Attitudes of clinicians following large-scale pharmacogenomics implementation.

Clinician attitudes toward multiplexed genomic testing may be vital to the success of translational programs. We surveyed clinicians at an academic medical center about their views on a large pharmacogenomics implementation, the PREDICT (Pharmacogenomic Resource for Enhanced Decisions in Care and Treatment) program. Participants were asked about test ordering, major factors influencing use of results, expectations of efficacy and responsibility for applying results to patient care. Virtually all respondents (99%) agreed that pharmacogenomics variants influence patients' response to drug therapy. The majority (92%) favored immediate, active notification when a clinically significant drug-genome interaction was present. However, clinicians were divided on which providers were responsible for acting on a result when a prescription change was indicated and whether patients should be directly notified of a significant result. We concluded genotype results were valued for tailoring prescriptions, but clinicians do not agree on how to appropriately assign clinical responsibility for actionable results from a multiplexed panel.The Pharmacogenomics Journal advance online publication, 11 August 2015; doi:10.1038/tpj.2015.57.

Public health impact of genetic tests at the end of the 20th century.

PURPOSE: To evaluate genetics tests available for clinical, research, and public health purposes in terms of their public health impact as measured by the number of people who could potentially be tested. METHODS: Genetic tests for the 751 inherited diseases or conditions listed in the GeneTests database as of November 2000, were classified on the basis of their use for population-based testing and the prevalence of the disease or condition being tested. The GeneTests database divides the tests into two groups: those offered for clinical use and those available for research only. RESULTS: Of the 423 clinical tests, 51 had potentially greater impact on public health because of their use in statewide newborn screening programs, other population screening programs, or testing for common diseases with a prevalence over 1 in 2,000 people. Among the 328 tests performed for research purposes only, 18 met the criteria for potentially greater public health impact. CONCLUSIONS: Our classification scheme indicated that fewer than 10% of the genetic tests listed in the GeneTests database at the end of 2000 are highly relevant to public health. The majority of genetic tests are used in diagnosis and/or genetic counseling for rare, single-gene disorders in a limited number of people. However, as more tests are being considered for newborn screening, and associations between genes and common diseases are being discovered, the impact of genetic testing on public health is likely to increase.

HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis.

CONTEXT: Hereditary hemochromatosis is recognized as one of the most common autosomal recessive disorders, with a prevalence of 1 in 200 to 400 in the white population. Early detection and treatment are completely effective in preventing pathology. It is anticipated that testing for hereditary hemochromatosis will increase, as will the need for a technology that can handle the demand. OBJECTIVE: To describe a high-throughput, single-tube, allele-specific multiplex polymerase chain reaction assay for identifying the 2 mutations in the HFE gene associated with hereditary hemochromatosis. DESIGN: Fluorescence-labeled polymerase chain reaction products from a multiplex polymerase chain reaction are analyzed by automated capillary electrophoresis. DATA ANALYSIS: The assay was validated by analysis of 25 blinded samples, and results were concordant with an established laboratory assay. CONCLUSION: The assay described offers a significant improvement over manual laboratory assays in throughput, reduced technologist time, and cost.

Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.

The human blood coagulation factor VIII C2 domain (Ser2173-Tyr2332) contains an epitope recognized by most polyclonal inhibitory anti-factor VIII alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine factor VIII and mapped a major determinant of the C2 epitope by using a series of active recombinant hybrid human/porcine factor VIII molecules. A series of five C2-specific human antibodies and a murine anti-factor VIII monoclonal antibody, NMC-VIII/5, inhibited a hybrid containing a substitution of porcine sequence for Glu2181-Val2243 significantly less than human factor VIII. In contrast, four of the five patient antibodies and NMC-VIII/5 inhibited a hybrid containing a substitution of porcine sequence for Thr2253-Tyr2332 equally well as human factor VIII. Thus, a major factor VIII inhibitor epitope determinant is bounded by Glu2181-Val2243 at the NH2-terminal end of the C2 domain. Because C2 inhibitors block the binding of factor VIII to phospholipid and von Willebrand factor, for which binding sites have been localized to Thr2303-Tyr2332, these results imply that the segment bounded by Glu2181-Val2243 also is involved in these macromolecular interactions.

Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis.

Antibodies directed to the A2 domain of factor VIII (fVIII) are usually an important component of the polyclonal response in patients who have clinically significant inhibitory antibodies to fVIII. A major determinant of the A2 epitope has been located by homolog scanning mutagenesis using recombinant hybrid human/porcine fVIII molecules to a sequence bounded by Arg484-Ile508 (Healey, J. F. , Lubin, I. M., Nakai, H., Saenko, E. L., Hoyer, L. W., Scandella, D. , and Lollar, P. (1995) J. Biol. Chem. 270, 14505-14509). Within this region, human residues Arg484, Pro485, Tyr487, Ser488, Arg489, Pro492, Val495, Phe501, and Ile508 differ from porcine fVIII. We stably expressed in mammalian cells nine active B-domainless human fVIII molecules containing single alanine substitutions at these sites. Their inhibition by a murine anti-A2 monoclonal antibody, monoclonal antibody (mAb) 413, and by three A2-specific alloimmune and two A2-specific autoimmune human inhibitor plasmas was measured by the Bethesda assay. The inhibition of Arg484 --> Ala, Tyr487 --> Ala, Arg489 --> Ala, and Arg492 --> Ala by mAb413 was reduced by greater than 90% compared with wild-type, B-domainless human fVIII. mAb413 inhibited the most severely affected mutant, Arg489 --> Ala, 0.01% as well as wild-type fVIII. For all five patient plasmas, the Tyr487 --> Ala mutant displayed the greatest reduction in inhibition. The inhibition of the Tyr487 --> Ala mutant by these antibodies ranged from 10% to 20% that of wild-type fVIII. The inhibition of the Ser488 --> Ala, Arg489 --> Ala, Pro492 --> Ala, Val495 --> Ala, Phe501 --> Ala, and Ile508 --> Ala mutants by most of the plasmas also was significantly reduced. In contrast, the Arg484 --> Ala and Pro485 --> Ala mutants were relatively unaffected. Thus, although mAb413 binds to the same region as human A2 inhibitors, it recognizes a different set of amino acid side chains. The side chains recognized by human A2 inhibitors appear to be similar, despite the differing immune settings that give rise to fVIII alloantibodies and autoantibodies.

The cDNA and derived amino acid sequence of porcine factor VIII.

The cDNA corresponding to 137 bp of the 5' untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337-372 and 1649-1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII.

The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.

Elimination of a major inhibitor epitope in factor VIII.

The A2 and C2 domains of human blood coagulation factor VIII (fVIII) contain the epitopes targeted by most inhibitory allo- and autoantibodies. Human inhibitors usually display limited or no reaction with porcine fVIII. We constructed an active, recombinant hybrid human/porcine fVIII molecule by replacing the putative human fVIII A2 domain epitope with the homologous porcine sequence. The hybrid retained full activity in the presence of antibodies with specificity restricted to the human A2 epitope. In contrast, the hybrid was neutralized by an anti-C2 antibody. These findings provide a basis for fine epitope mapping and for therapy of the inhibitor patient.

The tissue plasminogen activator finger domain confers fibrin-dependent enhancement of catalytic activity to single-chain urokinase-type plasminogen activator.

To determine whether the fibrin-binding domains of tissue plasminogen activator (tPA) can confer enhanced catalytic activity to single-chain urokinase-type plasminogen activator (scuPA), we constructed, expressed, and characterized the kinetics of five recombinant tPA/scuPA hybrid molecules. The hybrid molecules are: 1) tPA3-50 (tPA finger)/scuPA138-411, 2) tPA177-256 (tPA kringle2)/scuPA140-411 (scuPA catalytic), 3) tPA3-50/tPA177-256/scuPA140-411, 4) scuPA1-47 (scuPA growth factor)/tPA177-256/scuPA140-411, and 5) scuPA1-138 (scuPA growth factor and kringle)tPA127-256/scuPA139-411. The amidolytic activity of all hybrids was comparable, as were the kinetics for conversion from single-chain to two-chain plasminogen activator. We found that 1) the lag time prior to achieving maximal velocity among these hybrids varied, 2) hybrids 2, 3, 4, and 5 were 2-134-fold more potent (by kcat/Km) than hybrid 1, and 3) those hybrid proteins containing the tPA finger domain (hybrids 1 and 3) gave a 2-fold increase in catalytic efficiency in the presence of DESAFIB (reptilase-digested fibrinogen). These kinetic differences are likely mediated by changes in the tertiary structure of the scuPA catalytic domain resulting from interactions between catalytic and noncatalytic domains in the presence of fibrin.

Strategies for the design of novel thrombolytic and antithrombolytic agents.

Recent advances in our understanding of thrombosis and thrombolysis have led to the design of new thrombolytic agents and regimens that may offer improved efficacy. In general, these new approaches specifically target pivotal steps in thrombus formation or lysis. The goal is to reduce adverse side effects (such as bleeding complications) that result from development of a lytic state or that result from a failure to maintain patency (as characterized by rethrombosis). The points in the coagulation cascade that are susceptible to inhibition, as well as the proposed agents for intervention, are discussed in this review.

Conjugation of plasminogen activators and fibrin-specific antibodies to improve thrombolytic therapeutic agents.

Here we have reviewed chemical and recombinant approaches to the construction of hybrid molecules that combine a "targeting" antibody and an "effector" enzyme activity. There are advantages and disadvantages to both chemical and recombinant methods, and one goal of this review has been to elucidate these so that the appropriate method can be used by those interested in using hybrid molecules to study questions of basic or therapeutic importance. The system studied in greatest detail has as its goal the targeting of a plasminogen activator to an occlusive intravascular thrombus. We have, therefore, used this system as an example of currently available approaches. Now that these methodologies have been studied and put into use, it is anticipated that this principle will be generalized both to other therapeutic applications, as well as to the design and construction of molecules that will allow more basic questions to be addressed.

Import of rat liver mitochondrial transhydrogenase.

The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin.

Rhodamine 123 inhibits import of rat liver mitochondrial transhydrogenase.

Rhodamine 123, a laser dye, has been demonstrated to inhibit import of the precursor to pyridine dinucleotide transhydrogenase into mitochondria in rat liver cells. When rat hepatocytes were labeled with 35[S] methionine in the presence of 0.4 mM rhodamine 123, the precursor to transhydrogenase was found to have a half-life in the cytoplasm of 15 minutes as opposed to a half-life of 1-2 minutes when cells were radiolabeled in the absence of the dye. To clarify the mechanism of import inhibition, studies were initiated to assess the effect of rhodamine 123 on mitochondrial respiration. Upon addition of the dye to a mitochondrial suspension, respiration was initially enhanced, then inhibited. The inability of FCCP, a classical uncoupler, to enhance respiration during the inhibitory phase suggests that rhodamine 123 is primarily inhibiting respiration through the electron transport system rather than through the ATPase. These results suggest that rhodamine 123 may inhibit import of the transhydrogenase precursor into mitochondria by disrupting components in the mitochondrial membrane necessary for efficient import.

Biosynthesis of rat liver transhydrogenase in vivo and in vitro.

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.