Effects of germline DHFR and FPGS variants on methotrexate metabolism and relapse of leukemia

Abstract

Methotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10-8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10-9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms.

Graphical abstract

Figure 1.

GWASs identified DHFR and FPGS variants associated with MTX polyglutamates. (A) A total of 1 495 495 SNPs were tested for associations with _wmMTX_pg2 in 447 patients with ALL enrolled in the NOPHO ALL2008 protocol. The association P value is plotted on the y-axis against the respective chromosomal positions of each SNP on the x-axis. The red line indicates genome-wide significance (P = 5 × 10⁻⁸). DHFR loci are shown by the black arrow. (B) An allele at rs1382539 in DHFR was associated with increased levels of _wmMTX_pg2 in the discovery and validation cohorts. (C) Another GWAS was performed with _wmMTX_pg4 as the phenotype of interest, and FPGS loci (black arrow) reached genome-wide significance. (D) In the discovery cohort, _wmMTX_pg4 levels were significantly lower in patients who had the CT genotype at rs35789560 in FPGS than in those with the WT (CC) genotype. This trend was also confirmed in the validation cohort. (B,D) P values were estimated by linear regression, with adjustment for sex and risk group. Each box includes data between the 25th and 75th percentiles, with the horizontal line indicating the median. RN, rank normalized.

Figure 2.

Functional characterization of DHFR and FPGS. (A) The luciferase reporter assay confirmed that enhancer activity of DHFR was negatively influenced by the nucleotide substitution from G (WT) to A (rs1382539) in 293T cells. Bars represent means from 3 triplicate experiments, and T bars indicate standard deviations. (B) WT or variant FPGS R466C protein (5 ng/reaction) was studied by the polyglutamation assay, using the Phosphate Sensor Kit with MTX_pg1-6 as a substrate. The error bar shows the mean ± standard deviation. *P < .05 (Student t test).

Figure 3.

rs35789560 in FPGS is associated with elevated cumulative incidence of relapse. The cumulative incidence of relapse was higher in patients carrying the CT genotype (red) at rs35789560 in FPGS than in those with the CC genotype (blue). P value was calculated by Gray’s test.