Thomas J. Urban

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study

BACKGROUND & AIMSnWe performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors.nnnMETHODSnWe performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (nxa0= 137) and the United States (nxa0= 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs.nnnRESULTSnWe associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; Pxa0= 2.4xa0× 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; Pxa0= 9.7xa0× 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; Pxa0= 4.8xa0× 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; Pxa0= 7.1xa0× 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224.nnnCONCLUSIONSnIn a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors.

Features of Autoimmune Hepatitis in Patients With Drug-induced Liver Injury

BACKGROUND & AIMS: Drug‐induced liver injury (DILI) has features similar to those of other liver diseases including autoimmune hepatitis (AIH). We aimed to characterize the clinical and autoimmune features of liver injury caused by nitrofurantoin, minocycline, methyldopa, or hydralazine. METHODS: We analyzed data from 88 cases of DILI attributed to nitrofurantoin, minocycline, methyldopa, or hydralazine included in the Drug‐Induced Liver Injury Network prospective study from 2004 through 2014. Sera were collected from patients at baseline and follow‐up examination and tested for levels of immunoglobulin G (IgG), antibodies to nuclear antigen (ANA), smooth muscle (SMA), and soluble liver antigen (SLA). An autoimmune score was derived on the basis of increases in levels of IgG, ANA, SMA, and SLA (assigned values of 0, 1+, or 2+). AIH‐associated HLA‐DRB1*03:01 and HLA‐DRB1*04:01 allele frequencies were compared with those of the general population (controls). RESULTS: Of the 88 cases, 80 were women (91%), 74% had hepatocellular injury, and 25% had severe injury. At the onset of DILI, 39% of cases had increased levels of IgG, 72% had increased levels of ANA, 60% had increased levels of SMA, and none had increases in SLA. A phenotype of autoimmunity (autoimmune score ≥2) was observed in 82% of cases attributed to nitrofurantoin and 73% of cases attributed to minocycline (73%) but only 55% of cases attributed to methyldopa and 43% of cases attributed to hydralazine (P = .16 for nitrofurantoin and minocycline vs methyldopa and hydralazine). We observed a decrease in numbers of serum samples positive for ANA (P = .01) or SMA (P < .001) and in autoimmune scores (P < .001) between DILI onset and follow‐up. Similar percentages of patients with DILI had HLA‐DRB1*03:01 (15%) and HLA‐DRB1*04:01 (9%) as controls (12% and 9%, respectively). CONCLUSIONS: In analysis of data from the DILIN prospective study, we found that most cases of DILI attributed to nitrofurantoin or minocycline and about half of cases that were due to methyldopa and hydralazine have a phenotype of autoimmunity similar to AIH. These features decrease with recovery of the injury and are not associated with the typical HLA alleles found in patients with idiopathic AIH.

Toward Predicting Drug-Induced Liver Injury: Parallel Computational Approaches to Identify Multidrug Resistance Protein 4 and Bile Salt Export Pump Inhibitors

Drug-induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of multidrug resistance protein 4 (MRP4), in addition to bile salt export pump (BSEP), might be a risk factor for the development of cholestatic DILI. Recently, we demonstrated that inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. Here, we aimed to develop computational models to delineate molecular features underlying MRP4 and BSEP inhibition. Models were developed using 257 BSEP and 86 MRP4 inhibitors and noninhibitors in the training set. Models were externally validated and used to predict the affinity of compounds toward BSEP and MRP4 in the DrugBank database. Compounds with a score above the median fingerprint threshold were considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed for MRP4 and BSEP with LigandScout software using a training set of nine well characterized MRP4 inhibitors and nine potent BSEP inhibitors. Bayesian models for BSEP and MRP4 inhibition/noninhibition were developed with cross-validated receiver operator curve values greater than 0.8 for the test sets, indicating robust models with acceptable false positive and false negative prediction rates. Both MRP4 and BSEP inhibitor pharmacophore models were characterized by hydrophobic and hydrogen-bond acceptor features, albeit in distinct spatial arrangements. Similar molecular features between MRP4 and BSEP inhibitors may partially explain why various drugs have affinity for both transporters. The Bayesian (BSEP, MRP4) and pharmacophore (MRP4, BSEP) models demonstrated significant classification accuracy and predictability.

Minocycline Hepatotoxicity: Clinical characterization and identification of HLA-B* 35:02 as a risk factor

BACKGROUND & AIMSnMinocycline hepatotoxicity can present with prominent autoimmune features in previously healthy individuals. The aim of this study was to identify genetic determinants of minocycline drug-induced liver injury (DILI) in a well-phenotyped cohort of patients.nnnMETHODSnCaucasian patients with minocycline DILI underwent genome-wide genotyping and were compared to unexposed population controls. Human leukocyte antigen (HLA) binding of minocycline was assessed using AutoDock Vina.nnnRESULTSnAmong the 25 cases, 80% were female, median age was 19years and median latency from drug start to DILI onset was 318days. At presentation, 76% had acute hepatocellular liver injury, median ALT 1,077U/L (range: 63 to 2,333), median bilirubin 4.5mg/dl (range: 0.2 to 16.7), and 90% had a +ANA. During follow-up, 50% were treated with corticosteroids and no participants died or required a liver transplant. A significant association was noted between HLA-B∗35:02 and risk for minocycline DILI; a 16% carrier frequency in DILI cases compared to 0.6% in population controls (odds ratio: 29.6, 95% CI: 7.8-89.8, p=2.5×10-8). Verification of HLA-B∗35:02 imputation was confirmed by sequence-based HLA typing. HLA-B∗35:02 carriers had similar presenting features and outcomes compared to non-carriers. In silico modeling studies support the hypothesis that direct binding of minocycline to this novel HLA risk allele might be an important initiating event in minocycline DILI.nnnCONCLUSIONnHLA-B∗35:02 is a rare HLA allele that was more frequently identified in the 25 minocycline DILI cases compared to population controls. If confirmed in other cohorts, this HLA allele may prove to be a useful diagnostic marker of minocycline DILI.nnnLAY SUMMARYnDevelopment of liver injury following prolonged use of minocycline for acne is a rare but potentially severe form of drug-induced liver injury. Our study demonstrates that individuals who are HLA-B∗35:02 carriers are at increased risk of developing minocycline related liver injury. These results may help doctors more rapidly and confidently diagnose affected patients and possibly reduce the risk of liver injury in individuals receiving minocycline going forward.

IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance

Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3′ untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3′ UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217u2009G allele mRNA forms well-defined 3′ UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure.

Effects of Age, Sex, Body Weight, and Quantity of Alcohol Consumption on Occurrence and Severity of Alcoholic Hepatitis

BACKGROUND & AIMSnOnly a minority of heavy drinking individuals develop alcoholic hepatitis (AH), for unclear reasons. We analyzed data from the Translational Research and Evolving Alcoholic Hepatitis Treatment cohort, consisting of subjects who drink heavily with normal results from liver tests (controls) and patients with AH. We examined risk factors for the development of AH including body mass index (BMI), drinking pattern and quantity, and sex.nnnMETHODSnWe compared data from 145 patients with AH and 124 controls based on BMI when they joined the cohort; groups were matched for sex and race. Drinking patterns were assessed using the timeline followback method, the Alcohol Use Disorders Identification Test, and the National Institute of Alcohol Abuse and Alcoholism 6-question survey. We performed univariable and multivariable analyses to assess the effects of these factors and their interaction in increasing the risk for AH. We also explored the association between PNPLA3 variants and AH.nnnRESULTSnCases with AH were older (47 vs 44 y; Pxa0= .03). For nearly all measures of quantity of alcohol consumed or frequency of binge drinking, controls drank more heavily than cases with AH. We did not find an association between BMI, sex, drinking patterns, and the presence of AH. Age and BMI were independent predictors for the severity of AH. When we analyzed cases and controls of European ancestry, the PNPLA3 single-nucleotide polymorphism rs738409 was associated with risk for AH (odds ratio, 1.89; Pxa0= .007).nnnCONCLUSIONSnCompared with heavy drinkers without liver disease, subjects with AH consumed lower levels of alcohol and had less binge drinking, suggesting an increased sensitivity to the toxic effects of alcohol. The risk for AH may be associated with the PNPLA3 rs738409 polymorphism.

Prevalence of genetic variants of keratins 8 and 18 in patients with drug-induced liver injury

BackgroundKeratin 8 and 18 (K8/K18) cytoskeletal proteins protect hepatocytes from undergoing apoptosis and their mutations predispose to adverse outcomes in acute liver failure (ALF). All known K8/K18 variants occur at relatively non-conserved residues and do not cause keratin cytoskeleton reorganization, whereas epidermal keratin-conserved residue mutations disrupt the keratin cytoskeleton and cause severe skin disease. The aim of our study was to identify keratin variants in idiosyncratic drug-induced liver injury (DILI).MethodsGenomic DNA was isolated from 800 patients enrolled in an ongoing US multicenter study, with DILI attributed to a wide range of drugs. Specific K8/K18 exonic regions were PCR-amplified and screened by denaturing HPLC followed by DNA sequencing. The functional impact of keratin variants was assessed using cell transfection and immune staining.ResultsHeterozygous and compound amino acid-altering K8/K18 variants were identified in 86 DILI patients and non-coding variants in 15 subjects. Five novel amino acid-altering (K8 Lys393Arg, K8 Ala351Val, K8 Ala358Val, K8 Ile346Val, K18 Asp89His) and two non-coding variants were observed. Several variants segregated with specific ethnic backgrounds but were found at similar frequencies in DILI subjects and ethnically matched population controls. Notably, variants in highly conserved residues of K8 Lys393Arg (ezetimibe/simvastatin-related) and K18 Asp89His (isoniazid-related) were found in patients with fatal DILI. These novel variants also led to keratin network disruption in transfected cells.ConclusionsNovel K8/K18 cytoskeleton-disrupting variants were identified in two patients and segregated with fatal DILI. Other non-cytoskeleton-disrupting keratin variants did not preferentially associate with DILI.

Variant Inosine Triphosphatase Phenotypes Are Associated With Increased Ribavirin Triphosphate Levels.

Individuals with lower inosine triphosphatase (ITPA) enzyme activity have a reduced likelihood of experiencing hemolytic anemia during hepatitis C virus (HCV) treatment containing ribavirin (RBV). Because ITPA degrades purines and RBV is a purine analogue, it is conceivable that ITPA activity may affect intracellular RBV concentrations. Here we assessed the association between ITPA activity phenotype and concentrations of RBV triphosphate (RBV‐TP) in red blood cells (RBCs) during HCV treatment. RBV‐TP was quantified in the RBCs of 177 HCV‐infected individuals at a median (range) of 84 (19 to 336) days into HCV treatment that included RBV. Mean (SD) RBV‐TP concentrations were 92.8 (51.6), 101.3 (53.5), 184.8 (84.5), and 197.7 (64.6) pmol/106 cells for 100%, 60%, 30%, and ≤10% ITPA activity groups, respectively. Overall, RBV‐TP was approximately 2‐fold higher in patients with ≤30% ITPA activity compared to 100% activity (P < .0001). Despite higher RBV‐TP levels, individuals with variant ITPA phenotypes had less anemia. The 100% activity group had, on average, a –2.20 g/dL drop in hemoglobin vs –1.43 g/dL (P = .04) for 60% activity, –1.14 g/dL (P = .008) for 30% activity, and –0.70 g/dL (P = .06) for ≤10% activity. This finding of higher RBV‐TP concentrations in RBCs in ITPA variants was unexpected given that ITPA activity‐deficient individuals have a reduced likelihood of RBV‐induced anemia. It also refutes the hypothesis that the mechanism by which ITPA variants are protected against anemia is due to lower RBV‐TP levels in RBCs.

Genome-wide association study to characterize serum bilirubin elevations in patients with HCV treated with GS-9256, an HCV NS3 serine protease inhibitor.

BACKGROUNDnProtease inhibitors for the treatment of HCV can cause mild and reversible elevations of unconjugated bilirubin. We sought to characterize genetic determinants of bilirubin elevations using a genome-wide approach among patients with genotype 1 HCV who received combination therapy that included GS-9256, a novel potent inhibitor of HCV NS3 serine protease, as part of a Phase IIb trial.nnnMETHODSnOf the 200 patients sampled, 176 had confirmed European ancestry and were included in the analysis. Infinium HumanOmni5BeadChip (Illumina, Inc., San Diego, CA, USA) was used for genotyping. A categorical analysis of low (grade 0-1) versus high (grade 2-4) bilirubin toxicity grade and a quantitative trait locus mapping of peak bilirubin concentrations was performed.nnnRESULTSnA total of 4,466,809 genetic markers were analysed. No single variant showed a statistically significant association with observed bilirubin elevations in this patient population. In a targeted analysis of single nucleotide polymorphisms in genes known to be involved in bilirubin transport, no significant differences in allele frequency between high and low bilirubin toxicity grade were observed.nnnCONCLUSIONSnThese results indicate that risk for bilirubin elevation in patients receiving GS-9256 is unlikely to be strongly influenced by common genetic variants with large effects. The current study cannot rule out a role for common variants of weak effect, or a more complex model, including multiple contributing factors, such as rare variants and as yet unidentified environmental influences.

An exploratory genome-wide analysis of genetic risk for alcoholic hepatitis

Abstract Objectives: To elucidate the genetic variability between heavy drinkers with and without alcoholic hepatitis (AH). Materials and methods: An exploratory genome-wide association study (GWAS; NCT02172898) was conducted comparing 90 AH cases with 93 heavy drinking matched controls without liver disease in order to identify variants or genes associated with risk for AH. Individuals were genotyped using the multi-ethnic genotyping array, after which the data underwent conventional quality control. Using bioinformatics tools, pathways associated with AH were explored on the basis of individual variants, and based on genes with a higher ’burden’ of functional variation. Results: Although no single variant reached genome-wide significance, an association signal was observed for PNPLA3 rs738409 (pu2009=u2009.01, OR 1.9, 95% CI 1.1–3.1), a common single nucleotide polymorphism that has been associated with a variety of liver-related pathologies including alcoholic cirrhosis. Using the improved gene set enrichment analysis for GWAS tool, it was shown that, based on the single variants’ trait-association p-values, multiple pathways were associated with risk for AH with high confidence (false discovery rate [FDR]u2009<u20090.05), including several pathways involved in lymphocyte activation and chemokine signaling, which coincides with findings from other research groups. Several Tox Functions and Canonical Pathways were highlighted using Ingenuity Pathway Analysis, with an especially conspicuous role for pathways related to ethanol degradation, which is not surprising considering the phenotype of the genotyped individuals. Conclusion: This preliminary analysis suggests a role for PNPLA3 variation and several gene sets/pathways that may influence risk for AH among heavy drinkers.