Pharmacogene expression during progression of metabolic dysfunction-associated steatotic liver disease: Studies on mRNA and protein levels and their relevance to drug treatment.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is common worldwide. Genes and proteins contributing to drug disposition may show altered expression as MASLD progresses. To assess this further, we undertook transcriptomic and proteomic analysis of 137 pharmacogenes in liver biopsies from a large MASLD cohort. We performed sequencing on RNA from 216 liver biopsies (206 MASLD and 10 controls). Untargeted mass spectrometry proteomics was performed on a 103 biopsy subgroup. Selected RNA sequencing signals were replicated with an additional 187 biopsies. Comparison of advanced MASLD (fibrosis score 3/4) with milder disease (fibrosis score 0-2) by RNA sequencing showed significant alterations in expression of certain phase I, phase II and ABC transporters. For cytochromes P450, CYP2C19 showed the most significant decreased expression (30 % of that in mild disease) but significant decreased expression of other CYPs (including CYP2C8 and CYP2E1) also occurred. CYP2C19 also showed a significant decrease comparing the inflammatory form of MASLD (MASH) with non-MASH biopsies. Findings for CYP2C19 were confirmed in the replication cohort. Proteomics on the original discovery cohort confirmed decreased levels of several CYPs as MASLD advanced but this decrease was greatest for CYP2C19 where levels fell to 40 % control. This decrease may result in decreased CYP2C19 activity that could be problematic for prescription of drugs activated or metabolized by CYP2C19 as MASLD advances. More limited decreases for other P450s suggest fewer issues with non-CYP2C19 drug substrates. Negative correlations at RNA level between CYP2C19 and several cytokine genes provided initial insights into the mechanism underlying decreased expression.

A polygenic risk score for alcohol-associated cirrhosis among heavy drinkers with European ancestry.

BACKGROUND: Polygenic Risk Scores (PRS) based on results from genome-wide association studies offer the prospect of risk stratification for many common and complex diseases. We developed a PRS for alcohol-associated cirrhosis by comparing single-nucleotide polymorphisms among patients with alcohol-associated cirrhosis (ALC) versus drinkers who did not have evidence of liver fibrosis/cirrhosis. METHODS: Using a data-driven approach, a PRS for ALC was generated using a meta-genome-wide association study of ALC (N=4305) and an independent cohort of heavy drinkers with ALC and without significant liver disease (N=3037). It was validated in 2 additional independent cohorts from the UK Biobank with diagnosed ALC (N=467) and high-risk drinking controls (N=8981) and participants in the Indiana Biobank Liver cohort with alcohol-associated liver disease (N=121) and controls without liver disease (N=3239). RESULTS: A 20-single-nucleotide polymorphisms PRS for ALC (PRSALC) was generated that stratified risk for ALC comparing the top and bottom deciles of PRS in the 2 validation cohorts (ORs: 2.83 [95% CI: 1.82 -4.39] in UK Biobank; 4.40 [1.56 -12.44] in Indiana Biobank Liver cohort). Furthermore, PRSALC improved the prediction of ALC risk when added to the models of clinically known predictors of ALC risk. It also stratified the risk for metabolic dysfunction -associated steatotic liver disease -cirrhosis (3.94 [2.23 -6.95]) in the Indiana Biobank Liver cohort -based exploratory analysis. CONCLUSIONS: PRSALC incorporates 20 single-nucleotide polymorphisms, predicts increased risk for ALC, and improves risk stratification for ALC compared with the models that only include clinical risk factors. This new score has the potential for early detection of heavy drinking patients who are at high risk for ALC.

Relevance of PNPLA3, TM6SF2, HSD17B13, and GCKR Variants to MASLD Severity in an Egyptian Population.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a frequent clinical condition globally. Single nucleotide polymorphisms (SNPs) associated with NAFLD have been proposed in the literature and based on bioinformatic screening. The association between NAFLD and genetic variants in Egyptians is still unclear. Hence, we sought to investigate the association of some genetic variants with NAFLD in Egyptians. Egyptians have been categorized into either the MASLD group (n = 205) or the healthy control group (n = 187). The severity of hepatic steatosis and liver fibrosis was assessed by a Fibroscan device. TaqMan-based genotyping assays were employed to explore the association of selected SNPs with MASLD. PNPLA3 rs738409 C>G variant is associated with the presence of MASLD with liver fibrosis, the severity of both hepatic steatosis and liver fibrosis, increased systolic and diastolic blood pressure and increased alanine aminotransferase (all p < 0.05), while the TM6SF2 rs58542926 C>T, HSD17B13 rs9992651 G>A, and GCKR rs1260326 T>C variants were not (all p > 0.05). The TM6SF2 rs58542926 T allele is associated with increased fasting blood glucose and a decreased waist circumference. The GCKR rs1260326 C allele is associated with decreased aspartate transaminase and diastolic blood pressure (all p < 0.05). Only after adjusting for the risk factors (age, sex, BMI, WC, HDL, TG, diabetes mellitus, and hypertension) F2 liver fibrosis score is negatively correlated with the HSD17B13 rs9992651 GA genotype. This study offers evidence for the association of the PNPLA3 rs738409 C>G variant with MASLD among Egyptians and for the association of the PNPLA3 rs738409 G allele, the TM6SF2 rs58542926 T allele, and the GCKR rs1260326 C allele with some parameters of cardiometabolic criteria.

Drug-induced liver injury (DILI) ascribed to non-steroidal anti-inflammatory drugs (NSAIDs) in the USA-Update with genetic correlations.

OBJECTIVE: To describe patients with NSAID-DILI, including genetic factors associated with idiosyncratic DILI. METHODS: In DILIN, subjects with presumed DILI are enrolled and followed for at least 6 months. Causality is adjudicated by a Delphic approach. HLA sequencing of multiethnic NSAID-DILI patients and HLA allele imputation of matching population controls were performed following overall, class and drug-based association analysis. Significant results were tested in a non-Hispanic White (NHW) case-control replication cohort. RESULTS: Between September 2004 and March 2022, causality was adjudicated in 2498, and 55 (41 [75%] women) were assessed as likely due to NSAIDs. Median age at onset was 55 y (range 22-83 y). Diclofenac was the causative drug in 29, celecoxib in 7, ibuprofen in 5, etodolac and meloxicam each in 4. Except for meloxicam and oxaprozin (n = 2), the liver injury was hepatocellular with median R 15-25. HLA-DRB1*04:03 and HLA-B*35:03 were significantly more frequent in NSAID-DILI patients than in non-NSAID DILI controls. Interestingly, 85% of the HLA-DRB1*04:03 carriers developed DILI due to the use of acetic acid derivative NSAIDs, supporting the hypothesis that HLA-DRB1*04:03 could be a drug and/or class risk factor. HLA-B*35:03 but not HLA-DRB1*04:03 association was confirmed in the independent NHW replication cohort, which was largely driven by diclofenac. CONCLUSIONS: Despite prevalent use, NSAID-DILI is infrequent in the United States. Diclofenac is the most commonly implicated, and adherence to warnings of risk and close observation are recommended. The increased frequency of HLA-B*35:03 and DRB1*04:03, driven by diclofenac, suggests the importance of immune-mediated responses.

Impact of Genetic Variations on Thromboembolic Risk in Saudis with Sickle Cell Disease.

BACKGROUND: Sickle cell disease (SCD) is a Mendelian disease characterized by multigenic phenotypes. Previous reports indicated a higher rate of thromboembolic events (TEEs) in SCD patients. A number of candidate polymorphisms in certain genes (e.g., FVL, PRT, and MTHFR) were previously reported as risk factors for TEEs in different clinical conditions. This study aimed to genotype these genes and other loci predicted to underlie TEEs in SCD patients. METHODOLOGY: A multi-center genome-wide association study (GWAS) involving Saudi SCD adult patients with a history of TEEs (n = 65) and control patients without TEE history (n = 285) was performed. Genotyping used the 10× Affymetrix Axiom array, which includes 683,030 markers. Fisher's exact test was used to generate p-values of TEE associations with each single-nucleotide polymorphism (SNP). The haplotype analysis software tool version 1.05, designed by the University of Göttingen, Germany, was used to identify the common inherited haplotypes. RESULTS: No association was identified between the targeted single-nucleotide polymorphism rs1801133 in MTHFR and TEEs in SCD (p = 0.79). The allele frequency of rs6025 in FVL and rs1799963 in PRT in our cohort was extremely low (<0.01); thus, both variants were excluded from the analysis as no meaningful comparison was possible. In contrast, the GWAS analysis showed novel genome-wide associations (p < 5 × 10-8) with seven signals; five of them were located on Chr 11 (rs35390334, rs331532, rs317777, rs147062602, and rs372091), one SNP on Chr 20 (rs139341092), and another on Chr 9 (rs76076035). The other 34 SNPs located on known genes were also detected at a signal threshold of p < 5 × 10-6. Seven of the identified variants are located in olfactory receptor family 51 genes (OR51B5, OR51V1, OR51A1P, and OR51E2), and five variants were related to family 52 genes (OR52A5, OR52K1, OR52K2, and OR52T1P). The previously reported association between rs5006884-A in OR51B5 and fetal hemoglobin (HbF) levels was confirmed in our study, which showed significantly lower levels of HbF (p = 0.002) and less allele frequency (p = 0.003) in the TEE cases than in the controls. The assessment of the haplotype inheritance pattern involved the top ten significant markers with no LD (rs353988334, rs317777, rs14788626882, rs49188823, rs139349992, rs76076035, rs73395847, rs1368823, rs8888834548, and rs1455957). A haplotype analysis revealed significant associations between two haplotypes (a risk, TT-AA-del-AA-ins-CT-TT-CC-CC-AA, and a reverse protective, CC-GG-ins-GG-del-TT-CC-TT-GG-GG) and TEEs in SCD (p = 0.024, OR = 6.16, CI = 1.34-28.24, and p = 0.019, OR = 0.33, CI = 0.13-0.85, respectively). CONCLUSIONS: Seven markers showed novel genome-wide associations; two of them were exonic variants (rs317777 in OLFM5P and rs147062602 in OR51B5), and less significant associations (p < 5 × 10-6) were identified for 34 other variants in known genes with TEEs in SCD. Moreover, two 10-SNP common haplotypes were determined with contradictory effects. Further replication of these findings is needed.

Study design for development of novel safety biomarkers of drug-induced liver injury by the translational safety biomarker pipeline (TransBioLine) consortium: a study protocol for a nested case-control study.

A lack of biomarkers that detect drug-induced liver injury (DILI) accurately continues to hinder early- and late-stage drug development and remains a challenge in clinical practice. The Innovative Medicines Initiative's TransBioLine consortium comprising academic and industry partners is developing a prospective repository of deeply phenotyped cases and controls with biological samples during liver injury progression to facilitate biomarker discovery, evaluation, validation and qualification.In a nested case-control design, patients who meet one of these criteria, alanine transaminase (ALT) ≥ 5 × the upper limit of normal (ULN), alkaline phosphatase ≥ 2 × ULN or ALT ≥ 3 ULN with total bilirubin > 2 × ULN, are enrolled. After completed clinical investigations, Roussel Uclaf Causality Assessment and expert panel review are used to adjudicate episodes as DILI or alternative liver diseases (acute non-DILI controls). Two blood samples are taken: at recruitment and follow-up. Sample size is as follows: 300 cases of DILI and 130 acute non-DILI controls. Additional cross-sectional cohorts (1 visit) are as follows: Healthy volunteers (n = 120), controls with chronic alcohol-related or non-alcoholic fatty liver disease (n = 100 each) and patients with psoriasis or rheumatoid arthritis (n = 100, 50 treated with methotrexate) are enrolled. Candidate biomarkers prioritised for evaluation include osteopontin, glutamate dehydrogenase, cytokeratin-18 (full length and caspase cleaved), macrophage-colony-stimulating factor 1 receptor and high mobility group protein B1 as well as bile acids, sphingolipids and microRNAs. The TransBioLine project is enabling biomarker discovery and validation that could improve detection, diagnostic accuracy and prognostication of DILI in premarketing clinical trials and for clinical healthcare application.

Pharmacogenomics in practice: a review and implementation guide.

Considerable efforts have been exerted to implement Pharmacogenomics (PGx), the study of interindividual variations in DNA sequence related to drug response, into routine clinical practice. In this article, we first briefly describe PGx and its role in improving treatment outcomes. We then propose an approach to initiate clinical PGx in the hospital setting. One should first evaluate the available PGx evidence, review the most relevant drugs, and narrow down to the most actionable drug-gene pairs and related variant alleles. This is done based on data curated and evaluated by experts such as the pharmacogenomics knowledge implementation (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC), as well as drug regulatory authorities such as the US Food and Drug Administration (FDA) and European Medicinal Agency (EMA). The next step is to differentiate reactive point of care from preemptive testing and decide on the genotyping strategy being a candidate or panel testing, each of which has its pros and cons, then work out the best way to interpret and report PGx test results with the option of integration into electronic health records and clinical decision support systems. After test authorization or testing requirements by the government or drug regulators, putting the plan into action involves several stakeholders, with the hospital leadership supporting the process and communicating with payers, the pharmacy and therapeutics committee leading the process in collaboration with the hospital laboratory and information technology department, and healthcare providers (HCPs) ordering the test, understanding the results, making the appropriate therapeutic decisions, and explaining them to the patient. We conclude by recommending some strategies to further advance the implementation of PGx in practice, such as the need to educate HCPs and patients, and to push for more tests' reimbursement. We also guide the reader to available PGx resources and examples of PGx implementation programs and initiatives.

The Sixth European Society of Pharmacogenomics and Personalised Therapy Congress.

On 8-9 November 2022, the European Society of Pharmacogenomics and Personalised Therapy organized its sixth biennial congress, in Belgrade, Serbia (congress website: www.sspt.rs). The congress aimed to address the current status and future perspectives of pharmacogenomics, share latest knowledge in the field of precision medicine and showcase the implementation of clinical applications in pharmacogenomics/pharmacogenetics. The 2 day congress consisted of 17 lectures given by key-opinion leaders and included a poster session plus discussions. The meeting was a great success by generating an informal environment and enabling the exchange of information between 162 participants from 16 different countries.

A proteo-transcriptomic map of non-alcoholic fatty liver disease signatures.

Non-alcoholic fatty liver disease (NAFLD) is a common, progressive liver disease strongly associated with the metabolic syndrome. It is unclear how progression of NAFLD towards cirrhosis translates into systematic changes in circulating proteins. Here, we provide a detailed proteo-transcriptomic map of steatohepatitis and fibrosis during progressive NAFLD. In this multicentre proteomic study, we characterize 4,730 circulating proteins in 306 patients with histologically characterized NAFLD and integrate this with transcriptomic analysis in paired liver tissue. We identify circulating proteomic signatures for active steatohepatitis and advanced fibrosis, and correlate these with hepatic transcriptomics to develop a proteo-transcriptomic signature of 31 markers. Deconvolution of this signature by single-cell RNA sequencing reveals the hepatic cell types likely to contribute to proteomic changes with disease progression. As an exemplar of use as a non-invasive diagnostic, logistic regression establishes a composite model comprising four proteins (ADAMTSL2, AKR1B10, CFHR4 and TREM2), body mass index and type 2 diabetes mellitus status, to identify at-risk steatohepatitis.

Relevance of Pharmacogenomics to the Safe Use of Antimicrobials.

There has been widespread implementation of pharmacogenomic testing to inform drug prescribing in medical specialties such as oncology and cardiology. Progress in using pharmacogenomic tests when prescribing antimicrobials has been more limited, though a relatively large number of pharmacogenomic studies on aspects such as idiosyncratic adverse drug reactions have now been performed for this drug class. Currently, there are recommendations in place from either National Regulatory Agencies and/or specialist Pharmacogenomics Advisory Groups concerning genotyping for specific variants in MT-RNR1 and CYP2C19 before prescribing aminoglycosides and voriconazole, respectively. Numerous additional pharmacogenomic associations have been reported concerning antimicrobial-related idiosyncratic adverse drug reactions, particularly involving specific HLA alleles, but, to date, the cost-effectiveness of genotyping prior to prescription has not been confirmed. Polygenic risk score determination has been investigated to a more limited extent but currently suffers from important limitations. Despite limited progress to date, the future widespread adoption of preemptive genotyping and genome sequencing may provide pharmacogenomic data to prescribers that can be used to inform prescribing and increase the safe use of antimicrobials.

Identification of Reduced ERAP2 Expression and a Novel HLA Allele as Components of a Risk Score for Susceptibility to Liver Injury Due to Amoxicillin-Clavulanate.

BACKGROUND & AIMS: Drug-induced liver injury (DILI) due to amoxicillin-clavulanate (AC) has been associated with HLA-A∗02:01, HLA-DRB1∗15:01, and rs2476601, a missense variant in PTPN22. The aim of this study was to identify novel risk factors for AC-DILI and to construct a genetic risk score (GRS). METHODS: Transcriptome-wide association study and genome-wide association study analyses were performed on 444 AC-DILI cases and 10,397 population-based controls of European descent. Associations were confirmed in a validation cohort (n = 133 cases and 17,836 population-based controls). Discovery and validation AC-DILI cases were also compared with 1358 and 403 non-AC-DILI cases. RESULTS: Transcriptome-wide association study revealed a significant association of AC-DILI risk with reduced liver expression of ERAP2 (P = 3.7 × 10-7), coding for an aminopeptidase involved in antigen presentation. The lead eQTL single nucleotide polymorphism, rs1363907 (G), was associated with AC-DILI risk in the discovery (odds ratio [OR], 1.68; 95% CI, 1.23-1.66; P = 1.7 × 10-7) and validation cohorts (OR, 1.2; 95% CI, 1.04-2.05; P = .03), following a recessive model. We also identified HLA-B∗15:18 as a novel AC-DILI risk factor in both discovery (OR, 4.19; 95% CI, 2.09-8.36; P = 4.9 × 10-5) and validation (OR, 7.78; 95% CI, 2.75-21.99; P = .0001) cohorts. GRS, incorporating rs1363907, rs2476601, HLA-B∗15:18, HLA-A∗02:01, and HLA-DRB1∗15:01, was highly predictive of AC-DILI risk when cases were analyzed against both general population and non-AC-DILI control cohorts. GRS was the most significant predictor in a regression model containing known AC-DILI clinical risk characteristics and significantly improved the predictive model. CONCLUSIONS: We identified novel associations of AC-DILI risk with ERAP2 low expression and with HLA-B∗15:18. GRS based on the 5 risk variants may assist AC-DILI causality assessment and risk management.

Genetics of drug-induced liver injury: Current knowledge and future prospects.

Idiosyncratic drug-induced liver injury (DILI) remains an important clinical problem, both during drug development and the prescription of a range of licensed drugs. Although rare, the consequences are serious. Ongoing studies on genetic risk factors for DILI, especially genomewide association studies, have resulted in the identification of a number of genetic risk factors, including particular HLA alleles and a few non-HLA genes, both immune-related and metabolic. Some non-HLA associations, such as N-acetyltransferase 2 in isoniazid DILI and interferon regulatory factor 6 in interferon-beta DILI are likely to be drug-specific due to the role of the associated gene, but there is also evidence for polygenic susceptibility involving pathways such as oxidative and endoplasmic reticulum stress and mitochondrial function for DILI induced by multiple drugs. Increased knowledge of genetic risk factors should assist in better understanding underlying DILI mechanisms and help improve methods for identifying hepatotoxic drugs early in development. HLA allele-specific T cell proliferation together with in silico prediction of drug binding to specific HLA proteins have confirmed genetic findings for certain common causes of DILI. However, studies in hepatocytes exposed to high drug concentrations suggest toxicity that is not dependent on genotype also occurs. It seems likely that susceptibility to DILI involves several genetic risk factors combining with other factors that affect drug levels. Despite recent progress in detecting genetic risk factors for DILI, low positive predictive values mean that general implementation of genotyping prior to prescription of potentially hepatotoxic drugs is not useful currently.

Quantitative modeling of human liver reveals dysregulation of glycosphingolipid pathways in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent disease that is associated with multiple metabolic disturbances, yet the metabolic pathways underlying its progression are poorly understood. Here, we studied metabolic pathways of the human liver across the full histological spectrum of NAFLD. We analyzed whole liver tissue transcriptomics and serum metabolomics data obtained from a large, prospectively enrolled cohort of 206 histologically characterized patients derived from the European NAFLD Registry and developed genome-scale metabolic models (GEMs) of human hepatocytes at different stages of NAFLD. We identified several metabolic signatures in the liver and blood of these patients, specifically highlighting the alteration of vitamins (A, E) and glycosphingolipids, and their link with complex glycosaminoglycans in advanced fibrosis. Furthermore, we derived GEMs and identified metabolic signatures of three common NAFLD-associated gene variants (PNPLA3, TM6SF2, and HSD17B13). The study demonstrates dysregulated liver metabolic pathways which may contribute to the progression of NAFLD.

Pharmacogenetics of the cytochromes P450: Selected pharmacological and toxicological aspects.

With the availability of detailed genomic data on all 57 human cytochrome P450 genes, it is clear that there is substantial variability in gene product activity with functionally significant polymorphisms reported across almost all isoforms. This article is concerned mainly with 13 P450 isoforms of particular relevance to xenobiotic metabolism. After brief review of the extent of polymorphism in each, the relevance of selected P450 isoforms to both adverse drug reaction and disease susceptibility is considered in detail. Bleeding due to warfarin and other coumarin anticoagulants is considered as an example of a type A reaction with idiosyncratic adverse drug reactions affecting the liver and skin as type B. It is clear that CYP2C9 variants contribute significantly to warfarin dose requirement and also risk of bleeding, with a minor contribution from CYP4F2. In the case of idiosyncratic adverse drug reactions, CYP2B6 variants appear relevant to both liver and skin reactions to several drugs with CYP2C9 variants also relevant to phenytoin-related skin rash. The relevance of P450 genotype to disease susceptibility is also considered but detailed genetic studies now suggest that CYP2A6 is the only P450 relevant to risk of lung cancer with alleles associated with low or absent activity clearly protective against disease. Other cytochrome P450 genotypes are generally not predictors for risk of cancer or other complex disease development.

Epigenetics in drug disposition & drug therapy: symposium report of the 24th North American meeting of the International Society for the Study of Xenobiotics (ISSX).

The 24th North American International Society for the Study of Xenobiotics (ISSX) meeting, held virtually from September 13 to 17, 2021, embraced the theme of "Broadening Our Horizons." This reinforces a key mission of ISSX: striving to share innovative science related to drug discovery and development. Session speakers and the ISSX New Investigators Group, which supports the scientific and professional development of student and early career ISSX members, elected to highlight the scientific content presented during the captivating session titled, "Epigenetics in Drug Disposition & Drug Therapy." The impact genetic variation has on drug response is well established; however, this session underscored the importance of investigating the role of epigenetics in drug disposition and drug discovery. Session speakers, Drs. Ning, McClay, and Lazarus, detailed mechanisms by which epigenetic players including long non-coding RNA (lncRNAs), microRNA (miRNAs), DNA methylation, and histone acetylation can alter the expression of genes involved in pharmacokinetics, pharmacodynamics, and toxicity. Dr. Ning detailed current knowledge about miRNAs and lncRNAs and the mechanisms by which they can affect the expression of drug metabolizing enzymes (DMEs) and nuclear receptors. Dr. Lazarus discussed the potential role of miRNAs on UDP-glucuronosyltransferase (UGT) expression and activity. Dr. McClay provided evidence that aging alters methylation and acetylation of DMEs in the liver, affecting gene expression and activity. These topics, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are herein discussed, along with exciting future perspectives for epigenetics in drug disposition and drug discovery research.

Determination of Pleiotropic Effect of Warfarin in VKORC1 and CYP2C9 Genotypes in Patients With Heart Valve Replacement.

Warfarin has been widely used as an oral anticoagulant agent. In past, efforts have been done to study the contribution of genetic variation on warfarin dose requirements. The possible therapeutic dose determination of warfarin is very challenging, i.e., extremely low dose leading to unusable antithrombotic therapy or high dose causes particularly bleeding complications. Our study aimed to investigate these observations in more detail, we determined the correlation of interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) among VKORC1 and CYP2C9 genetic variants in patients with heart valve replacement who were treated with a range of warfarin doses and compared with levels in healthy controls. A total of 107 human subjects were recruited with low < 5 mg, medium 5-10 mg/day, and high > 10 mg/day warfarin doses. The genetic study of VKORC1-1639G/A, C1173T, 3730G > A, CYP2C9*2, and CYP2C9*3 was performed using TaqMan genotyping and DNA sequencing. The gene expression of IL-6, TNF-α, and COX-2 mRNA was analyzed. IL-6, TNF-α, and COX-2 protein expressions were determined by ELISA and Western blot analysis to evaluate the pro- and anti-inflammatory effects of warfarin. A statistically significant difference was found among the haplotypes of VKORC1 rs9934438 (C1173T), rs9923231 (-1639G > A), rs7294 (3730G > A) and CYP2C9 *2 p. Arg144 Cys (rs28371674), CYP2C9 *3 p. Ile359Leu (rs1057910) genotypes with warfarin dose requirements (p = 0.001). The increased levels of COX-2, IL-6, and TNF-α proteins were observed when a high dose of warfarin (>10 mg/ml) was administered. However, a lower concentration (1.0 mg/ml) was observed with decreased warfarin dose (<5 mg/day). The present study reported that in addition to its anticoagulant action, the genetic variants of warfarin may have a pleiotropic effect by influencing IL-6 depending on the dosing regimen and inducing the expression of COX-2.

Metabolic signatures across the full spectrum of non-alcoholic fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease with potentially severe complications including cirrhosis and hepatocellular carcinoma. Previously, we have identified circulating lipid signatures associating with liver fat content and non-alcoholic steatohepatitis (NASH). Here, we develop a metabolomic map across the NAFLD spectrum, defining interconnected metabolic signatures of steatosis (non-alcoholic fatty liver, NASH, and fibrosis). Methods: We performed mass spectrometry analysis of molecular lipids and polar metabolites in serum samples from the European NAFLD Registry patients (n = 627), representing the full spectrum of NAFLD. Using various univariate, multivariate, and machine learning statistical approaches, we interrogated metabolites across 3 clinical perspectives: steatosis, NASH, and fibrosis. Results: Following generation of the NAFLD metabolic network, we identify 15 metabolites unique to steatosis, 18 to NASH, and 15 to fibrosis, with 27 common to all. We identified that progression from F2 to F3 fibrosis coincides with a key pathophysiological transition point in disease natural history, with n = 73 metabolites altered. Conclusions: Analysis of circulating metabolites provides important insights into the metabolic changes during NAFLD progression, revealing metabolic signatures across the NAFLD spectrum and features that are specific to NAFL, NASH, and fibrosis. The F2-F3 transition marks a critical metabolic transition point in NAFLD pathogenesis, with the data pointing to the pathophysiological importance of metabolic stress and specifically oxidative stress. Clinical Trials registration: The study is registered at Clinicaltrials.gov (NCT04442334). Lay summary: Non-alcoholic fatty liver disease is characterised by the build-up of fat in the liver, which progresses to liver dysfunction, scarring, and irreversible liver failure, and is markedly increasing in its prevalence worldwide. Here, we measured lipids and other small molecules (metabolites) in the blood with the aim of providing a comprehensive molecular overview of fat build-up, liver fibrosis, and diagnosed severity. We identify a key metabolic 'watershed' in the progression of liver damage, separating severe disease from mild, and show that specific lipid and metabolite profiles can help distinguish and/or define these cases.