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. 2019 Oct;157(4):1138-1152.e14.
doi: 10.1053/j.gastro.2019.06.017. Epub 2019 Jun 19.

Gene Expression Signatures Associated With Survival Times of Pediatric Patients With Biliary Atresia Identify Potential Therapeutic Agents

Zhenhua Luo  1 , Pranavkumar Shivakumar  1 , Reena Mourya  1 , Sridevi Gutta  1 , Jorge A Bezerra  2
Affiliations

Gene Expression Signatures Associated With Survival Times of Pediatric Patients With Biliary Atresia Identify Potential Therapeutic Agents

Zhenhua Luo et al. Gastroenterology. 2019 Oct.

Abstract (V体育ios版)

Background & aims: Little is known about the factors that affect outcomes of patients with biliary atresia and there are no medical therapies that increase biliary drainage. VSports手机版.

Methods: Liver biopsies and clinical data were obtained from infants with cholestasis and from children without liver disease (controls); messenger RNA (mRNA) was isolated, randomly assigned to discovery (n = 121) and validation sets (n = 50), and analyzed by RNA sequencing V体育安卓版. Using the Superpc R package followed by Cox regression analysis, we sought to identify gene expression profiles that correlated with survival without liver transplantation at 24 months of age. We also searched for combinations of gene expression patterns, clinical factors, and laboratory results obtained at diagnosis and at 1 and 3 months after surgery that associated with transplant-free survival for 24 months of age. We induced biliary atresia in BALB/c mice by intraperitoneal administration of Rhesus rotavirus type A. Mice were given injections of the antioxidants N-acetyl-cysteine (NAC) or manganese (III) tetrakis-(4-benzoic acid)porphyrin. Blood and liver tissues were collected and analyzed by histology and immunohistochemistry. .

Results: We identified a gene expression pattern of 14 mRNAs associated with shorter vs longer survival times in the discovery and validation sets (P < . 001). This gene expression signature, combined with level of bilirubin 3 months after hepatoportoenterostomy, identified children who survived for 24 months with an area under the curve value of 0. 948 in the discovery set and 0. 813 in the validation set (P < . 001). Computer models correlated a cirrhosis-associated transcriptome with decreased times of transplant-free survival; this transcriptome included activation of genes that regulate the extracellular matrix and numbers of activated stellate cells and portal fibroblasts. Many mRNAs expressed at high levels in liver tissues from patients with 2-year transplant-free survival had enriched scores for glutathione metabolism. Among mice with biliary atresia given injections of antioxidants, only NAC reduced histologic features of liver damage and serum levels of aminotransferase, gamma-glutamyl transferase, and bilirubin. NAC also reduced bile duct obstruction and liver fibrosis and increased survival times. V体育ios版.

Conclusions: In studies of liver tissues from infants with cholestasis, we identified a 14-gene expression pattern that associated with transplant-free survival for 2 years. mRNAs encoding proteins that regulate fibrosis genes were increased in liver tissues from infants who did not survive for 2 years, whereas mRNAs that encoded proteins that regulate glutathione metabolism were increased in infants who survived for 2 years VSports最新版本. NAC reduced liver injury and fibrosis in mice with biliary atresia, and increased survival times. Agents such as NAC that promote glutathione metabolism might be developed for treatment of biliary atresia. .

Keywords: Biomarker; Cholestasis; Chronic Liver Disease; Risk Factor. V体育平台登录.

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"V体育平台登录" Figures

Figure 1.
Figure 1.. A 14-gene signature that predicts patient’s survival at 2 years of age.
Panel A shows the analytic pipeline that started with the random assignment of 171 human liver biopsies collected at the time of diagnosis into discovery (N=121) and validation (N=50) cohorts, then Supervised principal components for Cox regression was applied to dichotomize patients into low and high survival groups. Heatmaps in B and C show the expression of 14 genes in discovery and validation cohorts, respectively (levels of gene expression are shown as color variation from red [high] to blue [low]). Euclidean distance was used in hierarchical clustering of genes. Graphs in D and E show Kaplan-Meier plots dichotomizing individual cohorts into 2 groups based on high or low survival based on predicted scores generated by Superpc Cox regression. Log-rank test was used to compare the survival distributions of two groups. Graphs in F and G show PCA clustering of all 171 biliary atresia patients and 7 normal controls.
Figure 2.
Figure 2.. Prognostic index of 2-year survival with the native liver.
Panel A shows a strategy that combines the 14-gene signature and serum total bilirubin at 3 months after hepatoportoenterostomy to generate a prognostic index. Kaplan-Meier plots in B and C dichotomizes the cohort into groups of high or low survival based on prognostic index for individual patients. Log-rank test was used to compare the survival distributions of two groups. In D and E, ROC curves generated by the prognostic index at 2 years of age for D discovery and E validation cohorts. AUC of index is shown. In panel F, boxplots of expression levels of 14 genes in the low and high survival groups in discovery cohort and normal controls group. Expression levels are represented as mean ± SD of normalized expression (RPKM value). P value was calculated by Wilcoxon rank sum test. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3.
Figure 3.. Low survival group exhibits a prominent gene signatures of cirrhosis.
Panel A shows the use of differentially expressed genes between high and low survival groups compared with signatures of other liver diseases and animal models. The expression heatmaps of differentially expressed genes are shown for the discovery (B) and validation (C) cohorts (levels of gene expression are shown as color variation from red [high] to blue [low], with Euclidean distance used in hierarchical clustering). 14 genes are identified in the heatmaps. Pairwise overlapping comparisons were performed between upregulated or downregulated genes with gene signatures of human liver diseases (shown in D), animal models of human liver diseases (E), and hepatic stellate cells and portal fibroblasts (F). Overlaps were examined by Fisher’s exact test and shown as dots whose size is proportional to −log10(P value) and gradation of red is proportional to log2(odd ratio). Overlaps with −log10(P value) >1.3 and log2(odd ratio) >0 were defined as significant overlaps and are shown in red; the color black depicts overlaps with odds ratio ≤1 or P value > 0.05.
Figure 4.
Figure 4.. Extracellular matrix formation related gene signature and cellular source, CD8T and NK cells are enriched in the low survival group
Panel A shows an analytic pipeline identifying enriched pathways and cell types in the low survival group and the quantification of the relative abundance of individual cell types. Dotplot shown in B depicts pathways that are enriched in upregulated genes of the low survival group in the discovery cohort. Pathway terms were ranked according to their −log10(FDR) values and categorized into extracellular matrix (ECM), cell-cell interaction, inflammation and other. The dot sizes are proportional to number of genes. For panels C-G, boxplots show enrichment of activated hepatic stellate cells (aHSC, C), activated portal fibroblasts (aPFs, D), cholangiocytes (E), CD8 T cells (F), and NK cells (G). Enrichments are represented as mean ± SD of ssGSEA scores. P value was calculated by Wilcoxon rank sum test. Scatterplots shown in H-J represent linear correlation between NK cells and aHSCs, aPFs, and cholangiocytes. Pearson correlation coefficient r is shown, with the grey area representing 95% confidence limits. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5.
Figure 5.. Gene signature in the high survival group is enriched by glutathione metabolism.
Panel A shows an analytic pipeline identifying enriched pathways and cell types in the high survival group and the quantification of the oxidation signatures relative to the abundance of individual cell types. The dotplot shown in B depicts pathways that are enriched in upregulated genes of the high survival group in the discovery cohort. Pathway terms were ranked according to their −log10(FDR) values and categorized into oxidation, metabolism and other. The dot sizes are proportional to number of genes. For panels C-F, boxplots show ssGSEA scores for glutathione metabolism (C) and ratios of ssGSEA scores for glutathione metabolism and aHSCs (D), aPFs (E), and cholangiocytes (F). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6.
Figure 6.. N-acetyl-cysteine (NAC) suppresses liver injury in the standard mouse model of biliary atresia.
Panel A shows the experimental design of an RRV induced BA model, with 1.5×106 ffu of RRV injected intraperitoneally (i.p.) into newborn BALB/C mice, followed by the daily i.p. injection of PBS, NAC, or MnTBAP until day 14. Tissues and serum samples were harvest at days 7 and day 14 after RRV injection. Daily body weights (mean+ SD; N=18-26 mice per group from three independent experiments; shown in B), percent survival (C), and serum total bilirubin, ALT and GGT (D-F; mean ± SD; N=6 mice per group). **P < 0.01, ***P < 0.001. Representative EHBD (G) and liver (H) sections with H&E staining at Day 7 or Day 14 after RRV infection or wild type saline controls without RRV infection. In panel G, Arrows denote areas of periductal inflammation; asterisk denotes duct lumen. Representative liver sections with Sirius red (I) and anti-αSMA (J) staining at Day 7 or Day 14 after RRV infection or wild type saline controls without RRV infection.
Figure 7.
Figure 7.. N-acetyl-cysteine (NAC) suppresses liver injury and fibrosis in a modified mouse model of biliary atresia.
Panel A shows the experimental design of an RRV induced injury and liver fibrosis model, with 1.875×106 ffu of RRV injected intraperitoneally (i.p.) into newborn BALB/C mice at day 3 of life, followed by the daily i.p. injection of PBS, NAC, or MnTBAP until day 22 of life (19 doses), at which time tissues and serum samples were harvest. Daily body weights (mean+ SD; N=18-26 mice per group from three independent experiments; shown in B), percent survival (C), and serum total bilirubin, ALT and GGT (D-F; mean ± SD; N=6-12 mice per group). **P < 0.01, ***P < 0.001. Representative EHBD (G) and liver (H) sections with H&E staining at Day 7 or Day 14 after RRV infection or wild type saline controls without RRV infection. In panel G, arrows denote areas of periductal inflammation; asterisk denotes duct lumen. Representative liver sections with Sirius red (I) and anti-αSMA (J) staining at Day 7 or Day 14 after RRV infection or wild type saline controls without RRV infection. In panel J, arrows denote α-SMA positive cells.
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