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Valproic acid (VPA) is a widely used antiepileptic drug and first-line treatment in bipolar disorder, although the mechanisms underlying its therapeutic effects are largely unknown. Recently, the recognition of VPA as an epigenetic drug offers new opportunities for understanding its therapeutic actions. In a rat serotonergic cell line (RN46A) we observed that VPA exposure has a strong upregulatory effect on the gene for sepiapterin reductase (SPR), a key enzyme involved in the tetrahydrobiopterin (BH4) synthetic pathway. BH4 is an essential cofactor in the biosynthesis of neurotransmitters like serotonin, dopamine and noradrenalin, and the BH4 pathway may thus be important in mood biology. Using real-time quantitative PCR we show that VPA, at therapeutically relevant doses, increases the expression of the Spr gene by about 8-fold in RN46A cells. In addition, Spr protein levels in VPA-exposed cells were elevated, as were the intracellular BH4 levels. HDAC inhibitors (HDACI) trichostatin A and sodium butyrate also upregulated Spr, but this was not observed using the VPA-analogue valpromide, which lacks HDAC inhibitory activity. Further examination of this effect revealed that exposure to VPA increased the acetylated histone mark H3K9/K14ac at the Spr promoter. The DNMT inhibitor 5'aza-dC also upregulated Spr by over 8-fold. However, DNA methylation status across the Spr promoter did not change in response to VPA. The BH4 pathway is fundamental to the regulation of neurotransmitters relevant to mood disorders, and this epigenetic effect of VPA at the Spr promoter may represent a novel mechanism through which VPA achieves its therapeutic action.

Preferential conversion of azathioprine or 6-mercaptopurine into methylated metabolites is a major cause of thiopurine resistance. To seek potentially Mendelian causes of thiopurine hypermethylation, we recruited 12 individuals who exhibited extreme therapeutic resistance while taking azathioprine or 6-mercaptopurine and performed whole-exome sequencing (WES) and copy-number variant analysis by array-based comparative genomic hybridisation (aCGH). Exome-wide variant filtering highlighted four genes potentially associated with thiopurine metabolism (ENOSF1 and NFS1), transport (SLC17A4) or therapeutic action (RCC2). However, variants of each gene were found only in two or three patients, and it is unclear whether these genes could influence thiopurine hypermethylation. Analysis by aCGH did not identify any unusual or pathogenic copy-number variants. This suggests that if causative mutations for the hypermethylation phenotype exist they may be heterogeneous, occurring in several different genes, or they may lie within regulatory regions not captured by WES. Alternatively, hypermethylation may arise from the involvement of multiple genes with small effects. To test this hypothesis would require recruitment of large patient samples and application of genome-wide association studies.