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Biomedical subjects

Mark W Linder

Publications and source records attributed to Mark W Linder.

8 recordsLinked to original sources
PubMed2006

Simultaneous determination of 7 N-acetyltransferase-2 single-nucleotide variations by allele-specific primer extension assay.

BACKGROUND: Genotyping of N-acetyltransferase-2 (NAT2) is useful in predicting the risk for toxicity of NAT2 substrates. Current methods cannot detect the 7 most important single-nucleotide variations in NAT2 simultaneously in 1 tube. METHODS: We developed an assay that uses allele-specific primer extension (ASPE) and microsphere hybridization for the simultaneous detection of 7 single-nucleotide variations in NAT2. Using 12 samples previously genotyped by a TaqMan-based assay for method development and as positive controls, we amplified the genetic locus of NAT2 comprising the single-nucleotide variations of interest by PCR and then performed ASPE with allele-specific primers and biotinylated dCTP followed by bead hybridization and streptavidin-R-phycoerythrin binding. Genotypes were determined according to the allele-specific fluorescent signal ratios. RESULTS: The mean (SD) allelic ratios for homozygous common, heterozygous variant, and homozygous variant NAT2 genotypes were 0.0394 (0.0113) (n = 80), 0.4372 (0.0270) (n = 148), and 0.9331 (0.0127) (n = 325). The assay had 100% (95% confidence interval, 99%-100%) within-run reproducibility for 12 samples repeated 6 times and 100% (98%-100%) between-run reproducibility for a 5-sample subset run on 6 different days. NAT2 genotypes of 30 blinded samples determined by this assay were 100% (98%-100%) concordant with results obtained using the TaqMan method. CONCLUSIONS: The developed assay can simultaneously determine single-nucleotide variations in NAT2. The assay demonstrates no overlap in allele-specific signal ratios between homozygous common, heterozygous, and homozygous variant and shows agreement with a reference method and reproducibility of genotype identification.

Alleles↗
PubMed2006

Analysis of ligand binding by bioaffinity mass spectrometry.

BACKGROUND: Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach. METHODS: BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS. RESULTS: A protein with 141kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at approximately 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with approximately 3kDa to approximately 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts. CONCLUSIONS: Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.

Antibodies↗
PubMed2005

Prospective dosing of warfarin based on cytochrome P-450 2C9 genotype.

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.

Adult↗
PubMed2003

Suppression of cytochrome P450 2E1 promoter activity by interferon-gamma and loss of response due to the -71G>T nucleotide polymorphism of the CYP2E1*7B allele.

The CYP2E1*7B allele is defined by two nucleotide sequence polymorphisms, -71G>T and -333T>A. The CYP2E1 promoter sequence flanking the -71G nucleotide is consistent with a gamma-interferon activated sequence. Inflammation and interferon (IFN)-gamma suppress expression of CYP2E1 in vivo; however, the exact mechanism is not known. The objectives of this study were to determine whether the CYP2E1 promoter is regulated by IFN-gamma and to examine the influence of the nucleotide substitutions on this function. Treatment of HepG2 cells with IFN-gamma, after transient transfection with a luciferase reporter gene bearing the native CYP2E1 (-71G) promoter sequence resulted, in a dose-dependent reduction of luciferase activity. In contrast, no suppression was observed in cells transfected with the *7B allele promoter (-333A and -71T) nor a CYP2E1 plasmid containing only the -71T polymorphism. These data indicate that IFN-gamma suppresses native CYP2E1 promoter activity and that the -71G is critical for this response.

Alleles↗
PubMed2003

What is next in pharmacogenomics? Translating it to clinical practice.

Pharmacogenomics (PG) holds promise for transforming medical therapeutics but the details of how the promise will become reality are still vague. In this article, we focus on the role that laboratory medicine, as a discipline, might play in transitioning the application of pharmacogenomics into the healthcare system and begin to frame a perspective on how PG may be viewed in this context. Development of clinical diagnostic tests usually evolves as a continuum of information starting with the discovery of a potential biological marker through to its routine use in clinical practice. This process has traditionally been rooted in the practice of laboratory medicine and, importantly, includes the development of testing strategies to optimize the predictive value of single or a combination of biological markers. In this context, we also discuss a perspective on some future strategies that may prove useful in advancing the application of PG, including the need for an evidenced-based approach and the potential role of proteomics as a means to drive more comprehensive strategies.

Humans↗
PubMed2002

Warfarin dose adjustments based on CYP2C9 genetic polymorphisms.

BACKGROUND: The dose response relationship of warfarin is unpredictable. Polymorphism of the Cytochrome P4502C9 enzyme leads to warfarin hypersensitivity presumably due to decreased metabolism of the S-enantiomer. The purpose of this study was to further characterize the relationship between CYP2C9 genotype and phenotype and to develop a basis for guidelines to interpret CYP2C9 genotype for warfarin dosing. METHODS AND RESULTS: Patients stabilized on warfarin therapy were recruited from an anticoagulation clinic. Patients were genotyped for CYP2C9*2, CYP2C9*3 and CYP2C9*5 alleles by standard methods of polymerase chain reaction amplification and restriction endonuclease digestion. Phenotype was determined by; dose (mg/kg/d) required to maintain anticoagulation, (INR 2.0-3.0), oral plasma S-warfarin clearance, and the plasma S:R-warfarin ratio. In this cohort, no subjects were found to have the CYP2C9*5 allele. The plasma S-warfarin concentration did not differ with age, dose or CYP2C9 genotype. Both CYP2C9*2 and *3 alleles were associated with lower maintenance dosages, lower total and R-warfarin plasma concentrations, decreased oral clearance of S-warfarin, increased plasma S:R-warfarin ratio and extended S-warfarin elimination half-life. Advancing age was found to decrease Warfarin maintenance dose in subjects with the common active CYP2C9*1/*1 genotype but did not influence dose requirement of subjects with one or more variant CYP2C9 alleles. CONCLUSIONS: Subjects who have been titrated to a consistent target INR demonstrate comparable plasma S-warfarin concentrations independent of CYP2C9 genotype. The warfarin dose required to maintain a consistent target INR between subjects differs as a function of S-warfarin clearance which is decreased by both CYP2C9*2 and or CYP2C9*3 variant alleles. The variables of CYP2C9 genotype and age can be applied to restrict the dosage range considered for individual patients.

Adult↗