Studies on Novel Variants Associated with Co-amoxiclav-Induced Liver Injury Detected in Exome Sequencing Analysis

M Alshabeeb1,2, AK Daly1. 1Newcastle university, Newcastle upon Tyne, UK, 2King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia

Co-amoxiclav is one of the most common drugs associated with hepatic injury though such incidents are rare. Susceptibility shows strong associations with HLA class I and II alleles (1). To identify additional genetic risk factors, exome sequencing was performed on 66 UK drug-induced liver injury (DILI) cases due to co-amoxiclav and found possible associations for several rare variants, including rs117511121 in IL12RB1 (odds ratio (OR) 7.28 (95% CI 3.21-16.5; p=0.00012)) and rs145855109 in TPH1 (tryptophan hydroxylase) (OR 22.33 (95% CI 4.42-112.74; p=0.006)) (Goldstein, Daly and Daly, manuscript in preparation). The current study aimed to replicate and extend these associations in a larger numbers of co-amoxiclav DILI cases using a TaqMan SNP genotyping assay and European-American EVS controls (n=4300). Ethical approval for this study was obtained from the Leeds East Research Ethics committee. Additional cases (n=99) were genotyped for rs117511121, confirming the association (OR 3.26 (95% CI 1.29-8.28; p=0.025)) and giving a combined odds ratio of 4.81 (95% CI 2.55-9.07; p=0.000033). IL12RB1 has an important role in T cell responses, which involves interleukin-12 binding to IL12RB1/IL12RB2 receptor complexes on T cells. rs117511121 is associated with a R283Q amino acid change which is predicted to be non-damaging. Also R283 is not conserved within mammalian species. Further investigations on rs117511121 involving DNA sequencing of promoter regions in cases positive for this variant showed that it was in strong linkage disequilibrium with two more common promoter region SNPs (rs436875 and rs393548) previously reported to be risk factors for certain autoimmune diseases (2). Genotyping of cases and controls for rs436857 suggested borderline significance only (p=0.051). However, reporter gene studies revealed significantly lower luciferase activity for constructs positive for both promoter region variants (-2T and -111T) in HepG2 cells (p=0.014). These results are consistent with a previous study (2). The genotyping data suggests that the variant detected in the exome sequencing study is likely to be the more important risk factor but an additional contribution to risk from the promoter region SNPs cannot be ruled out.

The TPH1 rare variant (A300T which is predicted to be possibly damaging) was confirmed to be associated with co-amoxiclav DILI in a further 99 co-amoxiclav DILI cases (OR 14.73 (95% CI 2.94–73.92; p=0.013) with an odds ratio of 17.75 (95% CI 4.96-63.52; p=0.00032) for the combined cohort of 165 cases. The finding that TPH1 knockout mice are more susceptible to severe cholestatic liver injury and death after bile duct ligation (3) is of potential relevance to the association. Further studies on humans are needed to explore the relationship of this gene with DILI development to confirm our positive findings.

No associations with the IL12RB1 or TPH1 SNPs were seen for another cohort of DILI cases due to flucloxacillin. The results reported in this study make a small contribution to increasing understanding of the genetic basis of DILI due to co-amoxiclav and also suggest that exome sequencing is helpful in detecting rare mutations that contribute to adverse drug reactions.

Acknowledgement

We are grateful to the International Serious Adverse Event consortium for funding case recruitment.

(1) Lucena, M.I. et al (2011). Gastroenterology 141: 338-347

(2) Takahashi N et al. (2005). Hum Mol Genet 14:3149-59

(3) Jang et al. (2012). Hepatology 56:209-18