Functional analysis of the indoleamine 2,3 dioxygenase genetic variants

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Indoleamine 2, 3-dioxygenase (IDO) is a rate limiting enzyme in the catabolism of the essential amino acid, tryptophan. Degradation of tryptophan is plays an important role in the development of immune tolerance in several tissues, including cancer. Consequently, IDO inhibitors are now being tested for anticancer effects. Many clinical studies have demonstrated that interindividual IDO activity is highly variable. These include base line activity as well as therapy induced changes in IDO activity. This variability may contribute to the etiology of cancer. It may also cause variability in the efficacy of anticancer therapeutics.

We have previously shown that an exon 3 germline single nucleotide polymorphism (SNP) in the human IDO gene impairs the activity of the IDO protein. In the current study, we tested the functionality of a germline 9 bp deletion in the coding region of the human INDO gene. This allele was detected in 1 of 48 African American samples. The deletion of a 9 bp fragment in exon 7 replaces Ala-Leu-Leu-Glu with an Asp. We expressed the wild-type and variant cDNAs in human embryonic kidney 293 cells (HEK293) and determined IDO enzyme activity by measuring tryptophan and kynurenine concentrations in the cultured media 48 hours after transient transfection. The exon 3 and exon 7 variants cDNA had >90% reduction in activity compared to the wild-type. These variants did not appear to affect the steady state mRNA concentration following a transient transfection; however, even though the wild-type protein was easily detectable by western blot, the exon 3 and 9 bp deletion variant proteins were not detectable. We are currently studying the effect of these variants on the stability of the IDO protein. This data, together with our previous studies on the exon 3 variation, suggest that ~4% of the African American population are carriers of a nonfunctional INDO alleles.

We conclude that this 9 base pair deletion in the human germline DNA causes a nonfunctional IDO protein. This may have important implications for understanding the local immune response in tumors.