Antagonism of B cell enhancer networks by STAT5 drives leukemia and poor patient survival.

The transcription factor STAT5 has a critical role in B cell acute lymphoblastic leukemia (B-ALL). How STAT5 mediates this effect is unclear. Here we found that activation of STAT5 worked together with defects in signaling components of the precursor to the B cell antigen receptor (pre-BCR), including defects in BLNK, BTK, PKCß, NF-κB1 and IKAROS, to initiate B-ALL. STAT5 antagonized the transcription factors NF-κB and IKAROS by opposing regulation of shared target genes. Super-enhancers showed enrichment for STAT5 binding and were associated with an opposing network of transcription factors, including PAX5, EBF1, PU.1, IRF4 and IKAROS. Patients with a high ratio of active STAT5 to NF-κB or IKAROS had more-aggressive disease. Our studies indicate that an imbalance of two opposing transcriptional programs drives B-ALL and suggest that restoring the balance of these pathways might inhibit B-ALL.

The Pharmacogenomics Global Research Network Implementation Working Group: global collaboration to advance pharmacogenetic implementation.

Pharmacogenetics promises to optimize treatment-related outcomes by informing optimal drug selection and dosing based on an individual's genotype in conjunction with other important clinical factors. Despite significant evidence of genetic associations with drug response, pharmacogenetic testing has not been widely implemented into clinical practice. Among the barriers to broad implementation are limited guidance for how to successfully integrate testing into clinical workflows and limited data on outcomes with pharmacogenetic implementation in clinical practice. The Pharmacogenomics Global Research Network Implementation Working Group seeks to engage institutions globally that have implemented pharmacogenetic testing into clinical practice or are in the process or planning stages of implementing testing to collectively disseminate data on implementation strategies, metrics, and health-related outcomes with the use of genotype-guided drug therapy to ultimately help advance pharmacogenetic implementation. This paper describes the goals, structure, and initial projects of the group in addition to implementation priorities across sites and future collaborative opportunities.

Toxicity profile of high-dose methotrexate in young children with central nervous system tumors.

High-dose methotrexate (HD-MTX) is used in the treatment of children with central nervous system (CNS) tumors; however, toxicity information is limited. We characterized toxicities following 102 administrations of HD-MTX (4.6-13.5 g/m2) infused over 4 or 24 h in 38 children with a CNS tumor before 6 years of age (2010-2020). Delayed clearance of methotrexate occurred following 24% of infusions. Common Terminology Criteria for Adverse Events v5 grade 2-3 mucositis was observed in 47% of individuals, Grade 4 neutropenia in 76%, and grade 3-4 thrombocytopenia in 58%. No neurotoxicity was observed. HD-MTX can be safely used with supportive care and monitoring.

Gene-Based Dose Optimization in Children.

Pharmacogenetics is a key component of precision medicine. Genetic variation in drug metabolism enzymes can lead to variable exposure to drugs and metabolites, potentially leading to inefficacy and drug toxicity. Although the evidence for pharmacogenetic associations in children is not as extensive as for adults, there are several drugs across diverse therapeutic areas with robust pediatric data indicating important, and relatively common, drug-gene interactions. Guidelines to assist gene-based dose optimization are available for codeine, thiopurine drugs, selective serotonin reuptake inhibitors, atomoxetine, tacrolimus, and voriconazole. For each of these drugs, there is an opportunity to clinically implement precision medicine approaches with children for whom genetic test results are known or are obtained at the time of prescribing. For many more drugs that are commonly used in pediatric patients, additional investigation is needed to determine the genetic factors influencing appropriate dose.

Insights from a pharmacometric analysis of HDMTX in adults with cancer: Clinically relevant covariates for application in precision dosing.

AIMS: High-dose methotrexate (HDMTX) is an essential part of the treatment of several adult and paediatric malignancies. Despite meticulous supportive care during HDMTX administration, severe toxicities, including acute kidney injury (AKI), may occur contributing to patient morbidity. Population pharmacokinetics provide a powerful tool to predict time to clear HDMTX and adjust subsequent doses. We sought to develop and validate pharmacokinetic models for HDMTX in adults with diverse malignancies and to relate systemic exposure with the occurrence of severe toxicity. METHODS: Anonymized, de-identified data were provided from 101 US oncology practices that participate in the Guardian Research Network, a non-profit clinical research consortium. Modelled variables included clinical, laboratory, demographic and pharmacological data. Population pharmacokinetic analysis was performed by means of nonlinear mixed effects modelling using MonolixSuite. RESULTS: A total of 693 HDMTX courses from 243 adults were analysed, of which 62 courses (8.8%) were associated with stage 2/3 acute kidney injury (43 stage 2, 19 stage 3). A three-compartment model adequately fitted the data. Time-dependent serum creatinine, baseline serum albumin and allometrically scaled bodyweight were clinically significant covariates related to methotrexate clearance. External evaluation confirmed a satisfactory predictive performance of the model in adults receiving HDMTX. Dose-normalized methotrexate concentration at 24 and 48 hours correlated with AKI incidence. CONCLUSION: We developed a population pharmacometric model that considers weight, albumin and time-dependent creatinine that can be used to guide supportive care in adult patients with delayed HDMTX elimination.

Perspectives from the Society for Pediatric Research: pharmacogenetics for pediatricians.

This review evaluates the pediatric evidence for pharmacogenetic associations for drugs that are commonly prescribed by or encountered by pediatric clinicians across multiple subspecialties, organized from most to least pediatric evidence. We begin with the pharmacogenetic research that led to the warning of increased risk of death in certain pediatric populations ("ultrarapid metabolizers") who are prescribed codeine after tonsillectomy or adenoidectomy. We review the evidence for genetic testing for thiopurine metabolism, which has become routine in multiple pediatric subspecialties. We discuss the pharmacogenetic research in proton pump inhibitors, for which clinical guidelines have recently been made available. With an increase in the prevalence of behavioral health disorders including attention deficit hyperactivity disorder (ADHD), we review the pharmacogenetic literature on selective serotonin reuptake inhibitors, selective norepinephrine reuptake inhibitors, and ADHD medications. We will conclude this section on the current pharmacogenetic data on ondansetron. We also provide our perspective on how to integrate the current research on pharmacogenetics into clinical care and what further research is needed. We discuss how institutions are managing pharmacogenetic test results and implementing them clinically, and how the electronic health record can be leveraged to ensure testing results are available and taken into consideration when prescribing medications. IMPACT: While many reviews of pharmacogenetics literature are available, there are few focused on pediatrics. Pediatricians across subspecialties will become more comfortable with pharmacogenetics terminology, know resources they can use to help inform their prescribing habits for drugs with known pharmacogenetic associations, and understand the limitations of testing and where further research is needed.

Clinical implementation of pharmacogenetics and model-informed precision dosing to improve patient care.

Providing maximal therapeutic efficacy without toxicity is a universal goal of rational drug therapy. However, substantial between-patient variability in drug response often impedes such successful treatments and brings the necessity of tailoring drug dose to individual needs for more precise therapy. In many cases plenty of patient characteristics, such as body size, genetic makeup and environmental factors, need to be taken into consideration to find the optimal dose in clinical practice. A pharmacokinetics and pharmacodynamics (PK/PD) model-informed approach offers integration of various patient information to provide an expectation of drug response and derive practical dose estimates to support clinicians' dosing decisions. Such an approach was pioneered in the late 1970s, but its broad clinical acceptance and implementation have been hampered by the lack of widespread computer technology, including user-friendly software tools. This has significantly changed in recent years. With the advent of electronic health records (EHRs) and the ubiquity of user-friendly software tools, we now experience a convergence of clinical information, pharmacogenetics, systems pharmacology and pharmacometrics, and technology. Advanced pharmacometrics research is now more appliable and implementable to improve health care. This article presents examples of successful development and implementation of pharmacogenetics-guided and PK/PD model-informed decision support to facilitate precision dosing, including the development of an EHR-embedded decision support tool. Through the integration of clinical decision support tools in EHRs, clinical pharmacometrics support can be brought directly to the clinical team and the bedside.

Multisite investigation of strategies for the clinical implementation of pre-emptive pharmacogenetic testing.

PURPOSE: The increased availability of clinical pharmacogenetic (PGx) guidelines and decreasing costs for genetic testing have slowly led to increased utilization of PGx testing in clinical practice. Pre-emptive PGx testing, where testing is performed in advance of drug prescribing, is one means to ensure results are available at the time of prescribing decisions. However, the most efficient and effective methods to clinically implement this strategy remain unclear. METHODS: In this report, we compare and contrast implementation strategies for pre-emptive PGx testing by 15 early-adopter institutions. We surveyed these groups, collecting data on testing approaches, team composition, and workflow dynamics, in addition to estimated third-party reimbursement rates. RESULTS: We found that while pre-emptive PGx testing models varied across sites, institutions shared several commonalities, including methods to identify patients eligible for testing, involvement of a precision medicine clinical team in program leadership, and the implementation of pharmacogenes with Clinical Pharmacogenetics Implementation Consortium guidelines available. Finally, while reimbursement rate data were difficult to obtain, the data available suggested that reimbursement rates for pre-emptive PGx testing remain low. CONCLUSION: These findings should inform the establishment of future implementation efforts at institutions considering a pre-emptive PGx testing program.

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1-RAG-2 recombinase and was restored in Rag1-/- developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

Novel pharmacological treatments for generalized anxiety disorder: Pediatric considerations.

BACKGROUND: Pediatric anxiety disorders such as generalized anxiety disorder (GAD) are common, impairing, and often undertreated. Moreover, many youth do not respond to standard, evidence-based psychosocial or psychopharmacologic treatment. An increased understanding of the gamma-aminobutyric acid (GABA) and glutamate neurotransmitter systems has created opportunities for novel intervention development for pediatric GAD. METHODS: This narrative review examines potential candidates for pediatric GAD: eszopiclone, riluzole, eglumegad (LY354740), pimavanserin, agomelatine. RESULTS: The pharmacology, preclinical data, clinical trial findings and known side effects of eszopiclone, riluzole, eglumegad (LY354740), pimavanserin, agomelatine, are reviewed, particularly with regard to their potential therapeutic relevance to pediatric GAD. CONCLUSION: Notwithstanding numerous challenges, some of these agents represent potential candidate drugs for pediatric GAD. Further treatment development studies of agomelatine, eszopiclone, pimavanserin and riluzole for pediatric GAD also have the prospect of informing the understanding of GABAergic and glutamatergic function across development.

Asparaginase formulation impacts hypertriglyceridemia during therapy for acute lymphoblastic leukemia.

BACKGROUND: Glucocorticoids and asparaginase, used to treat acute lymphoblastic leukemia (ALL), can cause hypertriglyceridemia. We compared triglyceride levels, risk factors, and associated toxicities in two ALL trials at St. Jude Children's Research Hospital with identical glucocorticoid regimens, but different asparaginase formulations. In Total XV (TXV), native Escherichia coli l-asparaginase was front-line therapy versus the pegylated formulation (PEG-asparaginase) in Total XVI (TXVI). PROCEDURE: Patients enrolled on TXV (n = 498) and TXVI (n = 598) were assigned to low-risk (LR) or standard/high-risk (SHR) treatment arms (ClinicalTrials.gov identifiers: NCT00137111 and NCT00549848). Triglycerides were measured four times and were evaluable in 925 patients (TXV: n = 362; TXVI: n = 563). The genetic contribution was assessed using a triglyceride polygenic risk score (triglyceride-PRS). Osteonecrosis, thrombosis, and pancreatitis were prospectively graded. RESULTS: The largest increase in triglycerides occurred in TXVI SHR patients treated with dexamethasone and PEG-asparaginase (4.5-fold increase; P <1 × 10-15 ). SHR patients treated with PEG-asparaginase (TXVI) had more severe hypertriglyceridemia (>1000 mg/dL) compared to native l-asparaginase (TXV): 10.5% versus 5.5%, respectively (P = .007). At week 7, triglycerides did not increase with dexamethasone treatment alone (LR patients) but did increase with dexamethasone plus asparaginase (SHR patients). The variability in triglycerides explained by the triglyceride-PRS was highest at baseline and declined with therapy. Hypertriglyceridemia was associated with osteonecrosis (P = .0006) and thrombosis (P = .005), but not pancreatitis (P = .4). CONCLUSION: Triglycerides were affected more by PEG-asparaginase than native l-asparaginase, by asparaginase more than dexamethasone, and by drug effects more than genetics. It is not clear whether triglycerides contribute to thrombosis and osteonecrosis or are biomarkers of the toxicities.

Multi-site investigation of strategies for the clinical implementation of CYP2D6 genotyping to guide drug prescribing.

PURPOSE: A number of institutions have clinically implemented CYP2D6 genotyping to guide drug prescribing. We compared implementation strategies of early adopters of CYP2D6 testing, barriers faced by both early adopters and institutions in the process of implementing CYP2D6 testing, and approaches taken to overcome these barriers. METHODS: We surveyed eight early adopters of CYP2D6 genotyping and eight institutions in the process of adoption. Data were collected on testing approaches, return of results procedures, applications of genotype results, challenges faced, and lessons learned. RESULTS: Among early adopters, CYP2D6 testing was most commonly ordered to assist with opioid and antidepressant prescribing. Key differences among programs included test ordering and genotyping approaches, result reporting, and clinical decision support. However, all sites tested for copy-number variation and nine common variants, and reported results in the medical record. Most sites provided automatic consultation and had designated personnel to assist with genotype-informed therapy recommendations. Primary challenges were related to stakeholder support, CYP2D6 gene complexity, phenotype assignment, and sustainability. CONCLUSION: There are specific challenges unique to CYP2D6 testing given the complexity of the gene and its relevance to multiple medications. Consensus lessons learned may guide those interested in pursuing similar clinical pharmacogenetic programs.

Delayed methotrexate clearance in patients with acute lymphoblastic leukemia concurrently receiving dasatinib.

We aimed to determine whether patients receiving dasatinib or imatinib concurrently with high-dose methotrexate (HDMTX) had slower methotrexate clearance than patients not receiving a tyrosine kinase inhibitor (TKI) during the HDMTX infusion. Patients concurrently receiving dasatinib and HDMTX (N = 7) had significantly slower MTX clearance (P = 0.008) than patients not receiving a TKI (N = 111). Two patients receiving a TKI during a HDMTX infusion required glucarpidase. In vitro studies showed that dasatinib significantly inhibited methotrexate uptake by SLCO1B1-expressing cells (P = 0.009). There may be an interaction between dasatinib and HDMTX, mediated by the transporter SLCO1B1, that causes a delay in MTX clearance.

Consensus Guideline for Use of Glucarpidase in Patients with High-Dose Methotrexate Induced Acute Kidney Injury and Delayed Methotrexate Clearance.

Acute kidney injury due to high-dose methotrexate (HDMTX) is a serious, life-threatening toxicity that can occur in pediatric and adult patients. Glucarpidase is a treatment approved by the Food and Drug Administration for high methotrexate concentrations in the context of kidney dysfunction, but the guidelines for when to use it are unclear. An expert panel was convened to provide specific, expert consensus guidelines for the use of glucarpidase in patients who develop HDMTX-induced nephrotoxicity and delayed methotrexate excretion. The guideline provides recommendations to identify the population of patients who would benefit from glucarpidase rescue by more precisely defining the absolute methotrexate concentrations associated with risk for severe or life-threatening toxicity at several time points after the start of an HDMTX infusion. For an HDMTX infusion ≤24 hours, if the 36-hour concentration is above 30 µM, 42-hour concentration is above 10 µM, or 48-hour concentration is above 5 µM and the serum creatinine is significantly elevated relative to the baseline measurement (indicative of HDMTX-induced acute kidney injury), glucarpidase may be indicated. After a 36- to 42-hour HDMTX infusion, glucarpidase may be indicated when the 48-hour methotrexate concentration is above 5 µM. Administration of glucarpidase should optimally occur within 48-60 hours from the start of the HDMTX infusion, because life-threatening toxicities may not be preventable beyond this time point. IMPLICATIONS FOR PRACTICE: Glucarpidase is a rarely used medication that is less effective when given after more than 60 hours of exposure to high-dose methotrexate, so predicting early which patients will need it is imperative. There are no currently available consensus guidelines for the use of this medication. The indication on the label does not give specific methotrexate concentrations above which it should be used. An international group of experts was convened to develop a consensus guideline that was specific and evidence-based to identify the population of patients who would benefit from glucarpidase.

Genetic risk factors for the development of osteonecrosis in children under age 10 treated for acute lymphoblastic leukemia.

Osteonecrosis is a dose-limiting toxicity in the treatment of pediatric acute lymphoblastic leukemia (ALL). Prior studies on the genetics of osteonecrosis have focused on patients ≥10 years of age, leaving the genetic risk factors for the larger group of children <10 years incompletely understood. Here, we perform the first evaluation of genetic risk factors for osteonecrosis in children <10 years. The discovery cohort comprised 82 cases of osteonecrosis and 287 controls treated on Children's Oncology Group (COG) standard-risk ALL protocol AALL0331 (NCT00103285, https://clinicaltrials.gov/ct2/show/NCT00103285), with results tested for replication in 817 children <10 years treated on COG protocol AALL0232 (NCT00075725, https://clinicaltrials.gov/ct2/show/NCT00075725). The top replicated single nucleotide polymorphisms (SNPs) were near bone morphogenic protein 7 [BMP7: rs75161997, P = 5.34 × 10(-8) (odds ratio [OR] 15.0) and P = .0498 (OR 8.44) in the discovery and replication cohorts, respectively] and PROX1-antisense RNA1 (PROX1-AS1: rs1891059, P = 2.28 × 10(-7) [OR 6.48] and P = .0077 [OR 3.78] for the discovery and replication cohorts, respectively). The top replicated nonsynonymous SNP, rs34144324, was in a glutamate receptor gene (GRID2, P = 8.65 × 10(-6) [OR 3.46] and P = .0136 [OR 10.8] in the discovery and replication cohorts, respectively). In a meta-analysis, the BMP7 and PROX1-AS1 variants (rs75161997 and rs1891059, respectively) met the significance threshold of <5 × 10(-8). Top replicated SNPs were enriched in enhancers active in mesenchymal stem cells, and analysis of annotated genes demonstrated enrichment in glutamate receptor and adipogenesis pathways. These data may provide new insights into the pathophysiology of osteonecrosis.

Genetics of pleiotropic effects of dexamethasone.

OBJECTIVES: Glucocorticoids such as dexamethasone have pleiotropic effects, including desired antileukemic, anti-inflammatory, or immunosuppressive effects, and undesired metabolic or toxic effects. The most serious adverse effects of dexamethasone among patients with acute lymphoblastic leukemia are osteonecrosis and thrombosis. To identify inherited genomic variation involved in these severe adverse effects, we carried out genome-wide association studies (GWAS) by analyzing 14 pleiotropic glucocorticoid phenotypes in 391 patients with acute lymphoblastic leukemia. PATIENTS AND METHODS: We used the Projection Onto the Most Interesting Statistical Evidence integrative analysis technique to identify genetic variants associated with pleiotropic dexamethasone phenotypes, stratifying for age, sex, race, and treatment, and compared the results with conventional single-phenotype GWAS. The phenotypes were osteonecrosis, central nervous system toxicity, hyperglycemia, hypokalemia, thrombosis, dexamethasone exposure, BMI, growth trajectory, and levels of cortisol, albumin, and asparaginase antibodies, and changes in cholesterol, triglycerides, and low-density lipoproteins after dexamethasone. RESULTS: The integrative analysis identified more pleiotropic single nucleotide polymorphism variants (P=1.46×10(-215), and these variants were more likely to be in gene-regulatory regions (P=1.22×10(-6)) than traditional single-phenotype GWAS. The integrative analysis yielded genomic variants (rs2243057 and rs6453253) in F2RL1, a receptor that functions in hemostasis, thrombosis, and inflammation, which were associated with pleiotropic effects, including osteonecrosis and thrombosis, and were in regulatory gene regions. CONCLUSION: The integrative pleiotropic analysis identified risk variants for osteonecrosis and thrombosis not identified by single-phenotype analysis that may have importance for patients with underlying sensitivity to multiple dexamethasone adverse effects.

Genome-wide analysis links NFATC2 with asparaginase hypersensitivity.

Asparaginase is used to treat acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions can lead to suboptimal asparaginase exposure. Our objective was to use a genome-wide approach to identify loci associated with asparaginase hypersensitivity in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols Total XIIIA (n = 154), Total XV (n = 498), and Total XVI (n = 271), or Children's Oncology Group protocols POG 9906 (n = 222) and AALL0232 (n = 2163). Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome BeadChip array. In multivariate logistic regression, the intronic rs6021191 variant in nuclear factor of activated T cells 2 (NFATC2) had the strongest association with hypersensitivity (P = 4.1 × 10(-8); odds ratio [OR] = 3.11). RNA-seq data available from 65 SJCRH ALL tumor samples and 52 Yoruba HapMap samples showed that samples carrying the rs6021191 variant had higher NFATC2 expression compared with noncarriers (P = 1.1 × 10(-3) and 0.03, respectively). The top ranked nonsynonymous polymorphism was rs17885382 in HLA-DRB1 (P = 3.2 × 10(-6); OR = 1.63), which is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele we previously observed in a candidate gene study. The strongest risk factors for asparaginase allergy are variants within genes regulating the immune response.

HLA-DRB1*07:01 is associated with a higher risk of asparaginase allergies.

Asparaginase is a therapeutic enzyme used to treat leukemia and lymphoma, with immune responses resulting in suboptimal drug exposure and a greater risk of relapse. To elucidate whether there is a genetic component to the mechanism of asparaginase-induced immune responses, we imputed human leukocyte antigen (HLA) alleles in patients of European ancestry enrolled on leukemia trials at St. Jude Children's Research Hospital (n = 541) and the Children's Oncology Group (n = 1329). We identified a higher incidence of hypersensitivity and anti-asparaginase antibodies in patients with HLA-DRB1*07:01 alleles (P = 7.5 × 10(-5), odds ratio [OR] = 1.64; P = 1.4 × 10(-5), OR = 2.92, respectively). Structural analysis revealed that high-risk amino acids were located within the binding pocket of the HLA protein, possibly affecting the interaction between asparaginase epitopes and the HLA-DRB1 protein. Using a sequence-based consensus approach, we predicted the binding affinity of HLA-DRB1 alleles for asparaginase epitopes, and patients whose HLA genetics predicted high-affinity binding had more allergy (P = 3.3 × 10(-4), OR = 1.38). Our results suggest a mechanism of allergy whereby HLA-DRB1 alleles that confer high-affinity binding to asparaginase epitopes lead to a higher frequency of reactions. These trials were registered at www.clinicaltrials.gov as NCT00137111, NCT00549848, NCT00005603, and NCT00075725.

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

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.