A GAG trinucleotide-repeat polymorphism in the gene for glutathione biosynthetic enzyme, GCLC, affects gene expression through translation

A guanine-adenine-guanine (GAG) repeat polymorphism with 5 different alleles (4, 7, 8, 9, and 10 repeats) in the 5′ untranslated region (UTR) of GCLC has been associated with altered GCL activity and glutathione (GSH) levels. We investigated whether this polymorphism affects either transcription or translation using luciferase reporter constructs containing variant GCLC 5′ UTRs. Higher luciferase activity was observed in HepG2 and human embryonic kidney 293 (HEK293) cells transfected with constructs containing either 8 or 9 repeats than in constructs containing 4, 7, or 10 repeats (P<0.05). In cell-free lysates, GAG repeat number had no effect on luciferase mRNA yield. In vitro translation of mRNAs from luciferase constructs resulted in differences similar to those found in cell cultures (P<0.05). A similar association of GAG repeat with GCLC phenotype was observed in vivo in healthy adults, as individuals with GAG-7/7 genotype had lower GCL activity and GSH levels in lymphocytes compared to those with GAG-9/9 (P<0.05). Higher GCL activity and GSH levels observed in red blood cells (RBCs) from individuals with GAG-7/7 compared to GAG-9/9 are likely due to differences in GCL regulation in RBCs. Altogether, these results suggest that GAG polymorphism affects GCLC expression via translation, and thus may be associated with altered risk for GSHrelated diseases and toxicities.