Rare versus common variants in pharmacogenetics: SLCO1B1 variation and methotrexate disposition

Abstract

Methotrexate is used to treat autoimmune diseases and malignancies, including acute lymphoblastic leukemia (ALL). Inter-individual variation in clearance of methotrexate results in heterogeneous systemic exposure, clinical efficacy, and toxicity. In a genome-wide association study of children with ALL, we identified SLCO1B1 as harboring multiple common polymorphisms associated with methotrexate clearance. The extent of influence of rare versus common variants on pharmacogenomic phenotypes remains largely unexplored. We tested the hypothesis that rare variants in SLCO1B1 could affect methotrexate clearance and compared the influence of common versus rare variants in addition to clinical covariates on clearance. From deep resequencing of SLCO1B1 exons in 699 children, we identified 93 SNPs, 15 of which were non-synonymous (NS). Three of these NS SNPs were common, with a minor allele frequency (MAF) >5%, one had low frequency (MAF 1%-5%), and 11 were rare (MAF <1%). NS SNPs (common or rare) predicted to be functionally damaging were more likely to be found among patients with the lowest methotrexate clearance than patients with high clearance. We verified lower function in vitro of four SLCO1B1 haplotypes that were associated with reduced methotrexate clearance. In a multivariate stepwise regression analysis adjusting for other genetic and non-genetic covariates, SLCO1B1 variants accounted for 10.7% of the population variability in clearance. Of that variability, common NS variants accounted for the majority, but rare damaging NS variants constituted 17.8% of SLCO1B1's effects (1.9% of total variation) and had larger effect sizes than common NS variants. Our results show that rare variants are likely to have an important effect on pharmacogenetic phenotypes.

Figure 1.

Methotrexate clearance and presence of non-synonymous (NS) SNPs among outliers. (A) The average methotrexate clearance (adjusted for age, gender, ancestry, and treatment arm) is depicted for the lowest, middle, and highest tenth percentile groups (n = 70 patients per decile). The number of patients harboring NS SNPs did not differ among clearance decile groups (p = 0.3, Cochran-Armitage Trend Test) (B), whereas the number of patients harboring damaging NS SNPs differed by clearance decile group (p = 2.3 × 10⁻⁸, Cochran-Armitage Trend Test) (C), as did the number of patients harboring rare (MAF < 1%) damaging NS SNPs (p = 0.026, Cochran-Armitage Trend Test) (D).

Figure 2.

Patients with rare damaging NS SNPs have reduced methotrexate clearance. (A) Among the 542 patients who did not carry the variant C allele at rs4149056, median adjusted methotrexate clearance is lower in patients with rare damaging NS SNPs (n = 14) than those having no rare NS SNPs (n = 508, p = 8 × 10⁻⁴) or having rare tolerated NS SNPs (n = 20, p = 0.001, Wilcoxon rank-sum test). (B) SNPs with smaller MAFs have larger effect sizes. (Triangles) NS damaging SNPs; (squares) NS tolerated SNPs; (circles) other SNP types. Shapes filled in black indicate p < 0.05; others do not reach statistical significance. Effect size indicates the change in adjusted methotrexate clearance (in milliliters per minute per meter squared) attributable to each copy of the minor allele based on the general linear model, and error bars represent standard error within the model. Shading indicates area under a linear regression fit line for negative effect sizes (pink, lower clearance) or positive effect sizes (green, higher clearance) versus MAF.

Figure 3.

One-third of inter-patient variability in methotrexate clearance can be explained by clinical covariates and SLCO1B1 SNP genotypes. Forward variable selection was used to select covariates, including SNPs in each category (common NS, rare NS, synonymous [syn], 5′ upstream, or intronic) as input based on each covariate's increase in R² for explaining variability in clearance (see Methods). SNPs included in this model are listed in Supplemental Table 2. Of the variation in clearance explained by SLCO1B1 polymorphisms, the percent apportioned to each category of SNP (common NS, rare NS, synonymous, 5′ upstream, or intronic) is illustrated in the pie chart at right.

Figure 4.

SLCO1B1 haplotypes affect average adjusted methotrexate clearance. (A) Haplotypes are based on 15 NS SNPs. (Red boxes with D) Damaging SNPs; (T) tolerated SNPs; (blue boxes) the ancestral allele; (orange boxes) the variant allele. The univariate P-value for the association with methotrexate clearance is indicated after adjustment for age, gender, ancestry, and treatment arm. (Yellow P-values) The haplotype confers significantly lower methotrexate clearance; (green P-values) significantly higher methotrexate clearance; (purple frequencies) singletons; (pink) those that are rare (frequency < 1%). (B) Diplotypes (x-axis) are sorted by median adjusted methotrexate clearance (y-axis). (Yellow haplotypes) Associated with lower methotrexate clearance (p < 0.05); (green haplotypes) associated with higher methotrexate clearance (p < 0.05). (Horizontal line) Median; (boxes) 25th to 75th percentiles; (whiskers) non-outlier range; (circles) outliers. (C) Methotrexate (mtx) uptake by HEK293 cells stably transfected with SLCO1B1 coding variants, plotted as percentage of SLCO1B1*1a-expressing cells, which averaged 30.8 pmol/mg. (Empty) HEK293 cells expressing the empty vector; (asterisks) transport that differs significantly (at p < 0.05, Student's t-test) compared with the SLCO1B1*1a haplotype.

Figure 5.

Extracellular, transmembrane, and intracellular cytoplasmic domains predicted for SLCO1B1, indicating all 15 non-synonymous SNPs. Shown are SNPs associated with lower methotrexate clearance (p < 0.05) (yellow), higher clearance (green), or no association with clearance (black).