GABA TRANSAMINASE – CHARACTERISATION OF PHENOTYPE-GENOTYPE RELATIONSHIP

Prathibha Jose, Ana Alfirevich, Tim P Green P Green, Munir Pirmohamed Pirmohamed
Universityof Liverpool, Liverpool, United Kingdom

Vigabatrin is an antiepileptic drug that acts by irreversibly inhibiting GABA-transaminase (ABAT). Its use, however, has decreased since it was found to lead to visual field defects (VFD), in approximately 40% of patients. The activity of ABAT is known to vary but very little is known about how this relates to known polymorphisms in the gene encoding ABAT. Our objectives for this study were to (a) develop a robust assay for ABAT (b) identify common and putative functionally relevant SNPs in the ABAT gene from databases, develop genotyping assays for these SNPs and establish their frequency and (c) relate these polymorphisms to variation in activity of the enzyme.

ABAT is expressed in platelets and given their ready accessibility and ease of isolation; we used platelets to develop an ABAT assay. Platelet isolation was optimised using a density gradient method. ABAT activity was measured in the platelets by spectrophotometry. Putative functional SNPs in the ABAT gene were identified in silico using dbSNP, Ensemble, UCSC genome browser, PupaSNP and SNPper. The chosen SNPs (rs1731017, rs1641022, rs10261, rs1079348, rs1345300) were genotyped in a healthy population (n=139), using real time PCR and their allele frequencies estimated. The functional effect of these SNPs on ABAT activity was examined in 32 healthy multi-national individuals. Analysis was done by ANOVA. A p value <0.05 was considered significant.

Of the SNPs chosen, only rs1731017 is a non-synonymous SNP, while the others were in regulatory regions (rs10261, rs1345300) or affected potential splice sites (rs1641022, rs1079348). ABAT activity varied with rs1731017 which had a minor allele frequency of 0.4, as shown in figure 1. GG homozygotes at this position had significantly lower activity than AA or AG (p≤0.001, 0.01) individuals. No association was observed between the other genotyped SNPs and ABAT activity.

In conclusion, we have developed a novel assay using platelets to measure ABAT activity, and for the first time showed that variability in activity can be related to a non-synonymous SNP (rs1731017). The role of this SNP in vigabatrin-induced visual field constriction, and the effect it has on inhibition by vigabatrin requires further study.