Volume 134, Issue 3, Pages 716-726.e2 (March 2008) Regulatory Polymorphisms in the Promoter of CXCL10 Gene and Disease Progression in Male Hepatitis B Virus Carriers  Guohong 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  Gastroenterology  Volume 134, Issue 3, Pages 716-726.e2 (March 2008) DOI: 10.1053/j.gastro.2007.12.044 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 (A) Genomic location of SNPs identified in relation to the exon/intron structure of the human CXCL10 gene. The 4 exons are marked with boxes, in which white areas represents untranslated regions. Positions for SNPs are relative to the first nucleotide of open reading frame of the CXCL10 gene. The PCR fragments used for SNP screening are marked with lines below the gene. (B) Pairwise LD was measured by D′ and r2 among all of the 21 SNPs identified in 27 sequenced samples. The blocks are shaded corresponding to the LD values. Gastroenterology 2008 134, 716-726.e2DOI: (10.1053/j.gastro.2007.12.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Correlation of CXCL10 expression with the alleles and genotypes. (A) Binding affinity of nuclear proteins to biotin-labeled double-stranded oligonucleotides encompassing G-201A locus. Competitor, 200-fold unlabeled probe (corresponding cold probe); 1, positive control of AP-1; 2, Epstein-Barr virus nuclear antigen (EBNA)-positive control of LightShift EMSA kit (Pierce). (B) Luciferase activity in IFN-γ–stimulated HepG2 cells transfected with promoter-reporter vectors corresponding to -201G/G or A/A and -1596C-201G or -1596T-201A haplotype homozygotes, respectively (haplotype 1/1 and 3/3 homozygotes, see Supplementary Table 2; see supplemental material online at www.gastrojournal.org). The horizontal axis represents the relative proportion of luciferase activity to corresponding pGL3-enhancer vectors. Data represent mean ± SD. Representative data from 3 experiments performed in quadruplicate. P values were estimated by Student t test. (C) -201A/-201G ratio of unprecipitated (left) and immunoprecipitated DNA (right) using ChIP followed by real-time quantitative PCR assay in 5 -201G/A heterozygous cell samples from HBV-naive umbilical blood. Data represent mean ± SD. (D) Expression of CXCL10 measured by quantitative MGB-TaqMan PCR of RNAs purified from IFN-γ–stimulated peripheral blood mononuclear cells of 18 blood donors. Columns, mean from triplicate measurements; bars, SD. Gastroenterology 2008 134, 716-726.e2DOI: (10.1053/j.gastro.2007.12.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Serum CXCL10 levels in HBV carriers by enzyme-linked immunosorbent assay method. The average levels are marked as horizontal bars. (A and B) Variations of serum CXCL10 levels during inflammation flare-up in representative HBV carriers affected with moderate hepatitis B (patient A) and liver cirrhosis–based acute-on-chronic liver failure (patient B). (C and D) Serum CXCL10 levels between patients with different CXCL10 G-201A genotypes (C) and between men and women (D) in nonprogressed and progressed carriers. No significant difference was observed between different G-201A genotypes and between men and women in both nonprogressed carriers and progressed carriers by one-way ANOVA or Student t tests. (E) Serum CXCL10 levels in CXCL10 -201G/G homozygotic nonprogressed carriers, carriers with severe hepatitis B, carriers with liver cirrhosis, and carriers with hepatocellular carcinoma. The difference between groups was analyzed by one-way ANOVA tests. Gastroenterology 2008 134, 716-726.e2DOI: (10.1053/j.gastro.2007.12.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Immunohistochemical analysis of CXCL10 protein expression in liver tissues of HBV carriers. Brown chromogen indicates positive staining of CXCL10. Representative results for patients of the 3 different CXCL10 G-201A genotypes are shown (original magnification 400×). Ishak modified histologic activity index (HAI) scores were given by assessment of the corresponding H&E-stained section. (A) -201G/G homozygote, nonprogressed carrier; (B) -201G/G homozygote with mild hepatitis B, HAI score = 4; (C) -201G/G homozygote with severe hepatitis B, HAI score = 12; (D) -201G/G homozygote with liver cirrhosis–based acute-on-chronic liver failure, HAI score = 17; (E) -201G/A heterozygote with liver cirrhosis–based acute-on-chronic liver failure, HAI score = 14; (F) -201A/A homozygote with mild hepatitis B, HAI score = 4. Gastroenterology 2008 134, 716-726.e2DOI: (10.1053/j.gastro.2007.12.044) Copyright © 2008 AGA Institute Terms and Conditions