Genome-Wide Association Study for the Genetic Determinants of Thiopurine S-Methyltransferase Protein Expression in the Liver

HYPOTHESIS: Singe nucleotide polymorphisms will be associated with differential expression of Thiopurine S-Methyltransferase (TPMT) in the liver. METHODS: Normal human liver samples (n=288) were acquired from University of Minnesota Liver Tissue Cell Distribution System, the Cooperative Human Tissue Network, and XenoTech LCC (Lenexa, KS, USA). Absolute quantification of TPMT in liver microsomes was calculated using a DIA total protein approach (DIA-TPA). Human liver samples were genotyped using the Illumina Multi-Ethnic Global Assay (Illumina, Miami, USA). RESULTS: After accounting for population stratification, genome-wide association study (GWAS) analysis was conducted on 243 human liver samples. There were 30 genetic variants, all located on chromosome 6, that passed our genome-wide threshold (P < 5.0 x 10-8 ) after Bonferroni correction. Further GWAS analysis conditioning on rs1142345 (TPMT*3C/TPMT*3A), the strongest signal within the dataset and a known nonfunctional TPMT allele, showed no other independent signals. The presumed homozygous wild-type TPMT subjects had a 1.97-fold higher expression of TPMT than the TPMT*3A heterozygotes (0.1056 pmol/mg ± 0.0289 pmol/mg vs. 0.0536 pmol/mg ± 0.0143 pmol/mg, p-value: 8.4e-14 ). When comparing TPMT expression by ancestry, European ancestry donors (n= 214) had significantly higher expression than African ancestry donors (n= 34) when removing TPMT*3A carriers (0.1085 pmol/mg ± 0.0260 pmol/mg vs. 0.0860 pmol/mg ± 0.0416 pmol/mg, p-value: 0.004. CONCLUSION: For the first time, we quantified absolute TPMT protein expression in the liver using a liquid chromatography tandem mass spectrometry based DIA-TPA proteomics approach and conducted a GWAS to identify genetic variants affecting hepatic TPMT protein expression. The only independent genetic signal from the GWAS was from the TPMT*3A haplotype, which is comprised of two nonsynonymous SNPs rs1800460 and rs1142345. Previous in vitrostudies investigating the effect of common polymorphisms on TPMT expression in COS-1 cells demonstrated that TPMT*3A is almost a complete loss of detectable TPMT due to its rapid degradation rate.1 This is in line with our expression data, where heterozygous carriers of TPMT*3A had about half the amount of TPMT as wild-type subjects. We also discovered that donors with African ancestry had significantly less TPMT expression than donors with European ancestry after removing samples that had known TPMT polymorphisms (TPMT*3A/*3B/*3C). A previous study reported patients of Afro-Caribbean ancestry had significantly lower TPMT activity than those of European or South Asian descent.2 Future studies need to be conducted to determine if the difference in TPMT expression by ancestry is due to genetic polymorphisms or other factors. REFERENCES: 1. Ujiie, S., Sasaki, T., Mizugaki, M., Ishikawa, M. & Hiratsuka, M. Functional characterization of 23 allelic variants of thiopurine S-methyltransferase gene (TPMT*2 - *24). Pharmacogenet. Genomics 18, 887-893 (2008). 2. Cooper, S. C., Ford, L. T., Berg, J. D. & Lewis, M. J. Ethnic variation of thiopurine S-methyltransferase activity: a large, prospective population study. Pharmacogenomics 9, 303-309 (2008). © FASEB.