Genome-wide analysis of the inter-individual variability in in vitro cell viability on exposure to carbamazepine and its metabolites

Philippe Marlot1, Nour Abdo2, Ivan Rusyn2, Ana Alfirevic1, Munir Pirmohamed1. 1University of Liverpool, Liverpool, UK, 2University of North Carolina, Chapel Hill, NC, USA

Background

Carbamazepine (CBZ), an anticonvulsant and mood-stabilising drug used for the treatment of epilepsy, trigeminal neuralgia and bipolar disorder can cause hypersensitivity reactions. These reactions occur only in a minority of patients treated with the drug, but they may be severe and affect different organs including the skin and liver. Although genetic factors such as HLA-A*3101 and B*1502 have been associated with hypersensitivity reactions, the exact mechanisms of toxicity have not been fully elucidated. In this study we aimed to investigate the lymphoblastoid cell-viability on exposure to increasing concentrations of carbamazepine and its metabolites in vitro.

Methods

331 immortalised lymphoblast cell lines from unrelated individuals of European (British from England and Scotland, Utah residents, Tuscan in Italy) and African (Yoruban in Ibadan, Nigeria) ancestry were used in this study. The cells, which have been previously characterised through genome wide screen and RNA-seq expression analysis, were exposed to CBZ and its metabolites, CBZ-10,11 epoxide and 9-acredinecarboxaldehyde (9-AC) at eight concentrations. The cell viability, defined as ATP content, was measured using the previously validated CellTiter-Glo® assay (Promega). We derived a 10% cytotoxic effect level (EC10) for each compound and cell line using the measured concentration response data. The data were analysed using the MAGWAS software [1]. P-values above 1.0 x 10-5 were considered genome-wide suggestive and p-values above 5.0 x 10-8 were considered genome-wide significant.

Results

Considerable inter-individual variability in the cytotoxic response was observed for all three compounds. Drug concentrations that achieved EC10 varied from 0.3 µM to 200µM and above for the same compound. We found several genetic polymorphisms linked to cell toxicity at low concentrations of all three compounds. Candidate genes included TPP2 (p-value >3.2 x 10-6 [CBZ]), MiR572 (p-value: 2.0 x 10-5 [CBZ-10,11 epoxide], 7.8 x 10-5 [9-AC]), RP11-354|13.2 (p-value: 7.4 x 10-6 [CBZ], 4.5 x 10-5 [CBZ-10,11 epoxide], 5.0 x 10-7 [9-AC]), and DUOX1 (p-value: 8.5 x 10-6 [CBZ], 6.3 x 10-5 [CBZ-10,11 epoxide], 4.7 x 10-6 [9-AC]). Although none of these reached genome-wide significance, their p-values were above genome-wide suggestive level and biologically plausible signals which require further validation by genotyping hypersensitive patients and controls.

Conclusion

The study demonstrated large inter-individual variability in cytotoxicity on exposure to CBZ and its metabolites in vitro. We identified several candidate genes which are going to be investigated further in patient populations. This could contribute to our understanding of the underlying predisposing factors for CBZ hypersensitivity.

[1] Brown CC et al. (2012) BioData Min. 5