No abstract

Objectives Recent clinical trial data cast doubt on the utility of genotype-guided warfarin dosing, specifically showing worse dosing with a pharmacogenetic versus clinical dosing algorithm in African Americans. However, many genotypes important in African Americans were not accounted for. We aimed to determine if omission of the CYP2C9*5, *6, *8, *11 alleles and rs12777823 G>A genotype affects performance of dosing algorithms in African Americans. Methods In a cohort of 274 warfarin-treated African Americans, we examined the association between the CYP2C9*5, *6, *8, *11 alleles and rs12777823 G>A genotype and warfarin dose prediction error with pharmacogenetic algorithms used in clinical trials. Results The warfarindosing.org algorithm over-estimated doses by a median (IQR) of 1.2 (0.02 to 2.6) mg/day in rs12777823 heterozygotes (p<0.001 for predicted versus observed dose), 2.0 (0.6 to 2.8) mg/day in rs12777823 variant homozygotes (p=0.004), and 2.2 (0.5 to 2.9) mg/day in carriers of a CYP2C9 variant (p<0.001). The International Warfarin Pharmacogenetics Consortium (IWPC) algorithm under-dosed warfarin by 0.8 (−2.3 to 0.4) mg/day for patients with the rs12777823 GG genotype (p<0.001) and over-dosed warfarin by 0.7 (−0.4 to 1.9) mg/day in carriers of a variant CYP2C9 allele (p=0.04). Modifying the warfarindosing.org algorithm to adjust for variants important in African Americans led to better dose prediction than either the original warfarindosing.org (p<0.01) or IWPC (p<0.01) algorithm. Conclusions These data suggest that, when providing genotype-guided warfarin dosing, failure to account for variants important in African Americans leads to significant dosing error in this population.

No abstract

No abstract

The fluorescence emission spectrum for reduced diphosphopyridine nucleotide (DPNH) in Escherichia coli uridine diphosphate galactose 4-epimerase-DPNH complexes has a maximum at 435 nm, which is about twice as intense when the excitation is at 280 nm as at 340 nm. The fluorescence excitation spectrum monitored at 460 nm has two maxima, one at 340-345 nm and another about twice as intense at 280 nm. The polarization of DPNH fluorescence by these complexes is 0.43-0.44 compared with 0.46 for DPNH immobilized in propylene glycol at -20 degrees C. The small degree of fluorescence depolarization is due to rotational relaxation of the protein, relaxation time 205 ns. The excited-state lifetimes in epimerase-DPNH-nucleotide complexes are 3.5-4.2 ns. The fluorescence data show that the dihydropyridine ring in these complexes is highly immobilized and exhibits no detectable independent motion relative to rotational motions of the protein. The inhibition constants for uridine monophosphate (UMP) and 2,2,6,6-tetramethyl-4-piperidinyl-1-oxyl uridyl pyrophosphate acting as competitive reversible inhibitors of epimerase-DPN+ are 1.2 and 0.2 mM, respectively, at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5. A collection of Ki and Km values for uridine nucleotide inhibitors and substrates indicates that the principle substrate binding interactions involve the nucleotide moieties of substrates. Dissociation constants for uridine nucleotides dissociating from epimerase-DPNH-nucleotide complexes, measured by ultraviolet absorption and fluorescence techniques, are 12 muM for UMP, 14 muM for UDP-hexopyranoses, 4 muM for UDP-pentopyranoses, 27 muM for p-bromoacetamidophenyl uridyl pyrophosphate, 0.14 muM for UDP-4-ketohexopyranose intermediate, and 0.36 muM for UDP-4-ketopentopyranose intermediate at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5. Analysis of these data shows conclusively that the major part of the binding free energy for UDP-4-ketopyranose intermediates binding to epimerase-DPNH is attributable to the uridylpyrophosphoryl components and that the glycosyl-binding free energies are much smaller. The data show that the action of this enzyme does not require tight binding between the active site and glycosyl groups of either substrates or intermediates, although there is favorable binding of the uridylpyrophosphoryl components, particularly by epimerase-DPNH. It is postulated that nonstereospecific action results from and depends upon relatively weak, nonspecific active site binding of glycosyl groups in substrates and intermediates and that the uridylpyrophosphoryl groups serve as binding anchors in the epimerization process.

An expression vector, pIN‐GT, encoding the soluble form of beta 1,4‐galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN‐III‐ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN‐GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti‐GT antibodies. The recombinant GT was purified to homogeneity by N‐acetylglucosamine‐Sepharose affinity chromatography. The NH2‐terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP‐galactose analog, 4‐azido‐2‐nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site‐directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287––Phe remained as active as wild‐type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N‐acetyglucosamine binding and Tyr309 is involved in UDP‐galactose binding as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.

Key Points• A preclinical cell-based model identifies SNPs associated with cytarabine sensitivity that also associate with outcome in leukemia patients.• SNPs within the MCC gene were associated with cytarabine sensitivity in lymphoblastoid cell lines and leukemic blasts from patients.A whole-genome approach was used to investigate the genetic determinants of cytarabineinduced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7-to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10 25) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n 5 18) was significantly greater than expected by chance (P 5 .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P 5 1.31 3 10 26) and AA (vs GA or GG) genotype was associated with poorer OS (P 5 .015), likely as a result of greater treatment-related mortality (P 5 .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084. (Blood. 2013;121(21):4366-4376) IntroductionAcute myeloid leukemia (AML) is the most common form of acute leukemia in adults and also occurs in children. Despite the genetic heterogeneity of the disease, patients have been treated for decades with similar combinations of cytarabine and anthracyclines with little improvement in overall survival (OS).1 Although the majority of patients (50%-60%) under 60 years achieve complete remission with traditional anthracycline-and cytarabine-based induction regimens, the long-term survival rates continue to be around 30% to 40% for adults and 60% for children. [2][3][4][5][6] Outcomes are worse for patients >60 years, with complete response rates in the range of 40% to 55% and poor long-term survival rates. 7 The main reason for treatment failure among patients with AML is resistance to therapy. [8][9][10] In addition, treatment with cytarabine is associated with a number of adverse side effects including myelosuppression, infections, mucositis, neurotoxicity, and acute pulmonary syndrome. 11Cytarabine requires activation through intracellular phosphorylation to araC-triphosphate (ara-CTP). The mechanism of action of cytarabine involves the incorporation of ara-CTP in place of deoxycytidine triphosphate, resulting in chain termination, blocking DNA and RNA synthesis and causing leukemic cell de...