Michael J. Wagner

Estimation of the warfarin dose with clinical and pharmacogenetic data.

BACKGROUNDnGenetic variability among patients plays an important role in determining the dose of warfarin that should be used when oral anticoagulation is initiated, but practical methods of using genetic information have not been evaluated in a diverse and large population. We developed and used an algorithm for estimating the appropriate warfarin dose that is based on both clinical and genetic data from a broad population base.nnnMETHODSnClinical and genetic data from 4043 patients were used to create a dose algorithm that was based on clinical variables only and an algorithm in which genetic information was added to the clinical variables. In a validation cohort of 1009 subjects, we evaluated the potential clinical value of each algorithm by calculating the percentage of patients whose predicted dose of warfarin was within 20% of the actual stable therapeutic dose; we also evaluated other clinically relevant indicators.nnnRESULTSnIn the validation cohort, the pharmacogenetic algorithm accurately identified larger proportions of patients who required 21 mg of warfarin or less per week and of those who required 49 mg or more per week to achieve the target international normalized ratio than did the clinical algorithm (49.4% vs. 33.3%, P<0.001, among patients requiring < or = 21 mg per week; and 24.8% vs. 7.2%, P<0.001, among those requiring > or = 49 mg per week).nnnCONCLUSIONSnThe use of a pharmacogenetic algorithm for estimating the appropriate initial dose of warfarin produces recommendations that are significantly closer to the required stable therapeutic dose than those derived from a clinical algorithm or a fixed-dose approach. The greatest benefits were observed in the 46.2% of the population that required 21 mg or less of warfarin per week or 49 mg or more per week for therapeutic anticoagulation.

Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups

Warfarin-dosing algorithms incorporating CYP2C9 and VKORC1 -1639G>A improve dose prediction compared with algorithms based solely on clinical and demographic factors. However, these algorithms better capture dose variability among whites than Asians or blacks. Herein, we evaluate whether other VKORC1 polymorphisms and haplotypes explain additional variation in warfarin dose beyond that explained by VKORC1 -1639G>A among Asians (n = 1103), blacks (n = 670), and whites (n = 3113). Participants were recruited from 11 countries as part of the International Warfarin Pharmacogenetics Consortium effort. Evaluation of the effects of individual VKORC1 single nucleotide polymorphisms (SNPs) and haplotypes on warfarin dose used both univariate and multi variable linear regression. VKORC1 -1639G>A and 1173C>T individually explained the greatest variance in dose in all 3 racial groups. Incorporation of additional VKORC1 SNPs or haplotypes did not further improve dose prediction. VKORC1 explained greater variability in dose among whites than blacks and Asians. Differences in the percentage of variance in dose explained by VKORC1 across race were largely accounted for by the frequency of the -1639A (or 1173T) allele. Thus, clinicians should recognize that, although at a population level, the contribution of VKORC1 toward dose requirements is higher in whites than in nonwhites; genotype predicts similar dose requirements across racial groups.

Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study

Summary Background VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans. Methods We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 −1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10−8 in the discovery cohort and p<0·0038 in the replication cohort. Findings The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10−8). This association was confirmed in the replication cohort (p=5·04×10−5); analysis of the two cohorts together produced a p value of 4·5×10−12. Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement). Interpretation A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population. Funding National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.

Variation in the glucose transporter gene SLC2A2 is associated with glycemic response to metformin

Metformin is the first-line antidiabetic drug with over 100 million users worldwide, yet its mechanism of action remains unclear. Here the Metformin Genetics (MetGen) Consortium reports a three-stage genome-wide association study (GWAS), consisting of 13,123 participants of different ancestries. The C allele of rs8192675 in the intron of SLC2A2, which encodes the facilitated glucose transporter GLUT2, was associated with a 0.17% (P = 6.6 × 10−14) greater metformin-induced reduction in hemoglobin A1c (HbA1c) in 10,577 participants of European ancestry. rs8192675 was the top cis expression quantitative trait locus (cis-eQTL) for SLC2A2 in 1,226 human liver samples, suggesting a key role for hepatic GLUT2 in regulation of metformin action. Among obese individuals, C-allele homozygotes at rs8192675 had a 0.33% (3.6 mmol/mol) greater absolute HbA1c reduction than T-allele homozygotes. This was about half the effect seen with the addition of a DPP-4 inhibitor, and equated to a dose difference of 550 mg of metformin, suggesting rs8192675 as a potential biomarker for stratified medicine.

Exome resequencing identifies potential tumor-suppressor genes that predispose to colorectal cancer.

Inherited factors account for around one third of all colorectal cancers (CRCs) and include rare high penetrance mutations in APC, MSH2, MSH6, and POLE. Here, we sought novel tumor‐suppressor genes that predispose to CRC by exome resequencing 50 sporadic patients with advanced CRC (18 diagnosed ≤35 years of age) at a mean coverage of 30×. To help identify potentially pathogenic alleles, we initially sought rare or novel germline truncating mutations in 1,138 genes that were likely to play a role in colorectal tumorigenesis. In total, 32 such mutations were identified and confirmed, and included an insertion in APC and a deletion in POLE, thereby validating our approach for identifying disease alleles. We sought somatic mutations in the corresponding genes in the CRCs of the patients harboring the germline lesions and found biallelic inactivation of FANCM, LAMB4, PTCHD3, LAMC3, and TREX2, potentially implicating these genes as tumor suppressors. We also identified a patient who carried a germline truncating mutation in NOTCH3, part of the Notch signaling cascade that maintains intestinal homeostasis. Our whole exome analyses provided further gene lists to facilitate the identification of potential predisposition alleles.

Pharmacogenomic characterization of US FDA-approved cytotoxic drugs

AIMSnIndividualization of cancer chemotherapy based on the patients genetic makeup holds promise for reducing side effects and improving efficacy. However, the relative contribution of genetics to drug response is unknown.nnnMATERIALS & METHODSnIn this study, we investigated the cytotoxic effect of 29 commonly prescribed chemotherapeutic agents from diverse drug classes on 125 lymphoblastoid cell lines derived from 14 extended families.nnnRESULTSnThe results of this systematic study highlight the variable role that genetics plays in response to cytotoxic drugs, ranging from a heritability of <0.15 for gemcitabine to >0.60 for epirubicin.nnnCONCLUSIONnPutative quantitative trait loci for cytotoxic response were identified, as well as drug class-specific signatures, which could indicate possible shared genetic mechanisms. In addition to the identification of putative quantitative trait locis, the results of this study inform the prioritization of chemotherapeutic drugs with a sizable genetic response component for future investigation.

Genetic Predictors of Cardiovascular Mortality During Intensive Glycemic Control in Type 2 Diabetes: Findings From the ACCORD Clinical Trial

OBJECTIVE To identify genetic determinants of increased cardiovascular mortality among subjects with type 2 diabetes who underwent intensive glycemic therapy in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial. RESEARCH DESIGN AND METHODS A total of 6.8 million common variants were analyzed for genome-wide association with cardiovascular mortality among 2,667 self-reported white subjects in the ACCORD intensive treatment arm. Significant loci were examined in the entire ACCORD white genetic dataset (n = 5,360) for their modulation of cardiovascular responses to glycemic treatment assignment and in a Joslin Clinic cohort (n = 422) for their interaction with long-term glycemic control on cardiovascular mortality. RESULTS Two loci, at 10q26 and 5q13, attained genome-wide significance as determinants of cardiovascular mortality in the ACCORD intensive arm (P = 9.8 × 10−9 and P = 2 × 10−8, respectively). A genetic risk score (GRS) defined by the two variants was a significant modulator of cardiovascular mortality response to treatment assignment in the entire ACCORD white genetic dataset. Participants with GRS = 0 experienced a fourfold reduction in cardiovascular mortality in response to intensive treatment (hazard ratio [HR] 0.24 [95% CI 0.07–0.86]), those with GRS = 1 experienced no difference (HR 0.92 [95% CI 0.54–1.56]), and those with GRS ≥2 experienced a threefold increase (HR 3.08 [95% CI 1.82–5.21]). The modulatory effect of the GRS on the association between glycemic control and cardiovascular mortality was confirmed in the Joslin cohort (P = 0.029). CONCLUSIONS Two genetic variants predict the cardiovascular effects of intensive glycemic control in ACCORD. Further studies are warranted to determine whether these findings can be translated into new strategies to prevent cardiovascular complications of diabetes.

Application of next generation sequencing to CEPH cell lines to discover variants associated with FDA approved chemotherapeutics

BackgroundThe goal of this study was to perform candidate gene association with cytotoxicity of chemotherapeutics in cell line models through resequencing and discovery of rare and low frequency variants along with common variations. Here, an association study of cytotoxicity response to 30 FDA approved drugs was conducted and we applied next generation targeted sequencing technology to discover variants from 103 candidate genes in 95 lymphoblastoid cell lines from 14 CEPH pedigrees. In this article, we called variants across 95 cell lines and performed association analysis for cytotoxic response using the Family Based Association Testing method and software.ResultsWe called 2281 variable SNP genotypes across the 103 genes for these cell lines and identified three genes of significant association within this marker set. Specifically, ATP-binding cassette, sub-family C, member 5 (ABCC5), metallothionein 1A (MT1A) and NAD(P)H dehydrogenase quinone1 (NQO1) were significantly associated with oxaliplatin drug response. The significant SNP on NQO1 (rs1800566) has been linked with poor survival rates in patients with non-small cell lung cancer treated with cisplatin (which belongs to the same class of drugs as oxaliplatin). A SNP (rs1846692) near the 5′ region of MT1A was associated with arsenic trioxide.ConclusionsThe results from this study are promising and this serves as a proof-of-principle demonstration of the use of sequencing data in the cytotoxicity models of human cell lines. With increased sample sizes, such studies will be a fast and powerful way to associate common and rare variants with drug response; while overcoming the cost and time limitations to recruit cohorts for association study.

Common and rare genetic markers of lipid variation in subjects with type 2 diabetes from the ACCORD clinical trial

Background Individuals with type 2 diabetes are at an increased risk of cardiovascular disease. Alterations in circulating lipid levels, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides (TG) are heritable risk factors for cardiovascular disease. Here we conduct a genome-wide association study (GWAS) of common and rare variants to investigate associations with baseline lipid levels in 7,844 individuals with type 2 diabetes from the ACCORD clinical trial. Methods DNA extracted from stored blood samples from ACCORD participants were genotyped using the Affymetrix Axiom Biobank 1 Genotyping Array. After quality control and genotype imputation, association of common genetic variants (CV), defined as minor allele frequency (MAF) ≥ 3%, with baseline levels of TC, LDL, HDL, and TG was tested using a linear model. Rare variant (RV) associations (MAF < 3%) were conducted using a suite of methods that collapse multiple RV within individual genes. Results Many statistically significant CV (p < 1 × 10−8) replicate findings in large meta-analyses in non-diabetic subjects. RV analyses also confirmed findings in other studies, whereas significant RV associations with CNOT2, HPN-AS1, and SIRPD appear to be novel (q < 0.1). Discussion Here we present findings for the largest GWAS of lipid levels in people with type 2 diabetes to date. We identified 17 statistically significant (p < 1 × 10−8) associations of CV with lipid levels in 11 genes or chromosomal regions, all of which were previously identified in meta-analyses of mostly non-diabetic cohorts. We also identified 13 associations in 11 genes based on RV, several of which represent novel findings.

Modulation of GLP-1 levels by a genetic variant that regulates the cardiovascular effects of intensive glycemic control in ACCORD

OBJECTIVE A genome-wide association study in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial identified two markers (rs57922 and rs9299870) that were significantly associated with cardiovascular mortality during intensive glycemic control and could potentially be used, when combined into a genetic risk score (GRS), to identify patients with diabetes likely to derive benefit from intensive control rather than harm. The aim of this study was to gain insights into the pathways involved in the modulatory effect of these variants. RESEARCH DESIGN AND METHODS Fasting levels of 65 biomarkers were measured at baseline and at 12 months of follow-up in the ACCORD-Memory in Diabetes (ACCORD-MIND) MRI substudy (n = 562). Using linear regression models, we tested the association of the GRS with baseline and 12-month biomarker levels, and with their difference (Δ), among white subjects, with genotype data (n = 351) stratified by intervention arm. RESULTS A significant association was observed between GRS and ΔGLP-1 (glucagon-like peptide 1, active) in the intensive arm (P = 3 × 10−4). This effect was driven by rs57922 (P = 5 × 10−4). C/C homozygotes, who had been found to derive cardiovascular benefits from intensive treatment, showed a 22% increase in GLP-1 levels during follow-up. By contrast, T/T homozygotes, who had been found to experience increased cardiac mortality with intensive treatment, showed a 28% reduction in GLP-1 levels. No association between ΔGLP-1 and GRS or rs57922 was observed in the standard treatment arm. CONCLUSIONS Differences in GLP-1 axis activation may mediate the modulatory effect of variant rs57922 on the cardiovascular response to intensive glycemic control. These findings highlight the importance of GLP-1 as a cardioprotective factor.