Abstract

INTRODUCTION

Whole-genome studies have identified genetic variations associated with risk of complex diseases.^1–5 However, whole-genome pharmacogenetic studies are less common despite considerable speculation that such information will transform drug therapy.^6–10 Methotrexate is used to treat malignancies, including acute lymphoblastic leukemia (ALL), as well as autoimmune disorders.^11–15

The pharmacologic effects of methotrexate are well characterized, and its systemic exposure has been related to cure and toxicity in childhood ALL^16–19 and other diseases.^{20,21,19,22,23} The genetic origins of interindividual pharmacokinetic and pharmacodynamic variability of methotrexate remain poorly understood, with conflicting results on candidate pharmacogenetic predictors for methotrexate.^24,25 A genome-wide approach might identify new candidate genes whose polymorphisms forecast which patients might benefit from tailoring dosage on the basis of genomic variation. Herein, we performed a genome-wide association analysis of 434 patients with newly diagnosed ALL and surveyed 500,568 germline single-nucleotide polymorphisms (SNPs).

PATIENTS AND METHODS

RESULTS

In the discovery cohort, we estimated methotrexate clearance for 3,014 treatment courses given to 434 patients (Data Supplement Table 1). There was substantial interpatient variability in average (± standard deviation) methotrexate clearance (Fig 1), which differed among the 213 patients on the Total XIIIB (133 ± 25 mL/min/m²), the 114 patients on the Total XV low-risk (123 ± 28 mL/min/m²), and the 107 patients on the Total XV standard/high-risk (106 ± 22 mL/min/m²) treatment regimens (Data Supplement Fig 1; P < 2.2 × 10⁻¹⁶). The variability observed between treatment regimens is not surprising, given that dosage and the concomitant hydration and alkalinization (factors that can affect methotrexate clearance) differed (Data Supplement Table 2).²² Average methotrexate clearance differed by ancestral group (Data Supplement Fig 2; P = .005) with African American patients having the highest clearance. As previously noted,^19,22 methotrexate clearance decreased with increasing age (Data Supplement Fig 3; P = 1.1 × 10^-6). Thus, we adjusted all analyses for age, race, sex, and treatment regimen. Using a linear mixed effect model, the intrapatient variability in methotrexate clearance was 48%, while the interpatient variability was 52%.

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Fig 1.

Average methotrexate clearance and linkage disequilibrium structure of the SLCO1B1 single-nucleotide polymorphisms (SNPs) in 434 children with acute lymphoblastic leukemia. The average methotrexate clearance for each of the 434 children in the discovery cohort is plotted from the highest to lowest (gray shaded area), and each patient's genotype at each of the SLCO1B1 SNP loci listed is indicated (colored bars below the x axis). Patients homozygous for the major allele are coded in blue, heterozygotes are coded in green, and those homozygous for the minor allele are coded in yellow.

We tested the association between 398,699 SNPs and average methotrexate clearance in 434 children in the discovery cohort. The strongest association signal was on chromosome 12 (Fig 2). The two top SNPs associated with methotrexate clearance were annotated to the transporter gene, SLCO1B1/OATP1B1, located on chromosome 12 (Table 1; Data Supplement Tables 3-5). These two SLCO1B1 SNPs remained significant (rs11045879, P = 1.7 × 10⁻¹⁰ and rs4149081, P = 1.7 × 10⁻⁹) even after correction for multiple testing. These two SLCO1B1 SNPs were in complete LD (r² = 1) with each other (Fig 1) and were associated with methotrexate clearance across regimens (Fig 3).

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Fig 2.

Genome-wide P values showing the association of single-nucleotide polymorphisms (SNPs) with methotrexate clearance in the discovery cohort of 434 children with acute lymphoblastic leukemia. Shown is the distribution of P values (as –log₁₀ values) for the association of 398,699 SNP genotypes with methotrexate clearance in the discovery cohort. The P value of 1.7 × 10⁻¹⁰ is for the most significant association identified between a single SNP (located in SLCO1B1 on chromosome 12) and methotrexate clearance.

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Fig 3.

SLCO1B1 single-nucleotide polymorphism (SNP) rs11045879 is associated with methotrexate clearance and GI toxicity. (A) Association of SLCO1B1 SNP genotype (rs11045879) with methotrexate clearance in patients on Total XIIIB (TXIIIB), Total XV low-risk (TXVL), and Total XV standard/high-risk (TXVSH) treatment regimens. Numbers below each box plot indicate the sample size for each genotype. (B) Association of this SNP with GI toxicity. Logistic regression was used to analyze whether genotypes for SLCO1B1 SNPs were associated with toxicity as a dichotomous variable (yes or no) during the 2-week consolidation phase. The bar graphs display the percentage of patients (plotted on the y axis) per genotype (plotted on the x axis) who had grade 3 to 4 GI toxicity. Numbers above each bar represent the number of patients who had toxicity versus those who did not for the specified genotype. Similar relationships exist for SLCO1B1 SNP rs4149081 (data not shown).

We also used a linear mixed effects model to identify associations between SNP genotypes and methotrexate clearance; the results were similar to those assessing average clearance, with the same two top-ranked SNPs (P < 6.8 × 10⁻⁹) in SLCO1B1 (Data Supplement Table 4). Only 6.0% of the intracourse variability in clearance was accounted for by course number, even when accounting for the premethotrexate serum creatinine (Data Supplement Table 6).

Eight additional SLCO1B1 SNPs (Table 1) were associated with clearance (P < .05); these SNPs were not in LD (based on r²) with the top two SLCO1B1 SNPs (Fig 1) and were encompassed by multiple haplotype blocks (Data Supplement Figs 4A and ​and4B).4B). In a gene-level analysis, SLCO1B1 had a stronger association with methotrexate clearance (P < 1 × 10⁻⁶), with a higher r² and a lower Akaike information criterion, than any other gene (Data Supplement Tables 7 and 11).

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Fig 4.

Incidence of toxicity based on SLCO1B1 rs11045879 genotype. Cumulative incidence of the first episode of GI toxicity significantly differed (P = .024) by SLCO1B1 rs11045879 genotype during the continuation phase of treatment during the Total XIIIB treatment regimen. Cumulative incidences of GI toxicity were compared among genotypes using Gray's test with incorporation of competing risks.

We genotyped a validation cohort of 206 additional patients (Table 1). Seven of the 10 interrogated SLCO1B1 SNPs remained associated (P < .05) with methotrexate clearance (Table 1). Nine of the 10 SLCO1B1 SNPs were associated with clearance (P < .05) when the two cohorts (discovery and validation) were combined (Table 1), and the strongest signal remained in SLCO1B1 (Data Supplement Fig 5).

The percentage of methotrexate clearance variability that could be explained by SLCO1B1 genotypes compared with the variability explained by factors such as race, sex, age, treatment regimen, or serum creatinine was estimated in a multivariate analysis for both the discovery and validation cohorts (Table 2). In a stepwise linear regression analysis, five SNPs were retained in the model (Table 2), such that SLCO1B1 genetic variation accounted for 9.3% of the interpatient variability in the discovery cohort and 11.3% in the validation cohort. This superseded the proportion of variability contributed by the significant factors of race, serum creatinine, and sex; only treatment regimen accounted for a higher proportion (17.9%) of the variation in clearance (Table 2). Average serum creatinine concentrations explained 3.2% and 1.9% of the variability in methotrexate clearance in the discovery and validation cohorts, respectively; in a linear mixed effects model (Data Supplement Table 6), 2.4% of interpatient and 0.4% of intrapatient variability was accounted for by serum creatinine. Treatment regimen and SLCO1B1 SNP genotypes accounted for 18.6% and 11.0% of variability in clearance, respectively. Results were similar for the first cycle only of methotrexate (Data Supplement Table 8).

Table 2.

Multivariate Analysis of Factors Associated With Average Methotrexate Clearance in the Discovery and Validation Cohorts

Variables	Discovery Cohort (n = 434)				Validation Cohort With Serum Creatinine Data*(n = 125)				Validation Cohort† (n = 206)
	Coefficient‡	95% CI	P§	r2 (%)	Coefficient‡	95% CI	P§	r2 (%)	Coefficient‡	95% CI	P§	r2 (%)
Ancestry¶			.0007	2.1			.32	1.6			.021	4.1
African	Ref‡				Ref‡				Ref‡
European	−13.0	−19.8 to −6.3			−7.9	−18.5 to 2.8			−7.3	−16.7 to 2.0
Asian	−6.5	−25.0 to 12.0			2.4	−31.8 to 36.6			14.6	−4.3 to 33.6
Sex			.003	0.01			.02	3.3			.046	1.1
Female	Ref‡				Ref‡				Ref‡
Male	6.6	2.2 to 10.9			7.7	1.1 to 14.3			5.6	0.2 to 11.0
Serum creatinine, mg/dL‖	−60.2	−86.8 to −33.7	8.6 × 10−6	3.2	−33.6	−71.8 to 4.6	.09	1.9	NA		NA	NA
Age, years	0.39	−0.49 to 1.3	.39	0.1	0.98	−0.21 to 2.2	.10	1.7	−0.5	−1.1 to 0.1	.093	1.7
Treatment regimen			1.1 × 10−25	17.9			.00013	9.3			.001	2.8
Total XV low-risk	Ref‡				Ref‡				Ref‡
Total XV standard/high-risk	−15.9	−22.1 to −9.7			−15.0	−22.8 to −7.3			−7.2	−13.1 to −1.4
Total XIIIB	14.3	8.7 to 19.8			NA				NA
SLCO1B1 genotype#			2.3 × 10−10	9.3			.002	11.3			4.7 × 10−4	9.3
rs4149081 (A/G)	14.0	8.5 to 19.5			5.2	−3.3 to 13.8			6.6	0 to 13.2
rs10841753 (C/T)	−7.2	−13.6 to −0.8			0.07	−12.1 to 12.3			1.3	−6.8 to 9.4
rs11045818 (A/G)	−8.3	−19.4 to −2.9			−18.6	−34.7 to −2.4			−17.8	−29.7 to −5.8
rs2900476 (C/T)	3.4	−1.6 to 8.4			3.4	−4.2 to 11.0			2.9	−3.1 to 8.9
rs17328763 (C/T)	−8.4	−18.5 to 1.6			−6.0	−17.4 to 5.3			−6.2	−14.7 to 2.1

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Abbreviation: NA, Not applicable.

^*Results from a multivariate analysis in patients from the validation cohort who had evaluable serum creatinine levels available, n = 125. Eighty-one patients (of 206) had missing serum creatinine values.

^†Results from a multivariate analysis in the validation cohort without including serum creatinine as a covariate, n = 206.

^‡The coefficient or effect size represents the increase (positive value) or decrease (negative value) in methotrexate clearance (mL/min/m²) for each variable listed. For example in the discovery cohort, for a patient whose ancestry is 100% European, methotrexate clearance will decrease 13.0 mL/min/m² relative to someone whose ancestry is 100% African. Similarly, males will have methotrexate clearance that is 6.6 mL/min/m² higher than that of females. A patient with the G allele at rs4149081 would have a clearance of 14 mL/min/m² higher than a patient with the A allele. Ref denotes the baseline or reference variable in the multiple linear regression model.

^§The P value is derived from the χ² test comparing the deviance of the full model including all variables (eg, SLCO1B1 SNP genotypes, ancestry, sex, age) versus the model excluding each variable one at a time.

^¶Ancestry was determined using SNP genotypes from the 500-K mapping array set as described in Patients and Methods and the Data Supplement.

^‖The average serum creatinine concentration (mg/dL) was used as a measure for renal function and was included in the multivariate analysis.

^#Patients who carry the B allele (A/B) will have an increase or decrease in clearance based on the sign of the coefficient listed.

GI toxicity (grade 3 or 4 mucositis) and infection were the most common toxicities observed during the methotrexate-intensive consolidation and continuation phases of Total XIIIB therapy.²⁶ SNPs in SLCO1B1, rs11045879 T allele (OR, 16.4; 95% CI, 8.7 to 26.7; P = .004) and the G allele at rs4149081 (OR, 15.3; 95% CI, 7.9 to 24.6; P = .03; data not shown) were each associated with GI toxicity during consolidation (Fig 3). The same two SNPs were also associated with GI toxicity during the continuation phase (Fig 4). During the consolidation phase of Total XV, GI toxicity was less frequent (5% v 18% in Total XIIIB), and we found no association with SLCO1B1 SNP genotypes. Because all patients on Total XV had their methotrexate doses adjusted to achieve a common steady-state plasma concentration, relationships among toxicity and methotrexate exposure and SLCO1B1 SNPs may have been attenuated on this trial. We found no associations between SLCO1B1 SNP genotype and infectious toxicity in either trial (data not shown).

We also genotyped the nonsynonymous SLCO1B1 T521C (rs4149056) polymorphism in a subset of 489 patients; this subset included 387 patients from the discovery cohort and 102 patients from the validation cohort (Data Supplement Table 1). SLCO1B1 T521C was in high LD with the top SLCO1B1 SNPs rs11045879 (r² = 0.86, discovery cohort; r² = 0.89, validation cohort) and rs4149081 (r² = 0.86, discovery cohort; r² = 0.89, validation cohort). SLCO1B1 T521C was associated with methotrexate clearance in the discovery and the combined (discovery plus validation) patient cohorts (Data Supplement Table 9; P = 1.9 × 10⁻⁷ and P = 1.2 × 10⁻⁷, respectively). In addition, a marginal association (P = .07) was observed between this SNP genotype and the occurrence of GI toxicity during consolidation (Data Supplement Fig 6). In multivariate analysis, the T521C SNP was significantly associated with methotrexate clearance; however, when genotypes at T521C and at rs11045879 were allowed to compete, only the rs11045879 SNP remained in the model (Data Supplement Table 10).

DISCUSSION

Using a genome-wide interrogation of germline variation, we uncovered genetic variation that affects the disposition and effects of methotrexate in children with ALL. The strongest genetic variation associated with methotrexate pharmacokinetics and dynamics resided in a reasonable candidate gene, SLCO1B1, although this transporter was not previously studied as a candidate gene in clinical pharmacogenetic studies of methotrexate.^35–38

The SLCO1B1 locus maintained significance after adjusting for covariates such as age, race, serum creatinine, sex, and treatment regimen and was verified in an independent validation set. The top-ranked SNPs (Data Supplement Tables 3-5), rs11045879 and rs4149081, were located in the gene (SLCO1B1) encoding an organic anion transporter (OATP1B1). Eight additional SNPs in SLCO1B1 had P values less than .05 for their association with methotrexate clearance (Table 1). The fact that multiple SNPs in this locus, some of which were not in LD, were independently associated with methotrexate clearance provides further evidence of the functional importance of SLCO1B1.

SLCO1B1 is localized at the sinusoidal membrane of hepatocytes, and its transcript has been detected in enterocytes.^39–41 SLCO1B1 mediates uptake of substrates from sinusoidal blood, resulting in their net excretion from blood (likely via biliary excretion). SLCO1B1 has been shown to transport methotrexate in vitro,^42,43 as well as other compounds such as bilirubin, bile acids, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors,^44,45 benzylpenicillin, rifampicin, angiotensin-converting enzyme inhibitors, and the active metabolite of irinotecan, SN-38.^46,47

Previous studies have described functional consequences of nonsynonymous polymorphisms in SLCO1B1 in vitro^43,48 and in vivo (eg, statins).⁴⁹ The SLCO1B1 T521C SNP was in high LD (r² = 0.84) with the SNPs interrogated on the genome-wide arrays we used, and our independent genotyping confirmed an association of lower methotrexate clearance (higher systemic exposure) with the C allele. This is consistent with prior studies that demonstrate greater plasma exposure to multiple SLCO1B1 substrates in those carrying the 521C allele than those homozygous for the 521T allele.^49–51 Nonetheless, most of the SLCO1B1 SNPs that were associated with clearance (Table 1) were noncoding SNPs, not in LD with T521C, and in a multivariate model, SNPs other than T521C maintained an association with methotrexate clearance (Data Supplement Table 10). The fact that these SNPs resided throughout the gene suggest multiple mechanisms by which variations affect the function of SLCO1B1, perhaps by effects on transcription and post-transcriptional processing (eg, pre-mRNA splicing and mRNA translation). Moreover, noncoding SNPs may be in linkage with rare, nontyped coding SNPs. Thus it appears that the function of SLCO1B1 may be affected by several different polymorphisms.

SNPs in SLCO1B1 were associated not only with methotrexate clearance but also with GI toxicity (Fig 3) in Total XIIIB. Only 5% of patients in Total XV had toxicity, and this may in part be attributed to targeting of methotrexate to achieve a specific plasma concentration. The alleles that were associated with lower plasma exposure were always associated with increased GI toxicity. This finding is consistent with lower blood concentrations but higher GI tract or enterocyte concentrations of methotrexate being associated with GI toxicity. The fact that SLCO1B1 polymorphisms were not associated with the other common toxicity,²⁶ infection, is consistent with GI concentrations being a determinant of methotrexate GI toxicity^52,53 and being of relatively minor importance for infectious toxicity. Because 6-mercaptopurine was given during consolidation, toxicity (particularly infection) may have been partly due to 6-mercaptopurine. However, GI toxicity is more closely linked to methotrexate⁵³ than to thiopurines.^54,55

SLCO1B1 genotypes were associated with methotrexate clearance across three different treatment regimens (Fig 3) and were significant after adjusting for regimen differences and for sex, race, renal function, and age (Table 2 and Data Supplement Table 6). Moreover, clearance was measured on multiple occasions, and thus the average value of clearance is a composite that already accounts for intrapatient variability that may have been due to nongenetic causes. Accounting for each course independently in a mixed effects model, and accounting for variation in serum creatinine,⁵⁶ polymorphisms in SLCO1B1 remained significant predictors of interpatient variability in clearance. The fact that serum creatinine explained a small fraction (2.4%) of the interpatient variability in methotrexate clearance may be because serum creatinine was monitored before administration of the drug, and renal dysfunction was a contraindication to high-dose methotrexate. The proportion (9.3%) of interpatient variability in drug clearance attributable to germline variation in a single gene is on par with or greater than the variability accounted for by a single gene for other complex phenotypes.⁵⁷

Identifying patients at risk of low methotrexate clearance could be useful for monitoring and supportive care during high-dose methotrexate, particularly in settings in which rapid turnaround of plasma levels is not available. Moreover, identifying that SLCO1B1 accounts for a substantial degree of interpatient variability in clearance highlights that drug interactions are likely to occur if a potent SLCO1B1 substrate is given with methotrexate.

Methotrexate is metabolized to a 7-OH methotrexate metabolite.⁵⁸ This metabolite could interact with SLCO1B1. Other genetic variations may influence methotrexate clearance, such as in SLCO1A2 (Data Supplement Tables 3-5).⁵⁹ Moreover, there may be important insertion/deletion or germline copy number variations that were not adequately interrogated on the array we used.

Using a whole-genome approach, we identified a plausible candidate gene, SLCO1B1, that is associated with methotrexate pharmacokinetics and pharmacodynamics. This illustrates the proof of principle that genome-wide tools can lead to the discovery of important pharmacogenetic links between inherited genomic variation and drug response in humans.

Supplementary Material

Acknowledgment

Footnotes

REFERENCES