In silico analysis of HLA associations with drug-induced liver injury: use of a HLA-genotyped DNA archive from healthy volunteers

Abstract

Background: Drug-induced liver injury (DILI) is one of the most common adverse reactions leading to product withdrawal post-marketing. Recently, genome-wide association studies have identified a number of human leukocyte antigen (HLA) alleles associated with DILI; however, the cellular and chemical mechanisms are not fully understood.

Methods: To study these mechanisms, we established an HLA-typed cell archive from 400 healthy volunteers. In addition, we utilized HLA genotype data from more than four million individuals from publicly accessible repositories such as the Allele Frequency Net Database, Major Histocompatibility Complex Database and Immune Epitope Database to study the HLA alleles associated with DILI. We utilized novel in silico strategies to examine HLA haplotype relationships among the alleles associated with DILI by using bioinformatics tools such as NetMHCpan, PyPop, GraphViz, PHYLIP and TreeView.

Results: We demonstrated that many of the alleles that have been associated with liver injury induced by structurally diverse drugs (flucloxacillin, co-amoxiclav, ximelagatran, lapatinib, lumiracoxib) reside on common HLA haplotypes, which were present in populations of diverse ethnicity.

Conclusions: Our bioinformatic analysis indicates that there may be a connection between the different HLA alleles associated with DILI caused by therapeutically and structurally different drugs, possibly through peptide binding of one of the HLA alleles that defines the causal haplotype. Further functional work, together with next-generation sequencing techniques, will be needed to define the causal alleles associated with DILI.

Figure 1

Outline of the intended experimental strategy utilizing lymphocytes from HLA-genotyped volunteers.

Figure 2

Linkage disequilibrium and haplotype structure for HLA alleles associated with drug-induced liver injury in Caucasians. From the literature, we selected class I and class II HLA alleles that have been reported to be associated with DILI. Seven candidate gene and five genome-wide association studies were included in the analysis. Drugs and associated alleles that have been implicated in DILI are organized in frames. Colored boxes represent information gained from the England North West (ENW) cohort (red), dbMHC (green) and AFND (blue). Haplotype frequencies > 5% are depicted by connecting lines. Please note that the extended HLA class II haplotype associated with flucloxacillin-induced DILI comprises B*57:01, DRB1*07:01 and DQB1*03:03, rather than DQB1*02:01. Ambiguous codes A*33:03g, B*08:01g, DQB1*02:01g and DQB1*06:04g in the ENW population were allocated as A*33:03, B*08:01, DQB1*02:01 and DQB1*06:04, respectively. Haplotype frequencies in the ENW population are shown to three decimal places. Values in parentheses indicate the number of populations in dbMHC (green) and AFND (blue) that contain two loci haplotypes with frequencies over 5%. Table S6 in Additional file 1 lists the percentage of individuals from the ENW cohort who are carriers of alleles and haplotypes associated with DILI, as are represented in this figure.

Figure 3

Haplotype combinations between HLA-B*57:01 and HLA-DRB1 alleles. All haplotype combinations (colored lines) of the HLA-B*57:01 allele with HLA-DRB1 alleles reported in the dbMHC database are shown (n = 8,569). Haplotype frequencies were estimated using the Expectation Maximization algorithm described in PyPop [37]. Frequencies > 1% are shown in bold whereas frequencies < 1% are represented by dotted lines. Circles in red correspond to alleles that have been associated with DILI. As shown, high LD can be observed between the B*57:01 and DRB1*07:01 alleles, which have been associated with DILI. This picture was automatically generated using the GraphViz software [40] and is available as Scalable Vector Graphics (SVG) file.

Figure 4

Distance trees for HLA class I and class II alleles associated with DILI compared with most frequent HLA alleles selected as controls. (a) Class I alleles. (b) Class II alleles. Controls were selected based on high frequency alleles in Caucasians. HLA alleles associated with DILI are circled in red.