| Despite major advances in the treatment of diseased organs, transplantation is still the treatment of choice for patients with end-stage solid organ failure as it can drastically improve life span and quality of life for these patients. Graft failure because of rejection, despite advances in transplantation medicine, is still the main cause of mortality and morbidity. Rejection is prevented by the use of a cocktail of immunosuppressants, with mycophenolic acid (MPA) being used most widely. Although these drugs are safer and more effective than previously used agents, variable therapeutic response and significant adverse side effects continue to present issues. MPA therapy, for instance, reduced graft loss due to acute rejection by about 50% compared to older drugs, but a substantial portion of patients develop severe adverse reactions that require discontinuation of MPA, leading to suboptimal long-term outcomes. This variability in MPA drug response and toxicities is likely influenced, in part, by inheritance.;MPA is an antimetabolite that exerts its immunosuppressive effects by uncompetitively inhibiting inosine monophosphate dehydrogenase (IMPDH), which catalyzes the rate-limiting step in de novo purine biosynthesis. As lymphocytes cannot proliferate or mount an immune response without this process, while other cell types are relatively unaffected, inhibition of IMPDH leads to specific immunosuppressive effects. There are 2 isoforms of IMPDH encoded by two differentially regulated genes, IMPDH1 and IMPDH2. Genetic polymorphisms in IMPDH1 have also been associated with acute rejection, possibly as a result of an alteration in IMPDH1 expression. However, even though it is clear that genetic variation can affect levels of both MPA and the 2 isoforms of IMPDH, the underlying causes for the variability in clinical outcomes after MPA therapy remains unknown.;Specific Aim 1 of this thesis is to systematically compile a comprehensive catalogue of genetic variation present in IMPDH1 and IMPDH2, the genes that encode MPA's drug target, and then to define the functions, if any, of these variants so that future research might conclusively define the extent to which genetic variation in these two genes contribute to variation in MPA response. IMPDH1 and IMPDH2 were resequenced using DNA from 288 individuals from 3 ethnic groups and functional genomic studies of the sequence variants observed was performed. 73 single nucleotide polymorphisms (SNPs) were observed in IMPDH1 with 59 being novel. 25 SNPs, 24 novel, were observed in IMPDH2. One novel non-synonymous IMPDH1 variant, Ser275Leu (rs72624960), had 10.2% of the wildtype activity as a result of accelerated protein degradation, and a novel IMPDH2 intron 1 SNP, (rs72639214), was found to be associated with decreased mRNA quantity. These results provide insight into the nature and extent of sequence variation in the IMPDH1 and IMPDH2 genes, and provide a foundation for future translational studies designed to correlate sequence variation in these genes with outcomes in patients treated with MPA.;The in-depth functional characterization performed as part of Specific Aim 1 demonstrated that IMPDH1 Leu275 not only had low activity, but could also aggregate. The objective of Specific Aim 2 is to characterize these Leu275 aggregates to explore the possible range of IMPDH1 and IMPDH2 function. While IMPDH1 and IMPDH2 are typically cytosolic proteins, IMPDH aggregation has been reported in the form of MPA-induced IMPDH2 aggregation, and there is indirect evidence that aggregation of blindness-associated mutations in IMPDH1 might explain part of their pathophysiology. In this study, Leu275 was found to spontaneously form long, linear aggregates in the absence of MPA. In addition, these large aggregates are different in morphology than those formed by blindness-associated IMPDH1 mutations, and appear to induce aggregation of wildtype IMPDH1. Better understanding of the biochemical and biophysical properties of this unique, naturally-occurring IMPDH1 variant may be helpful in the design of better IMPDH1 inhibitors in addition to providing insight into the possible range of IMPDH1 structure and function.;Shifting the focus from the identification and functional validation of genes known to impact MPA response, the objective of Specific Aim 3 is to use a model cell line system-based pharmacogenomics approach to identify genes outside the canonical MPA pharmacokinetics and pharmacodynamics pathways that may also contribute to the wide variation in MPA drug response. While a portion of this variation can be explained by known pharmacokinetic and pharmacodynamic factors, including genetic variation in MPA-metabolizing UDP-glucuronyltransferase isoforms and the MPA targets, IMPDH1 and IMPDH2, much of the variation in MPA response presently remains unexplained. A genome-wide association study between basal gene mRNA expression profiles and a MPA cytotoxicity phenotype in 271 human lymphoblastoid cell lines was performed to identify genes that might contribute to variation in MPA response. 41 gene expression probe sets, corresponding to 35 genes, were found to be associated with MPA cytotoxicity at p < 1x10-6. siRNA-mediated knockdown of these 35 genes in various other cell lines revealed four candidate genes, C17orf108, CYBRD1, NASP, and RRM2, whose mRNA expression levels correlated with MPA cytotoxicity in the manner predicted by the association analysis. This study identified novel candidate genes that may contribute to variation in response to MPA therapy and, as a result, may help make it possible to move toward more highly individualized MPA-based immunosuppressive therapy.;All together, the work presented in this thesis illustrates how MPA pharmacogenetics is evolving into MPA pharmacogenomics, and the results provide stepping stones that will eventually lead to the ultimate goal of validating these findings in transplant patients and translating the novel insights gained to the clinic. |
