GWASs identified DHFR and FPGS variants associated with MTX polyglutamates. (A) A total of 1 495 495 SNPs were tested for associations with wmMTXpg2 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 wmMTXpg2 in the discovery and validation cohorts. (C) Another GWAS was performed with wmMTXpg4 as the phenotype of interest, and FPGS loci (black arrow) reached genome-wide significance. (D) In the discovery cohort, wmMTXpg4 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.