Initiation of warfarin therapy using trial-and-error dosing is problematic. our goal was to develop and validate a pharmacogenetic algorithm. in the derivation cohort of 1,015 participants, the independent predictors of therapeutic dose were: VKORC1 polymorphism −1639/3673 g>a (−28% per allele), body surface area (Bsa) (+11% per 0.25 m 2 ), CYP2C9*3 (−33% per allele), CYP2C9*2 (−19% per allele), age (−7% per decade), target international normalized ratio (inr) (+11% per 0.5 unit increase), amiodarone use (−22%), smoker status (+10%), race (−9%), and current thrombosis (+7%). This pharmacogenetic equation explained 53−54% of the variability in the warfarin dose in the derivation and validation (N = 292) cohorts. For comparison, a clinical equation explained only 17−22% of the dose variability (P < 0.001). in the validation cohort, we prospectively used the pharmacogenetic-dosing algorithm in patients initiating warfarin therapy, two of whom had a major hemorrhage. To facilitate use of these pharmacogenetic and clinical algorithms, we developed a nonprofit website, http://www.WarfarinDosing.org.Correspondence: BF Gage (E-mail: bgage@im.wustl.edu). CONFLICT OF INTEREST Dr Gage has consulted for Bristol-Myers Squibb on work unrelated to this article. Drs Rieder and Rettie report having applied for a patent (application serial no. 10/967,879) on the use of VKORC1 haplotypes and SNPs. The other authors declared no conflict of interest. NIH Public Access RESULTSIn the derivation cohort (N = 1,015), the daily therapeutic warfarin dose ranged from 1 to 18 mg/day. The mean age was 65 (range of 18−93); 83% were Caucasian, and 64% were male. The (geometric) mean daily warfarin dose was 4.8 mg ( Table 1). The most common indications for warfarin therapy were atrial fibrillation (N = 392) and prior venous thromboembolism (N = 376; 13 of whom also had atrial fibrillation). Patients in the validation cohort (N = 292) were younger, more often female, and had more often (77%) undergone joint replacement as their indication for warfarin therapy (Table 1).VKORC1 alleles were highly heterogeneous (Table 2), reflecting their original selection as common (>5% allele frequency), informative tagging SNPs (Table 2). 12 VKORC1 3673G>A was in high linkage disequilibrium with VKORC1 6853G>C (D' = 0.97). In both cohorts, all alleles were in Hardy-Weinberg equilibrium. Genotype data from all participants at Washington University and University of Florida have been submitted to the PharmGKB (accession numbers: PS207479 and PS207480 pending). Pharmacogenetic model developmentThe VKORC1 3673G>A SNP was the first variable to enter the stepwise regression model (Table 3); each VKORC1 3673A allele was associated with a 28% reduction (95% confidence interval 25−30%) in the therapeutic warfarin dose. Once VKORC1 3673G>A entered the model, none of the other VKORC1 SNPs was an independent predictor of warfarin dose. Body surface area (BSA) was the second variable to enter the model, and each 0.25 m 2 increase in BSA was associated with an 11% ...

Cytochrome P-450 2C9 (CYP2C9) polymorphisms (CYP2C9*2 and CYP2C9*3) reduce the clearance of warfarin, increase the risk of bleeding, and prolong the time to stable dosing. Whether prospective use of a retrospectively developed algorithm that incorporates CYP2C9 genotype and nongenetic factors can ameliorate the propensity to bleeding and delay in achieving a stable warfarin dose is unknown. We initiated warfarin therapy in 48 orthopedic patients tailored to the following variables: CYP2C9 genotype, age, weight, height, gender, race, and use of simvastatin or amiodarone. By using pharmacogenetics-based dosing, patients with a CYP2C9 variant achieved a stable, therapeutic warfarin dose without excessive delay. However compared to those without a CYP2C9 variant, patients with a variant continued to be at increased risk (hazard ratio 3.6, 95% confidence interval 1.4-9.5, p = 0.01) for an adverse outcome (principally INR > 4), despite pharmacogenetics-based dosing. There was a linear relationship (R(2) = 0.42, p < 0.001) between the pharmacogenetics-predicted warfarin doses and the warfarin maintenance doses, prospectively validating the dosing algorithm. Prospective, perioperative pharmacogenetics-based dosing of warfarin is feasible; however, further evaluation in a randomized, controlled study is recommended.

IntroductionWarfarin sodium is characterized by a narrow therapeutic range (eg, an international normalized ratio [INR]) of 2.0-3.0), a marked interindividual variation in dosing requirements, and an increased risk of adverse events when the dose is too high or low. 1,2 To minimize the high incidence of such events, [3][4][5] particularly during the first few weeks of initiating therapy, 1,6 most guidelines recommend prescribing warfarin at or near the anticipated maintenance dose and then adjusting the dose by trial and error. 1,7,8 While algorithms for predicting this maintenance dose a priori have improved, [9][10][11][12][13][14][15][16] there remains little guidance on how this starting dose should be adjusted a posteriori based on the subsequent INR values. We hypothesized that use of genetic markers could help optimize these dose refinements.Two common single nucleotide polymorphisms (SNPs) in the cytochrome P450 (CYP) 2C9 system are associated with impaired metabolism of warfarin, [3][4][5][6]11,17 while SNPs in the gene for vitamin K epoxide reductase complex 1 (VKORC1) correlate with warfarin sensitivity and resistance. 2,[18][19][20] No prior study has examined the impact of these SNPs on warfarin-dose adjustments. Given the current knowledge about these markers, we hypothesize that for a given INR, a patient who is a slow metabolizer of warfarin may need a more cautious adjustment in their dose than a similar patient who is a normal metabolizer. Failure to tailor dose refinements during warfarin induction in poor metabolizers may have contributed to the 3-fold increased risk of (laboratory or clinical) adverse events among poor metabolizers in our initial prospective study of pharmacogenetic-based warfarin therapy. 4 The purpose of this study was to develop a dose-refinement nomogram to guide clinicians in adjusting warfarin doses. This nomogram would be similar to prior algorithms, 21,22 but will have 2 advantages: (1) it will allow for, but not require, a first dose that is tailored to clinical and/or genetic factors and (2) it will incorporate genetics and clinical factors that are independent predictors of how much the dose should be refined. 1,11 If successful, the proposed warfarin nomogram would simplify and standardize warfarin initiation. Patients, materials, and methodsThe study was a retrospective analysis of 2 cohorts of orthopedic surgery patients who had participated in 2 prospective studies of pharmacogeneticbased warfarin therapy. The Human Research Protection Office at Washington University Medical Center approved these studies. PatientsFor patients in both cohorts, we offered participation if they were scheduled for primary or revision total knee or hip arthroplasty at Washington University Medical Center and if they were 18 years or older. We excluded patients who had previously taken warfarin or who had contraindications to warfarin treatment. To allow time for genotyping, we also excluded patients scheduled for surgery fewer than 7 days following referral to our anticoagulation s...

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Hepatitis C virus (HCV) infection is a major contributor to the development of end-stage liver disease, including cirrhosis and hepatocellular carcinoma (HCC). We have previously shown that HCV core protein promotes immortalization of primary human hepatocytes. To identify molecular changes involved in core protein-mediated immortalization, we have investigated differential gene expression by microarray analyses in primary human hepatocytes and HCV core gene introduced hepatocytes after senescence (early passage), immortalization (middle passage), and anchor-independent growth (late passage). Out of 33,000 human genes screened, 1918 transcripts were differentially expressed (>2-fold) in immortalized human hepatocytes (IHH) as compared to negative controls. Our analyses provided a molecular portrait of changes in gene expression associated with three distinct stages of hepatocytes after introduction of HCV core gene. Many of the overall changes were involved with important cellular pathways, including cell growth regulation, immune regulation, oxidative stress, and apoptosis. We focused on the Stat3 signaling pathway by further verifying selected genes at the protein level relevant to hepatocyte growth regulation. Our data suggested that the introduction of HCV core protein results in an increase in expression of IL-6, gp130, leptin receptor, and Stat3. Upregulation of these genes in turn may regulate c-myc and cyclin D1, downstream of the Stat3 signaling pathway. Identification of these modulated genes with potential roles may help in the selection of targets for therapies against HCV-mediated liver disease progression.

The 67-kDa elastin binding protein shares many immunological and structural properties with the high-affinity 67-kDa tumor cell laminin receptor. Taking advantage of these similarities, we have screened a bovine cDNA library with a partial cDNA probe for the laminin receptor and have isolated and characterized a cDNA clone of 1038 bp that hybridizes to a single-size mRNA of 1.3 kb. The clone encodes a protein with a predicted molecular weight of 33K that lacks an N-terminal leader sequence, shows no posttranslational processing when translated in vitro in the presence of microsomes, and does not bind to elastin affinity columns. Although the bovine clone is nearly identical with clones encoding human and mouse proteins proported to be 67-kDa laminin receptor, physical and functional characteristics of the encoded protein suggest that it is a cytoplasmic protein that does not bind elastin. This finding calls into question the earlier conclusion that the clone encodes the 67-kDa receptor.

Recent research suggests an increase in the incidence of hepatocellular carcinoma (HCC) in the United States, which may be related to an upsurge in the sequelae of chronic liver disease from hepatitis C virus. In addition to factors related to the underlying etiology of liver disease, a number of host factors such as age, gender, and ethnic background may be associated with this increased risk. The aim of this study was to evaluate a number of potential risk factors for HCC in patients with cirrhosis. Patients with biopsy proven HCC were identified from our pathology and cancer registry databases. All those without histologic or clinical cirrhosis and non-HCC hepatic malignancies were excluded. Cirrhotic patients without HCC were also selected from the Cleveland Clinic unified transplant database and were designated controls. Extensive clinicodemographic data were obtained from the databases and chart reviews. When available, paraffin-embedded liver biopsy blocks were obtained for HFE gene analysis. Univariate comparisons were made with chi-square and Fisher's exact test and multivariate analysis was carried out with logistic regression. A total of 760 patients were included in this study, 244 documented cases of HCC and 516 cirrhotic controls without HCC. Patients' age (RR = 3.1 [2.6-3.8]; P < 0.0001), male gender (RR = 3.4 [2.3-5.1]; P < 0.0001), African-American ethnicity (RR = 3.1 [1.6-5.8]; P = 0.0005), and other non-Caucasian ethnicity (RR = 6.9 [3.2-14.4]; P < 0.0001) were independently associated with HCC. Restricting the analysis to HCV-related cirrhosis, the same risk factors remained independently associated with HCC: age (decade; RR = 2.3 [1.6-3.4]; P < 0.0001), male gender (RR = 2.9 [1.2-7.0]; P = 0.02), African-American ethnicity (RR = 3.1 [1.3-7.4]; P = 0.009), and other non-Caucasian ethnicity (RR = 15.8 [1.9-134]; P = 0.01). Iron studies did not reveal an increased risk for iron overload or HFE mutation. Male gender, advancing age, and non-Caucasian ethnic background are independently associated with HCC.

Aim-To provide a more eYcient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis. Methods-The use of blood impregnated filter paper and Chelex-100 in DNA isolation was evaluated and compared with standard DNA isolation techniques. Results-In polymerase chain reaction (PCR) based assays of five point mutations, identical results were obtained with DNA isolated routinely from peripheral blood and isolated using the filter paper and Chelex-100 method. Conclusion-In the clinical setting, this method provides a useful alternative to conventional DNA isolation. It is easily implemented and inexpensive, and provides suYcient, stable DNA for multiple assays. The potential for specimen contamination is reduced because most of the steps are performed in a single microcentrifuge tube. In addition, this method provides for easy storage and transport of samples from the point of acquisition. (J Clin Pathol: Mol Pathol 1998;51:215-217)