, respectively. We systematically screened sequence variations in the CXCL10 gene and examined the association between the variations in this gene and susceptibility to disease progression of chronic HBV infection in Chinese populations from Beijing and Chongqing. Functional analyses were conducted to verify the biological significances of the associated genetic variation. Results: We identified that the polymorphism G-201A, located in the promoter region of CXCL10, was associated with susceptibility to disease progression in male HBV carriers (dominant model; odds ratio, 1.53; P = .001). Functional analyses show that the G-201A polymorphism alters the binding affinity of nuclear protein and regulates CXCL10 expression. We observed higher CXCL10 transcription in interferon gamma–stimulated peripheral blood mononuclear cells with the disease-susceptible genotypes. Enzyme-linked immunosorbent assay and immunohistochemical analysis showed augmented CXCL10 production in serum and liver tissues of progressed HBV carriers. Conclusions: The novel regulatory polymorphism G-210A in the promoter of CXCL10 gene could be a part of the genetic variation underlying the susceptibility of individuals to disease progression of chronic HBV infection. Background & Aims: The importance of expression of interferon gamma–inducible protein of 10 kilodaltons (IP-10, CXCL10) during chronic hepatitis B virus (HBV) infection has been recently emphasized. In this report, we investigated whether the naturally occurred sequence variations in the CXCL10 gene impact liver damage and disease progression of chronic HBV infection. Methods: A hospital-based case-control study was conducted, and a total of 613 and 1787 unrelated Han Chinese HBV carriers were recruited from Beijing and Chongqing, respectively. We systematically screened sequence variations in the CXCL10 gene and examined the association between the variations in this gene and susceptibility to disease progression of chronic HBV infection in Chinese populations from Beijing and Chongqing. Functional analyses were conducted to verify the biological significances of the associated genetic variation. Results: We identified that the polymorphism G-201A, located in the promoter region of CXCL10, was associated with susceptibility to disease progression in male HBV carriers (dominant model; odds ratio, 1.53; P = .001). Functional analyses show that the G-201A polymorphism alters the binding affinity of nuclear protein and regulates CXCL10 expression. We observed higher CXCL10 transcription in interferon gamma–stimulated peripheral blood mononuclear cells with the disease-susceptible genotypes. Enzyme-linked immunosorbent assay and immunohistochemical analysis showed augmented CXCL10 production in serum and liver tissues of progressed HBV carriers. Conclusions: The novel regulatory polymorphism G-210A in the promoter of CXCL10 gene could be a part of the genetic variation underlying the susceptibility of individuals to disease progression of chronic HBV infection. See Keeffe EB et al on page 268 in CGH. See Keeffe EB et al on page 268 in CGH. Hepatitis B virus (HBV) is the most common cause of acute and chronic liver diseases worldwide, especially in several areas of Asia and Africa.1Lee W.M. Hepatitis B virus infection.N Engl J Med. 1997; 337: 1733-1745Crossref PubMed Scopus (2200) Google Scholar, 2Lai C.L. Ratziu V. Yuen M.F. et al.Viral hepatitis B.Lancet. 2003; 362: 2089-2094Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar For subjects who are infected with HBV during early childhood, liver damage occurs mainly during the immune clearance phase.3Lok A.S. Lai C.L. Wu P.C. et al.Hepatitis B virus infection in Chinese families in Hong Kong.Am J Epidemiol. 1987; 126: 492-499PubMed Google Scholar, 4Coursaget P. Yvonnet B. Chotard J. et al.Age- and sex-related study of hepatitis B virus chronic carrier state in infants from an endemic area (Senegal).J Med Virol. 1987; 22: 1-5Crossref PubMed Scopus (110) Google Scholar During chronic HBV infection, the HBV-specific CD8 T-cell response, in the absence of viral clearance, may contribute to both liver cell damage and the recruitment of non–virus-specific T cells.5Maini M.K. Boni C. Lee C.K. et al.The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.J Exp Med. 2000; 191: 1269-1280Crossref PubMed Scopus (718) Google Scholar Such recruitment of non–antigen-specific CD4/CD8 cells mediated by interferon (IFN)-γ has been shown in HBV surface antigen (HBsAg) transgenic mice with fulminant hepatitis6Ando K. Moriyama T. Guidotti L.G. et al.Mechanisms of class I restricted immunopathology A transgenic mouse model of fulminant hepatitis.J Exp Med. 1993; 178: 1541-1554Crossref PubMed Scopus (418) Google Scholar and in the incubation phase of acute hepatitis B in humans.7Webster G.J. Reignat S. Maini M.K. et al.Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms.Hepatology. 2000; 32: 1117-1124Crossref PubMed Scopus (361) Google Scholar Two studies have identified that the IFN-γ–inducible protein of 10 kilodaltons (IP-10), also named as chemokine (C-X-C motif) ligand 10 (CXCL10), is a response gene induced in the livers of HBV transgenic mice8Kakimi K. Lane T.E. Wieland S. et al.Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes.J Exp Med. 2001; 194: 1755-1766Crossref PubMed Scopus (216) Google Scholar and chimpanzees acutely infected with HBV.9Wieland S. Thimme R. Purcell R.H. et al.Genomic analysis of the host response to hepatitis B virus infection.Proc Natl Acad Sci U S A. 2004; 101: 6669-6674Crossref PubMed Scopus (584) Google Scholar CXCL10 chemoattracts activated T lymphocytes and natural killer cells by binding to chemokine (C-X-C motif) receptor 3 (CXCR3), a G protein–coupled receptor.10Dufour J.H. Dziejman M. Liu M.T. et al.IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking.J Immunol. 2002; 168: 3195-3204PubMed Google Scholar, 11Liu L. Callahan M.K. Huang D. et al.Chemokine receptor CXCR3: an unexpected enigma.Curr Top Dev Biol. 2005; 68: 149-181Crossref PubMed Scopus (138) Google Scholar Recent reports have also shown that CXCL10 was expressed in the liver of patients with chronic hepatitis B; its expression on the transcription level was higher than those with liver disorders unrelated to viral infections but lower than those with chronic hepatitis C.12Patzwahl R. Meier V. Ramadori G. et al.Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization.J Virol. 2001; 75: 1332-1338Crossref PubMed Scopus (102) Google Scholar Harvey et al13Harvey C.E. Post J.J. Palladinetti P. et al.Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation.J Leukoc Biol. 2003; 74: 360-369Crossref PubMed Scopus (223) Google Scholar showed that CXCL10 could be induced within hepatocytes in liver tissue, and its expression correlated with histologic severity and lobular inflammation in patients with chronic hepatitis C virus infection. However, the role of CXCL10 in the progression of patients with chronic HBV infection has not been well established. Because CXCL10 seems to be important for the intrahepatic recruitment, antiviral effect, and pathogenetic effect of HBV-specific cytotoxic T lymphocytes and/or other nonspecific inflammatory cells during chronic HBV infection, it is expected that the naturally occurring sequence variations in the CXCL10 gene may result in altered expression and/or binding ability of the CXCL10 protein, thereby impacting liver damage and disease progression of patients with chronic HBV infection. In this report, we systematically screened sequence variations in the CXCL10 gene and then examined the association between the variations in this gene and susceptibility to disease progression of chronic HBV infection in Chinese populations. A total of 2400 unrelated Han Chinese HBV carriers were recruited at Infectious Disease Hospital (Beijing, China) and Southwest Hospital (Chongqing, China) between February 2001 and March 2006 (613 carriers were from Beijing and 1787 from Chongqing). All HBV carriers were positive for both HBsAg and antibody to HBV core antigen of the immunoglobulin G type for at least 12 months. All carriers were measured with liver function tests, serum immunologic marker screening, and liver ultrasonography/computed tomography imaging, and 376 (15.7%) were examined with histologic biopsy. All carriers had no serologic evidence for coinfection with hepatitis C virus, hepatitis D virus, and human immunodeficiency virus. Among the 2400 HBV carriers, 1147 progressed carriers and 1253 nonprogressed carriers were included, among which 312 patients with liver cirrhosis and 415 nonprogressed carriers were described in detail in our previous study.14Deng G. Zhou G. Zhai Y. et al.Association of estrogen receptor alpha polymorphisms with susceptibility to chronic hepatitis B virus infection.Hepatology. 2004; 40: 318-326Crossref PubMed Scopus (100) Google Scholar The nonprogressed carriers had normal serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin throughout the study, without any evidence for liver image/histologic change and previous history of hepatitis B. The progressed carriers were HBsAg positive, with severe hepatitis, liver cirrhosis, or hepatocellular carcinoma. Of the 1147 progressed carriers, 241 individuals were patients with severe hepatitis B who had serum total bilirubin levels >10 times the upper limit of normal without evidence for liver cirrhosis; among them, 185 (76.8%) were patients with acute liver failure (prothrombin index <40%, with an illness of 400 ng/mL. The normal control population consisted of 568 unrelated healthy Han Chinese (79.4% male; mean age, 34.4 ± 12.1 years) recruited from Beijing Blood Donor Centers (n = 253) and Southwest Hospital (n = 315) who were naive to HBV, with all HBV serum markers negative. All subjects provided informed consent to participate in the study, as approved by the ethical committee of the Chinese Human Genome, Beijing. Single nucleotide polymorphism (SNP) screening of the approximately 4.2-kilobase region of CXCL10 (referred to as GenBank accession no. NT_016354.16), continuously from the 5′ flanking to 3′ flanking genomic region (including all exons, relevant exon-intron boundaries, and ∼1.8-kilobase promoter region; see Figure 1), was performed with 11 pairs of overlapping primer sets (the primer sequences are listed in Supplementary Table 1; see supplemental material online at www.gastrojournal.org) by polymerase chain reaction (PCR) direct sequencing as described previously.14Deng G. Zhou G. Zhai Y. et al.Association of estrogen receptor alpha polymorphisms with susceptibility to chronic hepatitis B virus infection.Hepatology. 2004; 40: 318-326Crossref PubMed Scopus (100) Google Scholar, 15Zhou G. Zhai Y. Dong X. et al.Haplotype structure and evidence for positive selection at the human IL13 locus.Mol Biol Evol. 2004; 21: 29-35Crossref PubMed Scopus (31) Google Scholar SNP candidates were identified by the PolyPhred program and inspected by 2 observers. SNP positions and individual genotypes have been confirmed by reamplifying and resequencing the SNP site from the opposite strand. The screening panel included 27 unrelated individuals randomly selected from the 2400 HBV carriers regardless of disease status. The sample size gave us 95% probability of detecting alleles with a minimal frequency of 5.4%.16Kruglyak L. Nickerson D.A. Variation is the spice of life.Nat Genet. 2001; 27: 234-236Crossref PubMed Scopus (695) Google ScholarTable 1Primer Sequences Used in Screening for SNPs of the Human CXCL10 GeneFragment no.Primer sequencesaF, forward primer; R, reverse primer.Amplicon length (bp)Annealing temperature (°C)11F5′-TGTCACCATCTCTCATTTTGATTGT-3′499571R5′-GCAGATACTGTCTCAGAACCTGGTA-3′22F5′-CCATTTACCCCAAGCAACTCA-3′533572R5′-TTGGCACAATTTTAACTGGATGA-3′33F5′-TCAGGTTCAACATGTGTTCCAT-3′542563R5′-TGCTTTGATCATTCATAACACCATT-3′44F5′-TGTTTTCATTCAGGGACTGCT-3′537554R5′-AAGTTACGGAATTTCCCTCTGC-3′55F5′-TTCATGTTTTGGAAAGTGAAACCT-3′504555R5′-TGATCTGAGGGAATCTCTATTTATTTC-3′66F5′-GCACACTAGCCCCACGTTT-3′584576R5′-TGAGGAGGAATGGATCACATCA-3′77F5′-CAATGAACCACATGAACTGTGC-3′569577R5′-TGTTGGGTCAATAAAACAGATGG-3′88F5′-TCAGTAAAGGGTTTGTGATGATTC-3′453558R5′-TTCAAGGTTGACTGGTAATCAGG-3′99F5′-GGCAATCTTGGGAGTCAGAAA-3′537569R5′-CCATCATTGGTCACCTTTTAGTG-3′1010F5′-GCCAGAGTCAGACTATCACACTTTT-3′5705610R5′-TTCCTGTATGTGTTTGGAATTGTATG-3′1111F5′-GGTATGCAATCAAATCTGCTTTT-3′5425511R5′-TCATTTGTCTTCTGCCCATGC-3′a F, forward primer; R, reverse primer. Open table in a new tab CXCL10 haplotypes from the unrelated Chinese samples were assigned by the PHASE program.17Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6549) Google Scholar Program htSNP2 (Wellcome Trust, Cambridge, England) was used to determine the haplotype-tagged SNPs (htSNPs) as described previously.18Johnson G.C. Esposito L. Barratt B.J. et al.Haplotype tagging for the identification of common disease genes.Nat Genet. 2001; 29: 233-237Crossref PubMed Scopus (1027) Google Scholar The pairwise linkage disequilibrium (LD) measures were calculated using the Arlequin package (University of Geneva, Geneva, Switzerland). The values of pairwise D′ and r2 were plotted using the LDA program.19Ding K. Zhou K. He F. et al.LDA—a java-based linkage disequilibrium analyzer.Bioinformatics. 2003; 19: 2147-2148Crossref PubMed Scopus (116) Google Scholar We extracted genomic DNA from peripheral blood leukocytes of 5 mL whole blood using standard phenol/chloroform protocols. DNA samples were diluted to 8 ng/μL and distributed to 96-well plates (DNA panels), each of which contained 94 samples and 2 DNA-free control water. Two htSNPs, G-201A and C-1513T polymorphisms in the 5′ flanking region of the CXCL10 gene, were selected for genotyping in the study population using PCR/restriction fragment length polymorphism analysis. An amplification using forward primer 5′-GCAGATACTGTCTCAGAACCTGGTA-3′ and reverse primer 5′-TGTCACCATCTCTCATTTTGATTGT-3′ was performed with an annealing temperature of 57°C. The reaction yielded a 499–base pair amplicon. PCR products of 3 μL were digested with 4 U of XbaI and 4 U SacI (Takara BioTech, Dalian, China) for the C-1596T and the C-1513T polymorphisms, respectively. The homozygotes with -1596T and -1513C alleles, respectively, yield restriction fragments of 174/325 base pairs and 98/401 base pairs, respectively. Because the polymorphisms G-201A and C-1596T are in absolute LD (D′ = 1, r2 = 1, in a validation sample of 188 individuals), we infer the G-201A genotypes from the C-1596T genotypes. The accuracy of genotyping data for each SNP obtained from PCR/restriction fragment length polymorphism analysis was validated by direct sequencing of a 8% masked, random sample of patients. We performed electrophoretic mobility shift assays with IFN-γ– (100 ng/mL; BD Pharmingen, San Diego, CA) stimulated HepG2 cell nuclear extracts, biotin-labeled oligonucleotide probes corresponding to CXCL10 -201G and -201A alleles (synthesized by Shanghai Sangon Co, Shanghai, China), and LightShift EMSA kit (Pierce, Rockford, IL). The sequences of oligonucleotide probes for G and A alleles were 5′-TAAATAATACCTTCGAGTCTGCAACAT-3′ and 5′-TAAATAATACCTTCAAGTCTGCAACAT-3′, respectively. We chose genomic DNAs from haplotype 1/1 and 3/3 (see Supplementary Table 2; see supplemental material online at www.gastrojournal.org) homozygotes for reporter construction. DNA fragments corresponding to CXCL10 promoter region from nucleotides -1749 to −7 and -359 to −7 (relative to the first nucleotide of open reading frame of CXCL10 gene) were amplified and cloned into pGL3-enhancer vectors (Promega, Madison, WI). The sequences of all reporter constructs were verified by direct sequencing. IFN-γ– (100 ng/mL; BD Pharmingen) stimulated HepG2 cells (2 × 105) were transfected with 500 ng of one of the constructs together with 20 ng pRL-TK vector (Promega), an internal control for transfection efficiency, using JetPEI transfection reagent (PolyPlus-transfection Co, Illkirch, France). After 26 hours, cells were collected and their luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega). The relative luciferase levels were obtained from 3 independent experiments performed in quadruplicate.Table 2Estimated Frequencies of Haplotypes and htSNPs Observed at CXCL10 Gene LocusHaplotypeSNPsFrequency (%)1234567891011121314151617181920211-CGCGCCCGCG3G-TGGGGC-C40.72...T...T.............31.53.T........A..........13.04...T.................3.75...T...TT............3.76T.CTATT....AACA.ACGCT3.77...T.....A...........1.98...T...........A.....1.9Note. The no. of SNPs is referred to as in Table 1 Boxed SNPs represent the htSNPs that can capture the common haplotypes that are segregating in Chinese population. Dots represent the allele that is found on the most common haplotype. -, one-base deletion. Open table in a new tab Note. The no. of SNPs is referred to as in Table 1 Boxed SNPs represent the htSNPs that can capture the common haplotypes that are segregating in Chinese population. Dots represent the allele that is found on the most common haplotype. -, one-base deletion. HBV-naive umbilical blood mononuclear cells from newborn infants were stimulated with IFN-γ (100 ng/mL; BD Pharmingen). The CXCL10 G-201A genotypes of their genomic DNAs were determined by PCR/restriction fragment length polymorphism. Chromatin immunoprecipitations (ChIPs) were conducted on 5 heterozygous samples with monoclonal antibody against the Ser5 phosphorylated CTD of RNA polymerase II (MMS-134R clone H14; Covance, Princeton, NJ) as described previously.20Knight J.C. Keating B.J. Rockett K.A. et al.In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.Nat Genet. 2003; 33: 469-475Crossref PubMed Scopus (214) Google Scholar Quantitative allelic discrimination of the ChIP-isolated genome fragments was performed using Opticon Monitor 2 (MJ Research [BioRad, Hercules, CA]) with MGB-TaqMan probes corresponding to -201A and -201G alleles. RNAs of IFN-γ– (100 ng/mL; BD Pharmingen) stimulated peripheral blood mononuclear cells from adult blood donors were extracted by TriPure reagent (Roche, Basel, Switzerland). We performed real-time quantitative PCR using Opticon Monitor 2 (MJ Research) with MGB-TaqMan probes corresponding to CXCL10 and β-actin messenger RNAs. Relative quantification with MGB-TaqMan probes was performed by the 2-▵▵Ct method as described previously.21Schmittgen T.D. Zakrajsek B.A. Mills A.G. et al.Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.Anal Biochem. 2000; 285: 194-204Crossref PubMed Scopus (872) Google Scholar Primers and MGB-TaqMan probes used above are listed in Supplementary Table 3 (see supplemental material online at www.gastrojournal.org). All probes and primers were synthesized by GeneCore BioTechnologies Co (Shanghai, China), and the MGB-TaqMan probes were authorized by Applied Biosystems (Foster City, CA).Table 3Primers and MGB-TaqMan Probes Used in ChIPs and mRNA QuantificationPrimers and probesSequence (5′→3′)ChIPs Forward primer of PCRGCAAAACCTGCTGGCTGTTC Reverse primer of PCRAAATTCCGTAACTTGGAGGCTACA aMGB-TaqMan probe.FAM labeled -201G alleleTACCTTCGAGTCTGCA aMGB-TaqMan probe.VIC labeled -201A alleleAATACCTTCAAGTCTGCAmRNA quantification CXCL10 Forward primer of PCRTATTCCTGCAAGCCAATTTTGTC Reverse primer of PCRTCTTGATGGCCTTCGATTCTG aMGB-TaqMan probe.VIC labeled CXCL10 mRNA probeTGAGATCATTGCTACAATGA β-actin Forward primer of PCRACGGCCAGGTCATCACCAT Reverse primer of PCRAAGGCTGGAAGAGTGCCTCAG aMGB-TaqMan probe.FAM labeled β-actin mRNA probeCAATGAGCGGTTCCGa MGB-TaqMan probe. Open table in a new tab We examined serum CXCL10 levels in HBV carriers with human Quantikine CXCL10 Immunoassay kit (R&D Systems, Minneapolis, MN). Immunohistochemistry was performed to assess CXCL10 expression in the liver tissues of HBV carriers. Liver tissues were fixed in 4% buffered formaldehyde and embedded in paraffin. We used goat polyclonal anti-human CXCL10 immunoglobulin G (R&D Systems) and peroxidase-conjugated immune polymer reagents (Envision Plus Kit, Dako, Carpinteria, CA) for immunohistochemistry according to the manufacturer's instructions. Statistical analysis was performed using SPSS software (version 9.0; SPSS Inc, Chicago, IL). Allele frequencies for each SNP were determined by gene counting. χ2 tests were used to examine the differences in allele frequencies and genotype distributions between groups. Multivariable logistic regression analysis was performed to adjust risk factors (such as age, sex, and alcohol use). The association between genotyped polymorphisms and risk of disease was estimated by P values, odds ratios (ORs), and 95% confidence intervals (95% CIs). Student t tests were used to analyze the results of luciferase reporter assays, allele-specific ChIPs, and relative messenger RNA expressions. One-way analysis of variance (ANOVA) method was used to analyze serum levels of CXCL10 between patient groups. A P value of less than .05 was considered significant. Resequencing of the 4228 base pairs of the CXCL10 genomic region in the 27 samples (54 chromosomes) revealed 21 SNPs (Table 1 and Figure 1A), including 8 novel SNPs (2, 9, 10, 11, 13, 16, 20, and 21). None of the SNPs were in the coding region. No multiple-base insertion/deletion or short-tandem repeat variation was found. Pairwise LD measures (D′ and r2) are diagrammatically illustrated in Figure 1B. LD analysis showed that there was absolute LD (D′ = 1, r2 = 1) among the SNPs 1, 3, 5, 6, 7, 12, 13, 14, 15, 17, 18, 19, 20, and 21 and between the SNPs 2 and 11. Eight haplotypes were identified, among which 3 were with frequencies more than 10% (see Supplementary Table 2). Three SNPs (1, 4, and 11, ie, Del -1767T, C-1513T, and G-201A polymorphism, respectively), which captured more than 93% of the haplotype diversity observed within the gene region, were determined as htSNPs.Table 1Positions and Frequencies of SNPs Within the Human CXCL10 GeneNo.SNPaThe position of the SNPs is relative to the first nucleotide of open reading frame of the CXCL10 gene; the allele on the right is the minor allele.PositionbPositions are referred to as GenBank accession number NT_016354.16.FrequencycFrequencies of minor alleles. Allele frequencies for each SNP were determined by gene counting on the 27 unrelated samples (54 chromosomes).Region1Del -1767T14410560.0375′ Flank2C-1596T14408850.1305′ Flank3G-1555C14408440.0375′ Flank4C-1513T14408020.4635′ Flank5G-1415A14407040.0375′ Flank6C-1397T14406860.0375′ Flank7C-1101T14403900.0375′ Flank8C-938T14402270.3525′ Flank9G-448T14397370.0375′ Flank10C-202A14394910.0195′ Flank11G-201A14394900.1305′ Flank12G93A14391960.037Intron 113Del 819A14384700.037Intron 214T907C14383820.037Intron 215G1288T14380010.037Intron 316G1330A14379590.019Intron 317G1349A14379400.037Intron 318G1447C14378420.037Intron 319C1641G14376480.0373′ UTR20Del 1822C14374670.0373′ UTR21C2284T14370050.0373′ UTRDel, one-base deletion; UTR, untranslated region.a The position of the SNPs is relative to the first nucleotide of open reading frame of the CXCL10 gene; the allele on the right is the minor allele.b Positions are referred to as GenBank accession number NT_016354.16.c Frequencies of minor alleles. Allele frequencies for each SNP were determined by gene counting on the 27 unrelated samples (54 chromosomes). Open table in a new tab Del, one-base deletion; UTR, untranslated region. Because the no. 1 SNP (Del -1767T) has a very low frequency in the Chinese Han population (T allele frequency is 0.021, which was validated in 96 progressive carriers and 96 nonprogressive carriers), according to the common variant common diseases hypothesis,22Lohmueller K.E. Pearce C.L. Pike M. et al.Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease.Nat Genet. 2003; 33: 177-182Crossref PubMed Scopus (1647) Google Scholar it is unfit for genotyping analysis in a genetic association study. We selected the other 2 htSNPs as genetic markers for subsequent genotyping analysis; one was C-1513T (ie, SNP 4), which had the highest allele frequency, and the other was G-201A (ie, SNP 2), which had moderate variation frequency and was located between the nuclear factor κB1 and nuclear factor κB2 binding sites in the promoter region of the CXCL10 gene.23Majumder S. Zhou L.Z.H. Chaturvedi P. et al.Regulation of human IP-10 gene expression in astrocytoma cells by inflammatory cytokines.J Neurosci Res. 1998; 54: 169-180Crossref PubMed Scopus (64) Google Scholar The selected characteristics and genotype distributions of the C-1513T and G-201A in naive controls, nonprogressed carriers, and progressed carriers are summarized in Table 2. The genotype distributions for the 2 SNPs were in Hardy–Weinberg equilibrium in each group. Although an effort was made to obtain a good match on age and sex between progressed and nonprogressed carriers, progressed carriers were older (P < .001) and consisted of more men (P < .001) than nonprogressed carriers in our hospital-based case-control populations. The difference in alcohol consumption status (P < .001) between progressed and nonprogressed carriers may be due to a sex difference, because few women drink in China.Table 2Distribution of Selected Characteristics and CXCL10 Polymorphisms in Participants Included in the Current StudyParticipants from Beijing regionParticipants from Chongqing regionNaive controls(n = 253)Nonprogressed carriers (n = 283)Progressed carriers (n = 330)P valueaP values were for the comparison between progressed and nonprogressed HBV carriers; for sex, age, and alcohol consumption, P values were given by univariate logistic analysis; for the CXCL10 polymorphisms, P values were given by χ2 tests.Naive controls (n = 315)Nonprogressed carriers (n = 970)Progressed carriers (n = 817)P valueaP values were for the comparison between progressed and nonprogressed HBV carriers; for sex, age, and alcohol consumption, P values were given by univariate logistic analysis; for the CXCL10 polymorphisms, P values were given by χ2 tests.Men, no. (%)216 (85.4)199 (70.3)280 (84.8)<.001235 (74.6)548 (56.5)684 (83.7)<.001Age (y), mean (SD)31.2 (11.2)31.6 (8.5)46.3 (11.4)<.00137.0 (12.0)36.8 (10.8)41.6 (11.3)<.001Drinker, no. (%)—72 (25.4)100 (30.3).182—212 (21.9)250 (30.6)<.001HBsAg status—++—++HBeAg positive, no. (%)0 (0)162 (57.2)102 (30.9)0 (0)358 (36.9)264 (32.3)Had liver biopsy, no. (%)0 (0)22 (7.8)76 (23.0)0 (0)80 (8.2)198 (24.2)Known parent or sibling carriers, no. (%)bIndividuals who have known parent(s) or sibling(s) who are chronic HBV carriers(indicates early infection before childhood).—175 (61.8)199 (60.3)—645 (66.5)463 (56.7)CXCL10 polymorphisms G-201A G/G, no. (%)205 (82.0)241 (85.5)255 (77.7).014257 (81.6)793 (82.4)619 (76.5).001 G/A, no. (%)44 (17.6)40 (14.2)70 (21.4)55 (17.5)165 (17.1)178 (22.0) A/A, no. (%)1 (0.4)1 (0.3)3 (0.9)3 (0.9)5 (0.5)12 (1.5) A allele0.0920.0750.116.0150.0970.0910.125.001 C-1513T C/C, no. (%)67 (26.8)67 (23.7)83 (25.3).82581 (25.7)244 (25.3)206 (25.5).919 C/T, no. (%)127 (50.8)146 (51.8)170 (51.8)163 (51.8)480 (49.9)407 (50.3) T/T, no. (%)56 (22.4)69 (24.5)75 (22.9)71 (22.5)239 (24.8)196 (24.2) T allele0.4780.5040.488.6240.4840.4970.494.858NOTE. Total numbers for each polymorphism vary because not all samples were successfully genotyped. The genotype distribution in each group was in Hardy–Weinberg equilibrium. "Drinker" was defined as alcohol consumption of ≥40 g/wk for men and ≥20 g/wk for women, which included occasional drinkers and daily drinkers.a P values were for the comparison between progressed and nonprogressed HBV carriers; for sex, age, and alcohol consumption, P values were given by univariate logistic analysis; for the CXCL10 polymorphisms, P values were given by χ2 tests.b Individuals who have known parent(s) or sibling(s) w
Referência(s)