Discipline: Biological Sciences
Monica Saha - University of Washington / University of Kansas
Co-Author(s): Monica Saha, Ron Hause, Jen Milbank, Riza Daza, Lea Starita, and Jay Shendure, The University of Washington
Thiopurine drugs, such as 6 MP and azathioprine, are used to treat patients with acute lymphoblastic leukemia, autoimmune disease, irritable bowel syndrome, and organ transplant recipients. These agents are metabolized by S-methylation catalyzed by thiopurine methyltransferase (TPMT), in part, inactivating the drug. TPMT harbors considerable interindividual genetic variability resulting in variation in therapeutic efficiency. Patients with decreased catalytic activity and decreased expression of TPMT are at risk of myelosuppression. The first TPMT variant allele identified, TPMT*2, results in an ALa80Pro substitution that causes decreased function of TPMT. To date, 38 common, non-synonymous variants have been identified in TPMT in humans. Unfortunately, this represents a small population of all possible variants associated with this gene. We do not know the current phenotype for all possible mutations. We hypothesize we can evaluate all possible single residue substitutions in TPMT through Inverse PCR to create a site saturation mutagenesis. We pooled our library in a equimolar ratio for transformation and verified with Sanger Sequencing we had point mutations in the Nth position (85% success rate). Furthermore, we aimed to develop yeast growth assays to test the effects of mutations in TPMT on TPMT protein stability and organismal tolerance to 6-MP. To develop these assays, we first used wild-type TPMT and TPMT*3A, a known clinical variant resulting in non-functional TPMT, to derive a positive and negative effect magnitude for these assays. We successfully established baseline results to prove our assays are viable. TPMT*WT was able to express in SC-leu media and grow in the presence of 6MP, while TPMT*3A could not express in SCleu nor survive with 6MP treatment. We used an empty host plasmid, pRS416 as our control. Furthermore, we are in the process of barcoding our mutant library and will phenotype the library with both of these assays.
Funder Acknowledgement(s): This research was funded by the UW Genome Sciences: Summer Research Program and NRHGI training grant 5T32HG000035-19.
Faculty Advisor: Ron Hause,