Background & Aims Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting non-invasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. Methods We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0–1 (mild) and F3–4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. Results Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P<.05). Methylation at FGFR2, MAT1A, and CASP1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, whereas genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and under-expressed. Conclusions Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.
Clinicians rely upon the severity of liver fibrosis to segregate patients with well-compensated nonalcoholic fatty liver disease (NAFLD) into sub-populations at high versus low-risk for eventual liver-related morbidity and mortality. We compared hepatic gene expression profiles in high- and low-risk NAFLD patients to identify processes that distinguish the two groups and hence, might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low-risk, “mild” NAFLD patients (fibrosis stage 0–1, n=40) and high risk, “severe” NAFLD patients (fibrosis stage 3–4, n=32). Findings were validated in a second, independent cohort and confirmed by real time PCR and immunohistochemistry. As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically-indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64 gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20 gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism-related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and immunohistochemistry confirmed deregulation of metabolic and regenerative pathways in severe NAFLD, and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer. Conclusion By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD-related morbidity and mortality.
Determination of copy number variants (CNVs) inferred in genome wide single nucleotide polymorphism arrays has shown increasing utility in genetic variant disease associations. Several CNV detection methods are available, but differences in CNV call thresholds and characteristics exist. We evaluated the relative performance of seven methods: circular binary segmentation, CNVFinder, cnvPartition, gain and loss of DNA, Nexus algorithms, PennCNV and QuantiSNP. Tested data included real and simulated Illumina HumHap 550 data from the Singapore cohort study of the risk factors for Myopia (SCORM) and simulated data from Affymetrix 6.0 and platform-independent distributions. The normalized singleton ratio (NSR) is proposed as a metric for parameter optimization before enacting full analysis. We used 10 SCORM samples for optimizing parameter settings for each method and then evaluated method performance at optimal parameters using 100 SCORM samples. The statistical power, false positive rates, and receiver operating characteristic (ROC) curve residuals were evaluated by simulation studies. Optimal parameters, as determined by NSR and ROC curve residuals, were consistent across datasets. QuantiSNP outperformed other methods based on ROC curve residuals over most datasets. Nexus Rank and SNPRank have low specificity and high power. Nexus Rank calls oversized CNVs. PennCNV detects one of the fewest numbers of CNVs.
This is the largest TM expression study of POAG cases and controls performed to date and represents the first report of TM expression in a patient having POAG with a Q368X MYOC mutation. Our data suggest the potential role of endocytic and exosome pathways in the pathogenesis of POAG.
TRC105 is an endoglin-targeting drug that possesses anti-angiogenic and antitumor potential. Analysis of the initial phase I trial of TRC105 demonstrated good tolerability and efficacy in cancer patients. In this report, we analyzed multiple circulating biomarkers at baseline, cycle 2 day 1 (C2D1), and end of study (EOS) for each patient. The baseline level and the fold change from baseline to both C2D1 and EOS for each marker were statistically analyzed. At C2D1, seven markers were significantly downregulated (angiopoietin-2 [Ang-2], insulin-like growth factor-binding protein-3 [IGFBP-3], plasminogen activator inhibitor-1 [PAI-1] total, platelet-derived growth factor [PDGF]-AA, PDGF-BB, thrombospondin-1 [TSP-1], and vascular endothelial growth factor [VEGF]-D). Meanwhile, seven markers were upregulated by C2D1 (E-Cadherin, soluble Endoglin [sEnd], E-Selectin, interleukin-6 [IL-6], osteopontin [OPN], TSP-2, and von Willebrand factor [vWF]). At EOS, seven markers were upregulated including Ang-2, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), IGFBP-1, IL-6, TSP-2, and vascular cell adhesion molecule-1 (VCAM-1). A statistical trend was also seen for increases of VEGF-A and placenta growth factor (PlGF) at EOS. Throughout treatment, sEnd levels significantly increased, an observation that was recapitulated in cultured endothelial cells. This is the first report of plasma-based biomarkers in patients receiving TRC105. TRC105 treatment by C2D1 was associated with decreases in several angiogenic factors, including Ang-2, PDGF isoforms, and VEGF isoforms, offering insight into the mechanisms underlying TRC105's anti-angiogenic, antitumor function. Increases in sEnd were the most significant of all observed biomarker changes and may reflect direct drug effects. Additionally, biomarker changes in response to TRC105 are distinct from those seen in patients treated with VEGF-targeting drugs, suggesting the possible utility of combining these two classes of angiogenesis inhibitors in patients.
Targeting multiple regulators of tumor angiogenesis have the potential to improve treatment efficacy. Bevacizumab is a monoclonal antibody directed against vascular endothelial growth factor and ABT-510 is a synthetic analog of thrombospondin, an endogenous angiogenesis inhibitor. Dual inhibition may result in additional benefit. We evaluated the safety, tolerability, and efficacy of the combination of bevacizumab plus ABT-510 in patients with refractory solid tumors. We also explored the effects of these agents on plasma-based biomarkers and wound angiogenesis. Thirty-four evaluable subjects were enrolled and received study drug. Therapy was well tolerated; minimal treatment-related grade 3/4 toxicity was observed. One patient treated at dose level 1 had a partial response and five other patients treated at the recommended phase II dose had prolonged stable disease for more than 1 year. Biomarker evaluation revealed increased levels of D-dimer, von Willebrand factor, placental growth factor, and stromal-derived factor 1 in response to treatment with the combination of bevacizumab and ABT-510. Data suggest that continued evaluation of combination antiangiogenesis therapies may be clinically useful.
Background and Aims Trimethoprim (TMP)–sulfamethoxazole (SMX) is an important cause of idiosyncratic drug‐induced liver injury (DILI), but its genetic risk factors are not well understood. This study investigated the relationship between variants in the human leukocyte antigen (HLA) class 1 and 2 genes and well‐characterized cases of TMP‐SMX DILI. Approach and Results European American and African American persons with TMP‐SMX DILI were compared with respective population controls. HLA sequencing was performed by Illumina MiSeq (Illumina, San Diego, CA) for cases. The HLA genotype imputation with attribute bagging program was used to impute HLA alleles for controls. The allele frequency difference between case patients and controls was tested by Fisher’s exact tests for each ethnic group. For European Americans, multivariable logistic regression with Firth penalization was used to test the HLA allelic effect after adjusting for age and the top two principal components. Molecular docking was performed to assess HLA binding with TMP and SMX. The European American subset had 51 case patients and 12,156 controls, whereas the African American subset had 10 case patients and 5,439 controls. Four HLA alleles were significantly associated in the European American subset, with HLA‐B*14:01 ranking at the top (odds ratio, 9.20; 95% confidence interval, 3.16, 22.35; P = 0.0003) after covariate adjustment. All carriers of HLA‐B*14:01 with TMP‐SMX DILI possessed HLA‐C*08:02, another significant allele (P = 0.0026). This pattern was supported by HLA‐B*14:01–HLA‐C*08:02 haplotype association (P = 1.33 × 10−5). For the African American patients, HLA‐B*35:01 had 2.8‐fold higher frequency in case patients than in controls, with 5 of 10 patients carrying this allele. Molecular docking showed cysteine at position 67 in HLA‐B*14:01 and phenylalanine at position 67 in HLA‐B*35:01 to be the predictive binding sites for SMX metabolites. Conclusions HLA‐B*14:01 is associated with TMP‐SMX DILI in European Americans, and HLA‐B*35:01 may be a potential genetic risk factor for African Americans.
Background Neural tube defects (NTDs) are common human birth defects with a complex etiology. To develop a comprehensive knowledge of the genes expressed during normal neurulation, we established transcriptomes from human neural tube fragments during and after neurulation using long Serial Analysis of Gene Expression (long-SAGE). Methods Rostral and caudal neural tubes were dissected from normal human embryos aged between 26 and 32 days of gestation. Tissues from the same region and Carnegie stage were pooled (n>=4) and total RNA extracted to construct four long-SAGE libraries. Tags were mapped using the UniGene Homo sapiens 17 bp tag-to-gene best mapping set. Differentially expressed genes were identified by chi-square or Fisher’s exact test and validation was performed for a subset of those transcripts using in situ hybridization. In silico analyses were performed with BinGO and EXPANDER. Results We observed most genes to be similarly regulated in rostral and caudal regions, but expression profiles differed during and after closure. In silico analysis found similar enrichments in both regions for biological process terms, transcription factor binding and miRNA target motifs. Twelve genes potentially expressing alternate isoforms by region or developmental stage, and the miRNAs miR-339-5p, miR-141/200a, miR-23ab, and miR-129/129-5p, are among several potential candidates identified here for future research. Conclusions Time appears to influence gene expression in the developing central nervous system more than location. These data provide a novel complement to traditional strategies of identifying genes associated with human NTDs, and offer unique insight into the genes associated with normal human neurulation.
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