Polymerase γ gene POLG determines the risk of sodium valproate-induced liver toxicity

Abstract

Sodium valproate (VPA) is widely used throughout the world to treat epilepsy, migraine, chronic headache, bipolar disorder, and as adjuvant chemotherapy. VPA toxicity is an uncommon but potentially fatal cause of idiosyncratic liver injury. Rare mutations in POLG, which codes for the mitochondrial DNA polymerase γ (polγ), cause Alpers-Huttenlocher syndrome (AHS). AHS is a neurometabolic disorder associated with an increased risk of developing fatal VPA hepatotoxicity. We therefore set out to determine whether common genetic variants in POLG explain why some otherwise healthy individuals develop VPA hepatotoxicity. We carried out a prospective study of subjects enrolled in the Drug Induced Liver Injury Network (DILIN) from 2004 to 2008 through five US centers. POLG was sequenced and the functional consequences of VPA and novel POLG variants were evaluated in primary human cell lines and the yeast model system Saccharomyces cerevisiae. Heterozygous genetic variation in POLG was strongly associated with VPA-induced liver toxicity (odds ratio = 23.6, 95% confidence interval [CI] = 8.4-65.8, P = 5.1 × 10⁻⁷). This was principally due to the p.Q1236H substitution which compromised polγ function in yeast. Therapeutic doses of VPA inhibited human cellular proliferation and high doses caused nonapoptotic cell death, which was not mediated through mitochondrial DNA depletion, mutation, or a defect of fatty acid metabolism.

Conclusion: These findings implicate impaired liver regeneration in VPA toxicity and show that prospective genetic testing of POLG will identify individuals at high risk of this potentially fatal consequence of treatment.

Figure 1

(a) Gene structure of POLG showing the location of the (b) c.911T>G/p.L304R, c.1399G>A/p.A467T, (c) c.3708G>T/p.Q1236H, and (d) c.3428A>G/p.E1143G substitutions. The exonuclease domain extends from amino acid residue 1 to 418. The polymerase domain extends from amino acid residue 756 to 1239. The linker region lies between amino acid residues 418 and 756.

Figure 2

(a) Alignment of C-terminal stretch of human polγ and the corresponding stretch of yeast Mip1. Q1236 amino acid is in bold; the region which is changed in Mip1C is underscored. (b) Linear representation of polγ, Mip1, Mip1C and Mip1C^Q1236H organization. Q1236 amino acid is in bold. (c) Petri dish images showing the normal and petite colonies from the parental (Mip1C) and the Mip1C^Q1236H strain (bottom).

Figure 3

(a) Ratio of mtDNA to nuclear DNA for each cell line following ten days of treatment with either 2mM VPA, 10mM VPA, 50ng/ml Ethidium bromide (EtBr) or untreated. AHS = p.A467T/p.K1191N compound heterozygote. (b) Relationship between mtDNA level and cell viability after ten days of treatment. For comparison, mtDNA ratios (mtDNA/nDNA) are represented as columns on the left hand y-axis and cell count (×10⁶) as a line plot on the right hand y-axis. VPA = sodium valproate. EtBr = ethidium bromide. AHS = p.A467T/p.K1191N compound heterozygote. (c) Ratio of mtDNA to nuclear DNA for each myotube cell line following 9 days of treatment with/without 300 μM didanosine (D) or stavudine (S) and additionally with/without 10mM VPA. MB= myoblasts, MT=myotubes, AHS = p.A467T/p.K1191N compound heterozygote. VPA = sodium valproate. Error bars = SD in all panels.