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Clinical Trial
. 2014 Mar;15(4):433-47.
doi: 10.2217/pgs.13.226.

Thiopurine pharmacogenomics: association of SNPs with clinical response and functional validation of candidate genes

Alice Matimba  1 , Fang Li, Alina Livshits, Cher S Cartwright, Stephen Scully, Brooke L Fridley, Gregory Jenkins, Anthony Batzler, Liewei Wang, Richard Weinshilboum, Lynne Lennard
Affiliations
Clinical Trial

Thiopurine pharmacogenomics: association of SNPs with clinical response and functional validation of candidate genes

Alice Matimba et al. Pharmacogenomics. 2014 Mar.

Abstract

Aim: We investigated candidate genes associated with thiopurine metabolism and clinical response in childhood acute lymphoblastic leukemia.

Materials & methods: We performed genome-wide SNP association studies of 6-thioguanine and 6-mercaptopurine cytotoxicity using lymphoblastoid cell lines. We then genotyped the top SNPs associated with lymphoblastoid cell line cytotoxicity, together with tagSNPs for genes in the 'thiopurine pathway' (686 total SNPs), in DNA from 589 Caucasian UK ALL97 patients. Functional validation studies were performed by siRNA knockdown in cancer cell lines.

Results: SNPs in the thiopurine pathway genes ABCC4, ABCC5, IMPDH1, ITPA, SLC28A3 and XDH, and SNPs located within or near ATP6AP2, FRMD4B, GNG2, KCNMA1 and NME1, were associated with clinical response and measures of thiopurine metabolism. Functional validation showed shifts in cytotoxicity for these genes.

Conclusion: The clinical response to thiopurines may be regulated by variation in known thiopurine pathway genes and additional novel genes outside of the thiopurine pathway.

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Figures

Figure 1
Figure 1. Genome-wide association studies for SNP associations with thiopurine IC50 values in lymphoblastoid cell lines
Manhattan plots for lymphoblastoid cell line GWAS for IC50 values for (A) 6-TG and (B) 6-MP. (C) Overlap of top SNPs for 6-TG and 6-MP with p-values of <10−4. The blue line denotes p < 1 × 10−4; SNPs with an association <1 × 10−4 were considered for further study. Red dots denote thiopurine pathway SNPs. 6-MP: 6-mercaptopurine; 6-TG: 6-thioguanine; GWAS: Genome-wide association study.
Figure 2
Figure 2. Selection of SNPs for genotyping samples from acute lymphoblastic leukemia patients
(A) Top SNPs associated with 6-TG and 6-MP IC50 values in LCLs. Additional SNPs in a region of chromosome 17 were selected from the CEU Hapmap2 data over approximately 380 kb extending across a SNP ‘signal’ on chromosome 17 that was observed during the LCL GWAS for 6-MP IC50 value. (B) Thiopurine pathway SNPs. These SNPs were in or near genes (<200 kb) encoding proteins known to be involved in the thiopurine pathway as defined by PharmGKB. 6-MP: 6-mercaptopurine; 6-TG: 6-thioguanine; GWAS: Genome-wide association study; LCL: Lymphoblastoid cell lines.
Figure 3
Figure 3. Functional validation of candidate genes in cancer cell lines
(A) ‘Nonthiopurine pathway’ genes. (B) Thiopurine pathway genes. Cytotoxicity curves after gene knockdown followed by drug treatment are shown for top candidate genes that showed consistent ‘shifts’ in at least two cell lines. In this screening test best fit parameters of a four parameter nonlinear regression model were calculated and statistical significance was calculated. p-values are indicated if they were <0.05. In cases where the curves did not fit the model (GNG2 and KCNMA1 in U87M cells and NME1 in U251 cells, all for 6-TG cytotoxicity) the curves could not be compared therefore p-values could not be calculated although a clear curve shift can be observed. 6-MP: 6-mercaptopurine; 6-TG: 6-thioguanine.
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References

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Website

    1. PharmGKB. www.pharmgkb.org.

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