Background-Pharmacogenetic-guided dosing of warfarin is a promising application of "personalized medicine" but has not been adequately tested in randomized trials. Methods and Results-Consenting patients (nϭ206) being initiated on warfarin were randomized to pharmacogeneticguided or standard dosing. Buccal swab DNA was genotyped for CYP2C9 *2 and CYP2C9 *3 and VKORC1 C1173T with a rapid assay. Standard dosing followed an empirical protocol, whereas pharmacogenetic-guided dosing followed a regression equation including the 3 genetic variants and age, sex, and weight.

AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer.

The ligand-binding head region of integrin ␤ subunits contains a von Willebrand factor type A domain (␤A). Ligand binding activity is regulated through conformational changes in ␤A, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from ␤A is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the ␤ 1 subunit hybrid domain, which is connected to the lower face of ␤A. Changes in the expression of these epitopes are induced by conformational changes in ␤A caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated ␣ 5 ␤ 1 with a mutation L358A in the ␣7 helix of ␤A has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the ␣ subunit. Taken together, these data suggest that the transduction of conformational changes through ␤A involves shape shifting in the ␣7 helix region, which is linked to a swing of the hybrid domain away from the ␣ subunit.Integrins mediate a wide variety of essential cell-matrix and cell-cell interactions and also participate in many common disease processes (1, 2). Integrins are heterodimers containing non-covalently associated ␣ and ␤ subunits; each subunit has a large extracellular domain linked to a transmembrane segment and a short cytoplasmic tail. Integrins participate in bi-directional signaling; ligand recognition is dynamically regulated by "inside-out" signaling, and ligand occupancy leads to "outsidein" signals that affect cell migration, growth, differentiation, and survival (3-5). Modulation of integrin activity is essential in such processes as leukocyte migration to sites of tissue injury and the aggregation of platelets to form a hemostatic plug. Integrin activation can be mimicked in vitro by divalent cations such as Mn 2ϩ or Mg 2ϩ (6). Three major conformational states of integrins can be distinguished using monoclonal antibodies (mAbs) 1 : an inactive (resting or low affinity) state, an active (or high affinity) state, and a ligand-occupied state (7). The conformations of the inactive and active states are discriminated by low and high expression, respectively, of activation epitopes (such as those recognized by 12G10, 15/7, and 9EG7 for the ␤ 1 subunit, see Refs. 8 -10). The ligand-occupied conformer expresses high levels of ligand-induced binding site (LIBS) epitopes (which are generally also activation epitopes) and shows decreased expression of ligand-attenuated binding site (LABS) epitopes (such as mAb 13 for the ␤ 1 subunit, see Ref. 11). The conformational states are in equilibrium; therefore, antibodies that recognize activation epitopes or LIBS tend to cause activation and stabilize the ligand-occupied state. Conversely, antibodies that recognize LABS appear to block ligand binding by preventing conformati...

Background-Warfarin is characterized by marked variations in individual dose requirements and a narrow therapeutic window. Pharmacogenetics (PG) could improve dosing efficiency and safety, but clinical trials evidence is meager. Methods and Results-A Randomized and Clinical Effectiveness Trial Comparing Two Pharmacogenetic Algorithms andStandard Care for Individualizing Warfarin Dosing (CoumaGen-II) comprised 2 comparisons: (1) a blinded, randomized comparison of a modified 1-step (PG-1) with a 3-step algorithm (PG-2) (Nϭ504), and (2) a clinical effectiveness comparison of PG guidance with use of either algorithm with standard dosing in a parallel control group (Nϭ1866). A rapid method provided same-day CYP2C9 and VKORC1 genotyping. Primary outcomes were percentage of out-of-range international normalized ratios at 1 and 3 months and percentage of time in therapeutic range. Primary analysis was modified intention to treat. In the randomized comparison, PG-2 was noninferior but not superior to PG-1 for percentage of out-of-range international normalized ratios at 1 month and 3 months and for percentage of time in therapeutic range at 3 months. However, the combined PG cohort was superior to the parallel controls (percentage of out-of-range international normalized ratios 31% versus 42% at 1 month; 30% versus 42% at 3 months; percentage of time in therapeutic range 69% versus 58%, 71% versus 59%, respectively, all PϽ0.001). Differences persisted after adjustment for age, sex, and clinical indication. There were fewer percentage international normalized ratios Ն4 and Յ1.5 and serious adverse events at 3 months (4.5% versus 9.4% of patients, PϽ0.001) with PG guidance. Conclusions-These findings suggest that PG dosing should be considered for broader clinical application, a proposal that is being tested further in 3 major randomized trials. The simpler 1-step PG algorithm provided equivalent results and may be preferable for clinical application. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00927862. Key Words: anticoagulants Ⅲ clinical trial Ⅲ genetics Ⅲ pharmacogenetics Ⅲ warfarin P harmacogenomics, the study of interactions of genetics with pharmacotherapy, is a promising area for applying genetics to personalized or precision medicine. [1][2][3] Warfarin is prescribed to over 2 million patients in the United States for prevention of thromboembolic events associated with atrial fibrillation, venous and arterial thrombosis, orthopedic surgery, and prosthetic heart valves. Unfortunately, clinical management is difficult because of a narrow therapeutic index and marked interpatient variability in drug pharmacokinetics and pharmacodynamics, which lead to unpredictable and variable (up to 10-fold or greater) dosing requirements. 4 Anticoagulation trials for nonrheumatic atrial fibrillation have determined the optimal prothrombin time international normalized ratio (INR) range to be 2 to 3 with ratios Ͻ2 increasing thrombotic events and those Ͼ4 increasing hemorrhagic events. 5...

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cationbinding sites are found in both ␣ and ␤ integrin subunits. A key cation-binding site that lies in the ␤ subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin ␣ V ␤ 3 have identified a novel cation binding site in this domain, known as the ADMIDAS (adjacent to MIDAS). The role of this novel site in ligand recognition has yet to be elucidated. Using the interaction between ␣ 5 ␤ 1 and fibronectin as a model system, we show that mutation of residues that form the ADMIDAS site inhibits ligand binding but this effect can be partially rescued by the use of activating monoclonal antibodies. The ADMIDAS mutants had decreased expression of activation epitopes recognized by 12G10, 15/7, and HUTS-4, suggesting that the ADMIDAS is important for stabilizing the active conformation of the integrin. Consistent with this suggestion, the ADMIDAS mutations markedly increased the dissociation rate of the integrin-fibronectin complex. Mutation of the ADMIDAS residues also reduced the allosteric inhibition of Mn 2؉ -supported ligand binding by Ca 2؉ , suggesting that the ADMIDAS is a Ca 2؉ -binding site involved in the inhibition of Mn 2؉ -supported ligand binding. Mutations of the ADMIDAS site also perturbed transduction of a conformational change from the MIDAS through the C-terminal helix region of the ␤A domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.Integrins constitute a large family of ␣/␤ heterodimeric transmembrane receptors found in all metazoa (1). Cell-matrix and cell-cell interactions mediated by integrins are central to many fundamental biological processes such as embryonic morphogenesis, leukocyte trafficking, and platelet aggregation. Integrins can exist in either active (ligand competent) or inactive conformational states; the equilibrium between these two states is regulated intracellularly by the binding of cytoskeletal and signaling molecules (2, 3). Integrin-ligand interactions also require divalent cations and are regulated in a complex manner by changes in the concentrations of these ions. The effects of activation can be mimicked in vitro by cations such as Mn 2ϩ or Mg 2ϩ , whereas Ca 2ϩ typically favors the inactive state. The different effects of these cations are related to their differential abilities to induce the integrin to undergo the shape changes involved in activation (4 -6).The molecular basis of integrin function has been greatly elucidated by x-ray crystal structures of the extracellular domains of ␣ V ␤ 3 in the unliganded and liganded states (7,8). The overall structure of the heterodimer is that of a "head" on two "legs." The head region (where ligand binding takes place) comprises a seven-bladed ␤-propeller in the ␣ subunit and a von Willebrand factor A-type domain in the ␤ subunit (␤A 1 ; also referred to as "I-like domain"), an ␣,␤-fold, which is inserted by short N-a...

The ligand-binding region of integrin ␤ subunits contains a von Willebrand factor type A-domain: an ␣/␤ "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the ␤A-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the ␤ 1 A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). 2؉ acts as a potent activator of ␤ 1 integrins because it can promote a shift in the position of this helix. The mechanism of ␤ subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin ␣ subunits.

Ellis-van Creveld syndrome (EvC) is caused by mutations in EVC and EVC2, genes in a divergent orientation separated by only 2.6 kb. We systematically sought mutations in both genes in a panel of 65 affected individuals to assess the proportion of cases resulting from mutations in each gene. We PCR amplified and sequenced the coding exons of both genes. We investigated mutations that could affect splicing by in vitro splicing assays and cDNA analysis. We have identified EVC mutations in 20 cases (31%); in all of these we have detected the mutation on each allele. We have identified EVC2 mutations in 25 cases (38%); in 22 of these we have isolated a mutation on each allele. The majority of the mutations introduce a premature termination codon. We sequenced the region between the two genes in 10 of the 20 cases in which we had not identified a mutation in either gene, revealing only one SNP that was not a common polymorphism. As we have not identified mutations in either gene in 20 cases (31%) it is possible that there is further genetic heterogeneity.

Background Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. Methods We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). Results Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. Conclusions Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.