Genetic variations in drug-induced liver injury (DILI): resolving the puzzle

Introduction: Idiosyncratic drug-induced liver injury (DILI) is a challenging condition with widespread implications. The underlying mechanism of DILI is not yet fully elucidated, but genetic predispositions are believed to contribute to DILI susceptibility. The identification of genetic risk factors has been a goal in DILI research for more than two decades. Areas covered: Here we provide an overview of genetic studies in DILI performed to date and outline polymorphisms identified to have a potential role in DILI development. This review covers both earlier candidate gene studies and more recent genome-wide association studies. The clinical applications of these findings are also discussed. Expert opinion: Various polymorphisms have been identified as associated with DILI susceptibility, but all of these have not been confirmed in independent studies or contradictive findings are available. Genome-wide significant associations between distinct HLA risk alleles and DILI due to specific causative agents strengthen the hypothesis that DILI is partially immune-mediated. These HLA alleles generally have low positive predictive value and are therefore not useful in preemptive tests to reduce DILI incidences, but can aid DILI diagnosis and clinical decision-making.

Drug-induced liver injury: Is it time for genetics to change our clinical practice?

Drug induced liver injury and its relationship to autoimmune hepatitis

Chapter 22 - Drug-Induced Liver Injury

There is considerable evidence that susceptibility to idiosyncratic drug-induced liver injury (DILI) is genetically determined. Though genetic associations with DILI have been reported since the 1980s, the development of genome-wide association studies has enabled genetic risk factors for DILI, in common with other diseases, to be detected and confirmed more confidently. Human leukocyte antigen (HLA) genotype has been demonstrated to be a strong risk factor for development of DILI with a range of drugs and the underlying mechanism, probably involving presentation of a drug-peptide complex to T cells is increasingly well understood. However, specific HLA alleles are not associated with all forms of DILI and non-HLA genetic risk factors, especially those relating to drug disposition, also appear to contribute. For some drugs, there is evidence of a dual role for HLA and drug metabolism genes. Though the associations with non-HLA genes have been less well replicated than the HLA associations, there is increasing evidence that drug metabolism genes such as NAT2 and UGT2B7 contribute to some forms of DILI. Translating current genetic findings on DILI susceptibility to the clinic has been relatively slow, but some progress is now being made. In the future, DNA sequencing may lead to the identification of rare variants that contribute to DILI. Developments in the related area of epigenomics and in the development of improved models for DILI by use of genetically defined induced pluripotent stem cells should improve understanding of the biology of DILI and inform drug development.

A Genome-Wide Association Study Identifies Potential Susceptibility Loci for Hepatotoxicity Due to Various Drugs

Novel Susceptibility Loci for Primary Sclerosing Cholangitis Identified by Genome-Wide Association and Replication Analysis

The late event onset of a fraction of idiosyncratic drug-induced liver injury (DILI) cases and the link observed by genome-wide association studies (GWASs) of certain human leucocyte antigen (HLA) alleles with DILI due to specific drugs support the crucial role of the immune system (both innate and adaptive) in the pathogenesis of DILI. Recent advances in both flow and mass cytometry have allowed the profiling of all major immune cell types in a given sample. Therefore, determining the lymphocyte populations in samples from patients with DILI would facilitate the development of specific biomarkers for DILI diagnosis and prognosis. To date, a few studies have explored the immune landscape in DILI. In a recent study of leukocyte immunophenotyping using flow cytometry from the Spanish DILI Registry, an important role of adaptive immune response in DILI is suggested. DILI patients had significantly higher levels of T helper 1 (Th1) cells and activated helper and cytotoxic T cells than healthy controls. Furthermore, the increased expression of negative immune checkpoints and ligands in DILI patients could reflect a restoration of the immune homeostasis. Differences in the profile of cytokines in DILI patients from the Drug-Induced Liver Injury Network (DILIN) also suggest an involvement of both innate and adaptive immune systems in DILI development and prognosis. Moreover, several studies based on immunophenotyping of liver infiltrates showed a distinctive pattern of cellular infiltrates in patients with immune checkpoint inhibitors (ICIs)-DILI, with lower levels of plasma cells, CD20+ B cells and CD4+ T cells than in autoimmune hepatitis (AIH) patients. These pioneering studies highlight the importance of immunophenotyping for the mechanistic understanding of DILI. In this review, available data on immunophenotyping in DILI are gathered, and the potential clinical applications of cutting-edge, novel immunophenotyping techniques are discussed.

Flupirtine is a nonopioid analgesic with regulatory approval in a number of European countries. Because of the risk of serious liver injury, its use is now limited to short-term pain management. We aimed to identify genetic risk factors for flupirtine-related drug-induced liver injury (DILI) as these are unknown. Six flupirtine-related DILI patients from Germany were included in a genome-wide association study (GWAS) involving a further 614 European cases of DILI because of other drugs and 10,588 population controls. DILI was diagnosed by causality assessment and expert review. Human leucocyte antigen (HLA) and single nucleotide polymorphism genotypes were imputed from the GWAS data, with direct HLA typing performed on selected cases to validate HLA predictions. Four replication cases that were unavailable for the GWAS were genotyped by direct HLA typing, yielding an overall total of 10 flupirtine DILI cases. In the six flupirtine DILI cases included in the GWAS, we found a significant enrichment of the DRB1*16:01-DQB1*05:02 haplotype compared with the controls (minor allele frequency cases 0.25 and minor allele frequency controls 0.013; P=1.4 × 10(-5)). We estimated an odds ratio for haplotype carriers of 18.7 (95% confidence interval 2.5-140.5, P=0.002) using population-specific HLA control data. The result was replicated in four additional cases, also with a haplotype frequency of 0.25. In the combined cohort (six GWAS plus four replication cases), the haplotype was also significant (odds ratio 18.7, 95% confidence interval 4.31-81.42, P=6.7 × 10(-5)). We identified a novel HLA class II association for DILI, confirming the important contribution of HLA genotype towards the risk of DILI generally.

Drug-induced liver injury (DILI) is an increasing health prob-lem and a challenge for physicians, regulatory bodies, and thepharmaceutical industry, mainly due to the potential severity andundefined pathogenesis of this condition. In addition, DILI is ofteninaccurately diagnosed, missed entirely, or not reported due to thelack of reliable diagnostic biomarkers. Idiosyncratic DILI is generallyconsidered as unpredictable in terms of dose administration ordrug pharmacology. Hence, genetic variations are believed to playan important role in DILI susceptibility. Polymorphisms in genesinvolved in drugmetabolism, that is, phase I (bioactivation), phase II(detoxification), and phase III (elimination), and the immune systemhave been targeted for genetic DILI studies to date. This chapterreviews the major findings in genetic DILI studies initially throughcandidate gene approachmethods andmore recently using genome-wide association (GWA) studies.

Biomarkers for metabolic drug activation : towards an integrated risk assessment for drug-induced liver injury (DILI)

Avoiding idiosyncratic DILI: Two is better than one

Nasopharyngeal carcinoma (NPC) is a common malignancy in East and Southeast Asia, especially in South China. The etiology of NPC has been linked to genetic susceptibility, Epstein-Barr virus (EBV) infection, and environmental factors. Accumulated evidence including multiple genome-wide association studies (GWASs) has revealed robust genetic predisposition of NPC. However, GWAS-identified genetic variants collectively account for only 8.2% of NPC heritability [1]. The underlying inherited predisposition is largely undetermined. The strongest genetic signal for NPC consistently hits the human leukocyte antigen (HLA) region on 6p21 [2]. However, the highly polymorphic nature and complicated long-range linkage disequilibrium (LD) in the HLA region particularly obscure the causal variants driving the association. In addition, most genetic variants located in introns or intergenic regions. The causal genes mediating genetic effects on NPC risk have rarely been ascertained by GWAS alone. Recently, transcriptome-wide association study (TWAS) has been proposed as an attractive approach to identify novel gene-trait associations and prioritize causal genes for complex traits [3]. By integrating GWAS and gene expression data, TWAS can effectively and economically assess associations between genetically predicted gene expression levels and disease risks in large populations. Hence, using the cis-regulated expression in addition to genetic variants to explore NPC susceptibility genes could be promising and reasonable for mechanistic and functional inference. Nevertheless, neither public data of nasopharyngeal tissue were available, nor TWAS for NPC had been conducted. Herein, we integrated genome and transcriptome data of 89 nasopharyngeal tumor tissues and investigated the associations between predicted gene expression levels and NPC risk using multicenter GWAS data involving 4506 NPC cases and 5384 cancer-free subjects (defined as controls) from South China. Given the close relationship between EBV infection and NPC, a cis-regulated expression weight matrix from EBV-transformed lymphocytes (n = 117) in the GTEx project was used for further evaluation. Study populations and detailed methodology are described in the Supplementary file of methods. We predicted the expression levels of 2505 and 2411 genes in the GWAS population by constructing the models for the prediction of gene expression in nasopharyngeal tissues (NP models) and EBV-transformed lymphocytes (lymphocyte models), respectively (Supplementary Table S1), and 377 genes overlapped (Supplementary Figure S1). Thirty-three genes were associated with NPC at a Bonferroni-corrected threshold, and all were located in the HLA region (Figure 1A). Among them, 11 of 13 previously reported genes were replicated. Our results were consistent with the studies focusing on the HLA region in South China [4, 5], where most of the reported genes available in TWAS were replicated. The predicted expression levels of ZFP57 (NP models), MICA (both models), and HLA-C (lymphocyte models) were significantly higher in cases than in controls, while the expression levels of MOG, HCG27, HLA-DQB1, HLA-H, HLA-U (NP models), HLA-F (both models), HLA-A, and HLA-DRB1 (lymphocyte models) were lower in cases than in controls. The two overlapping genes showed similar associations with NPC (HLA-F: Z score = -10.28 and -8.95; MICA: Z score = 7.82 and 6.60, for NP and lymphocyte models, respectively) (Supplementary Table S2). Interestingly, half of the previously reported genes belonged to HLA class I. Most of them showed lower levels of predicted expression in cases than in controls, possibly because EBV transcripts in NPC tumors were involved in the inhibition of HLA class I gene expression [6]. It is rational to assume that the low expression levels of these genes may affect the anti-EBV immune response in presenting peptides to cytotoxic T cells, facilitating immune evasion of tumor cells or EBV-mediated oncogenic action. TWAS-identified susceptibility genes and pathways for NPC. (A) Manhattan plot of TWAS in NP models and lymphocyte models. The blue lines represent the Bonferroni-corrected significance threshold. The red dots above or below the blue line represent the genes passed the Bonferroni threshold in the association analysis. The genes with green labels have been reported to be associated with NPC by previous genome-wide or candidate pathway association studies. The genes with black labels were newly identified as NPC susceptibility genes by our study. The genes in different chromosomes were exhibited in light and dark grey dots. (B) Expression quantitative trait locus analysis for the seven putative causal genes in the expression data of 89 nasopharyngeal tissue samples. The Kruskal-Wallis test was used to compare medians among three genotypes for most of the variants. In a certain homozygote group, the P values were recalculated using only the wild-type and heterozygous groups for the expression of MICD, HCG27 and HLA-DOB by excluding the groups with a sample size less than 5. (C) GO pathway enrichment analysis of NPC. (D) KEGG pathway enrichment analysis of NPC. "Gene Ratio" refers to the percentage of total significant genes in the given pathway. All 354 significant genes (P < 0.05) in TWAS were used in the enrichment analysis. Abbreviations: TWAS, Transcriptome-wide association analysis; NPC, Nasopharyngeal carcinoma; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes Although the significant signals consistently hit the HLA region, 22 additional genes not previously reported were identified in TWAS. Among them, the predicted expression levels of 9 genes were significantly higher in NPC cases than in controls, including HLA-DOB, HCG4B, RPL23AP1, HLA-J in NP models and HCG4, CCHCR1, STK19, C4B, IFITM4P in lymphocyte models, while 13 other genes showed significantly lower expression levels in cases than in controls, including HCP5, ZSCAN23, HCG4P11, HCG4P7, MICD, MICB-DT, SNHG32 in NP models and NOTCH4, C4A, HCG22, POU5F1, MICE, HLA-S in lymphocyte models (Figure 1A). We performed conditional analyses to determine whether the associations between predicted gene expression levels and NPC were influenced by the GWAS signals. After conditioning on the respective GWAS index SNP, the associations for HLA-DOB, NOTCH4, ZSCAN23, STK19, C4B, HLA-J, HLA-S, and MICB-DT remained significant. After conditioning on all previously reported SNPs, NOTCH4, HCG4, HCG22, POU5F1, HCG4B, HCG4P11, MICB-DT, STK19 and IFITM4P remained significant. It indicated that their associations were partially independent of the GWAS signals (Supplementary Table S3). Due to the complicated structure with high LD and co-expression networks in the HLA region, we conducted fine-mapping analyses to prioritize the causal genes. Using posterior inclusion probability (PIP) analysis, we prioritized 7 causal genes: MICA, HLA-DQB1, HLA-DOB, ZSCAN23, HCG27, MICD, and HLA-U. HLA-DOB, ZSCAN23, and MICD were newly identified as NPC susceptibility genes (Supplementary Table S4). Furthermore, we conducted expression quantitative trait locus (eQTL) analyses to identify whether the genetic variants could influence the expression levels of these genes. We found that individuals with relevant risk SNPs (the GWAS index SNPs) exhibited higher expression of HLA-DQB1, MICA, MICD and HLA-U, or lower expression levels of ZSCAN23, HCG27, and HLA-DOB. These results indicated that the risk alleles affected the expression levels of the causal genes (Figure 1B). Two HLA class II genes (HLA-DQB1 and HLA-DOB) were prioritized as causal genes. Both genes were associated with other virus-associated cancers, such as cervical cancer [7]. A comprehensive TWAS exploring genetic susceptibility for antiviral immune response using 7924 subjects from the UK Biobank cohort revealed that the genetic determinants for EBV infection were predominantly located on HLA class II genes. The most significant signals associated with the antibody level of BamHI Z EBV replication activator (ZEBRA) hit HLA-DQB1 [8]. HLA-DOB may impact viral clearance capacity and persistent infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) [9]. Since EBV reactivation with elevated EBV DNA load or antibodies was observed at the preclinical phase of NPC, we hypothesized that HLA class II genes, especially HLA-DQB1and HLA-DOB, participate in the early stage of NPC tumorigenesis by influencing EBV infection. Besides, some identified pseudogenes, such as IFITM4P [10], may function by regulating their parental genes. However, their biological mechanisms remain unclear, and further researches are needed. Gene Ontology (GO) enrichment analysis confirmed that TWAS-identified genes (354 genes with P < 0.05) were enriched in the pathways of cell-mediated immune response, antigen processing and presentation (Figure 1C). Similarly, the top pathways annotated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database focused on infection of herpes simplex virus type 1, human T-cell leukemia virus type 1, EBV, and autoimmune disorders such as graft-versus-host disease (Figure 1D). In summary, using a TWAS approach, we corroborated the central role of HLA genes in NPC susceptibility. Apart from HLA class I genes, we propose critical roles of HLA class II genes and other nonclassical HLA genes. Seven genes, including HLA-DQB1 and HLA-DOB, were prioritized as causal genes. Recent evidence indicated that these genes are pivotal in the metastable equilibrium between host and virus. Our findings provide additional evidence for a better understanding of the genetic etiology of NPC and clues to further advance this field. We thank the staffs from Sun Yat-sen University Cancer Center biorepository. We thank all the study participants and research staff who recruited participants and collected samples in this study. This study was funded by the National Key Research and Development Program of China (2021YFC2500400), the Basic and Applied Basic Research Foundation of Guangdong Province, China (2021B1515420007), Sino-Sweden Joint Research Programme (81861138006), the Science and Technology Planning Project of Guangzhou, China (201804020094), the Special Support Program for High-level Professionals on Scientific and Technological Innovation of Guangdong Province, China (2014TX01R201), National Natural Science Foundation of China (81973131, 81903395, 81803319, 82003520), National Science Fund for Distinguished Young Scholars of China (81325018). The authors have no potential conflicts of interest to declare. The Institutional Review Board of Sun Yat-sen University Cancer Center approved this study. Informed consent was obtained from all study participants. WHJ and YQH devised the project and the main conceptual ideas; YQH, WQX, DHL, and TMW wrote the original draft; DHL and TMW performed the computational analyses; TMW, DWY, CMD, and WLZ contributed to implementation of data processing and analyses; DWY, CMD, YL, WLZ, RWX, LL, HD, XT, YW, TZ, XZL, PFZ, XHZ, SDZ, YZH, MT, YZ, YC and JBZ contributed to the sample preparation; TMW and WLZ contributed to the RNA-seq quantification and quality control pipeline; ETC, ZZ, GH, SMC, QL, LF, YS, MLL, HOA, WY, and THL contributed to the interpretation of the results; YQH, WQX, and TMW revised and wrote the final version of the manuscript; verified the analytical methods; WHJ supervised the project. All authors read and approved the final manuscript. Methods and materials are available in the supplementary file. The datasets generated and used during the current study are available at Research Data Deposit (RDD) public platform (www.researchdata.org.cn) with the approval RDD number of RDDB2021406340. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Minimal Risk of Drug-Induced Liver Injury With Molnupiravir and Ritonavir-Boosted Nirmatrelvir

Background: Idiosyncratic drug-induced liver injury (DILI) is a serious uncommon disease that may develop as a result of the intake of certain drugs such as the antimicrobials flucloxacillin and co-amoxiclav. The reported cases showed significant associations between DILI and various human leukocyte (HLA) markers. The solute carrier organic anion transporter 1B1 (SLCO1B1), a non-HLA candidate gene, was previously reported as a risk factor for liver injury induced by rifampin and methimazole. This study presumed that SLCO1B1 may play a general role in the DILI susceptibility and therefore investigated the association of rs4149056 (SLCO1B1*5, T521C) polymorphism with flucloxacillin- and co-amoxiclav–induced liver injury.Methodology: We recruited 155 and 165 DILI cases of white ancestral origin from various European countries but mainly from the United Kingdom owing to flucloxacillin and co-amoxiclav, respectively. Only adult patients (≥18 years) who were diagnosed with liver injury and who showed i) clinical jaundice or bilirubin >2x the upper limit of normal (ULN), ii) alanine aminotransferase (ALT) >5x ULN or iii) alkaline phosphatase (ALP) >2x ULN and bilirubin > ULN were selected. The population reference sample (POPRES), a European control group (n = 282), was used in comparison with the investigated cases. TaqMan SNP genotyping custom assay designed by Applied Biosystems was used to genotype both DILI cohorts for SLCO1B1 polymorphism (rs4149056). Allelic discrimination analysis was performed using a step one real-time PCR machine. Genotype differences between cases and controls were examined using Fisher’s exact test. GraphPad Prism version 5.0 was used to determine the p-value, odds ratio, and 95% confidence interval. Compliance of the control group with Hardy–Weinberg equilibrium was proven using a web-based calculator available at https://wpcalc.com/en/equilibrium-hardy-weinberg/.Results: A small number of cases failed genotyping in each cohort. Thus, only 149 flucloxacillin and 162 co-amoxiclav DILI cases were analyzed. Genotyping of both DILI cohorts did not show evidence of association with the variant rs4149056 (T521C) (OR = 0.71, 95% CI = 0.46–1.12; p = 0.17 for flucloxacillin cases and OR = 0.87, 95% CI = 0.56–1.33; p = 0.58 for co-amoxiclav), although slightly lower frequency (22.8%) of positive flucloxacillin cases was noticed than that of POPRES controls (29.4%).Conclusion: Carriage of the examined allele SLCO1B1*5 is not considered a risk factor for flucloxacillin DILI or co-amoxiclav DILI as presumed. Testing a different allele (SLCO1B1*1B) and another family member gene (SLCO1B3) may still be needed to provide a clearer role of SLCO1B drug transporters in DILI development–related to the chosen antimicrobials.