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

Abstract

Background: Keratin 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).

Methods: Genomic 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.

Results: Heterozygous 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.

Conclusions: Novel 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.

Fig. 1

Distribution of the identified keratin 8/18 (K8/K18) variants within the protein backbone. The schematics depict the tripartite keratin structure consisting of head, rod and tail domains with their corresponding amino acid annotations. The rod subdomains IA, IB and II are connected by the corresponding linker (L) sequences, L1, L1-2, L2, and the shaded regions at the beginning and end of the rod domain correspond to particularly conserved helix initiation/termination motifs (HIM/HTM) that represent the mutational hotspots in epidermal keratins. The exonic structure of both genes is also depicted (E1–E8 for K8 and E1–E7 for K18). The exons that were analyzed in the present study are highlighted in bold. The coding variants are denoted by position and alteration of the affected amino acid displayed by the single-letter code. The relation of the intronic variants to the coding sequences is also shown. For annotation of intronic variants, the intervening sequences are labeled as ‘IVS’ and the position of a single variant located in the 5′UTR of K18 is denoted by its nucleotide (‘nt’) distance from the starting codon. The novel variants which were not previously described are highlighted in bold and the absolute count of all variants is listed in parentheses. Note that the novel K8 K393R and K18 D89H represent the first described K8/K18 variants localized in the most conserved HTM/HIM regions

Fig. 2

K8 K393R and K18 D89H are the first identified simple epithelial keratin variants located in the most conserved regions of the rod domain. Conservation of the novel K8 K393R/K18 D89H variants among (a,c) species and b type II or d type I keratins (standard single-letter amino acid abbreviations are used). Sequences surrounding the novel K8/K18 variants are depicted. Dots highlight conserved amino acids. e Distribution of simple epithelial and epidermal keratin variants within the keratin backbone. The schematic shows the characteristic tripartite structure (N-terminal ‘head’, central ‘rod’ and C-terminal ‘tail’ domains) of all IFs including keratins. The rod domain is subdivided into IA, IB, IIA, and IIB subdomains that in turn are separated through linker (L1, L1-2, L2) sequences. The shaded regions are the most conserved segments of the rod domain (termed helix initiation motif ‘HIM’ and helix termination motif ‘HTM’) and constitute mutation ‘hot spots’ in severe epidermal keratins and other IF mutations. Note that unlike epidermal keratins, the frequent human K8/K18 variants are located outside HIM/HTM. IF, intermediate filament

Fig. 3

K8 K393R and K18 D89H variants result in keratin network disruption. a NIH 3T3 cells were transiently transfected with wild-type K8/K18 (WT) or a combination of K8/K18 variants and a WT partner keratin and stained with an anti-K18 antibody. Transfections were performed in triplicate. b The percentage of cells with disrupted keratin filaments was quantified. At least 100 cells were counted in each experiment