A Scale of Agonism and Allosteric Modulation for Assessment of Selectivity, Bias, and Receptor Mutation.

An index of agonism is described that can be used to quantify agonist receptor selectivity, bias, cell-based agonism, and the effects of receptor mutation on signaling. The parameter is derived from agonist concentration-response curves and comprises the maximal response to the agonist (max) and the EC50 in the form of Δlog(max/EC50). This parameter is derived from equations describing agonists as positive allosteric facilitators of receptor-signaling protein interaction. A similar index is also derived to quantify the potentiating effects of positive allosteric modulators, which can be used to quantify in situ positive allosteric modulator activity in vivo. These indices lend themselves to statistical analysis and are system-independent in that the effects of the system processing of agonist response and differences in assay sensitivity and receptor expression are cancelled. The various applications of the Δlog(max/EC50) scale are described for each pharmacologic application.

The route of HIV escape from immune response targeting multiple sites is determined by the cost-benefit tradeoff of escape mutations.

Cytotoxic T lymphocytes (CTL) are a major factor in the control of HIV replication. CTL arise in acute infection, causing escape mutations to spread rapidly through the population of infected cells. As a result, the virus develops partial resistance to the immune response. The factors controlling the order of mutating epitope sites are currently unknown and would provide a valuable tool for predicting conserved epitopes. In this work, we adapt a well-established mathematical model of HIV evolution under dynamical selection pressure from multiple CTL clones to include partial impairment of CTL recognition, [Formula: see text], as well as cost to viral replication, [Formula: see text]. The process of escape is described in terms of the cost-benefit tradeoff of escape mutations and predicts a trajectory in the cost-benefit plane connecting sequentially escaped sites, which moves from high recognition loss/low fitness cost to low recognition loss/high fitness cost and has a larger slope for early escapes than for late escapes. The slope of the trajectory offers an interpretation of positive correlation between fitness costs and HLA binding impairment to HLA-A molecules and a protective subset of HLA-B molecules that was observed for clinically relevant escape mutations in the Pol gene. We estimate the value of [Formula: see text] from published experimental studies to be in the range (0.01-0.86) and show that the assumption of complete recognition loss ([Formula: see text]) leads to an overestimate of mutation cost. Our analysis offers a consistent interpretation of the commonly observed pattern of escape, in which several escape mutations are observed transiently in an epitope. This non-nested pattern is a combined effect of temporal changes in selection pressure and partial recognition loss. We conclude that partial recognition loss is as important as fitness loss for predicting the order of escapes and, ultimately, for predicting conserved epitopes that can be targeted by vaccines.

Autosomal dominant vasovagal syncope: clinical features and linkage to chromosome 15q26.

OBJECTIVE: To establish the occurrence of an autosomal dominant form of vasovagal syncope (VVS) by detailed phenotyping of multiplex families and identification of the causative locus. METHODS: Patients with VVS and a family history of syncope were recruited. A standardized questionnaire was administered to all available family members and medical records were reviewed. Of 44 families recruited, 6 were suggestive of autosomal dominant inheritance. Genome-wide linkage was performed in family A using single nucleotide polymorphism genotyping microarrays. Targeted analysis of chromosome 15q26 with microsatellite markers was implemented in 4 families; 1 family was too small for analysis. RESULTS: Family A contained 30 affected individuals over 3 generations with a median onset of 8 to 9 years. The other families comprised 4 to 14 affected individuals. Affected individuals reported typical triggers of VVS (sight of blood, injury, medical procedures, prolonged standing, pain, frightening thoughts). The triggers varied considerably within the families. Significant linkage to chromosome 15q26 (logarithm of odds score 3.28) was found in family A. Linkage to this region was excluded in 2 medium-sized families but not in 2 smaller families. Sequence analysis of the candidate genes SLCO3A1, ST8SIA2, and NR2F2 within the linkage interval did not reveal any mutations. CONCLUSIONS: Familial VVS, inherited in an autosomal dominant manner, may not be rare and has similar features to sporadic VVS. The chromosome 15q26 locus in family A increases the susceptibility to VVS but does not predispose to a particular vasovagal trigger. Linkage analysis in the remaining families established likely genetic heterogeneity.

Frequency, spectrum, and nonzero fitness costs of resistance to myxopyronin in Staphylococcus aureus.

The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 × 10(-8) to 7 × 10(-8) per generation, which is equal within error to the resistance rate for Rif (3 × 10(-8) to 10 × 10(-8) per generation). Substitutions conferring Myx resistance were obtained in the RNAP ß subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127(1322)P] and the RNAP ß' subunit [five substitutions: K334(345)N, T925(917)K, T925(917)R, G1172(1354)C, and G1172(1354)D] (residues numbered as in Staphylococcus aureus RNAP and, in parentheses, as in Escherichia coli RNAP). Sites of substitutions conferring Myx resistance map to the RNAP switch region SW1/SW2 subregion and do not overlap the binding site on RNAP for Rif, and, correspondingly, Myx-resistant mutants exhibit no cross-resistance to Rif. All substitutions conferring Myx resistance exhibit significant fitness costs (4 to 15% per generation). In contrast, at least three substitutions conferring Rif resistance exhibit no fitness costs (≤0% per generation). The observation that all Myx-resistant mutants have significant fitness costs whereas at least three Rif-resistant mutants have no fitness costs, together with the previously established inverse correlation between fitness cost and clinical prevalence, suggests that Myx resistance is likely to have lower clinical prevalence than Rif resistance.

[The cost of antibiotic resistance: analysis and consequences]. / Coût biologique de la résistance aux antibiotiques : analyse et conséquences.

Antimicrobial resistance, either by mutation or acquisition of resistance determinants harbored by mobile genetic elements, may confer a biological cost for the bacteria. This biological cost can be evaluated by comparing the resistant mutant to the wild susceptible strain, in the absence of antibiotic selection. This fitness cost can affect the growth rate in vitro or the survival in the host or in the environment or the virulence capacity. Various studies have evidenced this cost, either in vitro or in vivo, in different analysis models. However, bacteria can evolve and adapt to reduce this cost, by compensatory mutations or fine regulation of resistance expression. This compensatory evolution allows resistant bacteria to persist even in the absence of antibiotic selection pressure.

Myelin protein zero Arg36Gly mutation with very late onset and rapidly progressive painful neuropathy.

Mutations in myelin protein zero (MPZ) protein result in a wide spectrum of peripheral neuropathies, from congenital hypomyelinating to late onset sensory and motor axonal forms. In some patients, neuropathic pain can be a prominent symptom, making the diagnosis challenging mainly in those with other risk factors for neuropathy. We describe a 77-year-old woman with impaired glucose tolerance presenting with rapidly progressive axonal neuropathy leading to excruciating pain and severe weakness of lower limbs within 2 years from the onset. Her son abruptly complained of similar painful symptoms at the age of 47 years. Molecular analysis revealed a novel heterozygous missense mutation (c.106A>G) in MPZ exon 2, causing the substitution of arginine-36 with glycine in the extracellular domain. Our observation suggests that MPZ-related neuropathy should be considered in the diagnostic work up of patients with painful axonal neuropathy even presenting with rapid progression and at a very late age of onset.

Chronic methadone treatment shows a better cost/benefit ratio than chronic morphine in mice.

Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading µ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.

Kinetic modeling of Nav1.7 provides insight into erythromelalgia-associated F1449V mutation.

Gain-of-function mutations of the voltage-gated sodium channel (VGSC) Na(v)1.7 have been linked to human pain disorders. The mutation F1449V, located at the intracellular end of transmembrane helix S6 of domain III, induces the inherited pain syndrome erythromelalgia. A kinetic model of wild-type (WT) and F1449V Na(v)1.7 may provide a basis for predicting putative intraprotein interactions. We semiautomatically constrained a Markov model using stochastic search algorithms and whole cell patch-clamp recordings from human embryonic kidney cells transfected with Na(v)1.7 and its F1449V mutation. The best models obtained simulated known differences in action potential thresholds and firing patterns in spinal sensory neurons expressing WT and F1449V. The most suitable Markov model consisted of three closed, one open, and two inactivated states. The model predicted that the F1449V mutation shifts occupancy of the closed states closer to the open state, making it easier for the channel pore to open. It also predicted that F1449V's second inactivated state is more than four times more likely to be occupied than the equivalent state in WT at hyperpolarized potentials, although the mutation still lowered the firing threshold of action potentials. The differences between WT and F1449V were not limited to a single transition. Thus a point mutation in position F1449, while phenotypically most probably affecting the activation gate, may also modify channel functions mediated by structures in more distant areas of the channel protein.

Inclusive fitness from multitype branching processes.

I use multitype branching processes to study genetic models for the evolution of social behaviour, i.e. behaviours that, when acted out, affect the success of the actor's neighbours. Here, I suppose an individual bearing a mutant copy of a gene influences the reproductive success of a neighbour by altering its own competitive ability. Approximations based on assumptions about the rareness of the mutant allele and the strength of selection allow me to formulate statements concerning the probability of mutant extinction in terms of inclusive fitness. Inclusive fitness is an idea well known to biologists and can be thought of as a sum of an individual's fitness and the fitness of each of its relatives, weighted by some measure of genetic relatedness. Previous work has led to some confusion surrounding the definition of the inclusive-fitness effect of a mutant allele when individuals carrying that allele experience demographic conditions that fluctuate randomly. In this paper, I emphasise the link between inclusive fitness and the probability of mutant extinction. I recover standard results for populations of constant size, and I show that inclusive fitness can be used to determine the short-term fate of mutants in the face of stochastic demographic fluctuations. Overall, then, I provide a connection between certain inclusive-fitness-based approaches routinely applied in theoretical studies of social evolution.

BRCA carriers' thoughts on risk management in relation to preimplantation genetic diagnosis and childbearing: when too many choices are just as difficult as none.

Women with a BRCA mutation have unique concerns about childbearing and future fertility. In a focus group conducted among unaffected carriers, the majority of women held positive attitudes toward preimplantation genetic diagnosis to reduce transmission to future offspring and further identified unmet needs for education and support for decision making.

A report of fulminant malignant hyperthermia in a patient with a novel mutation of the CACNA1S gene.

PURPOSE: To report the identification of a novel mutation in the CACNA1S gene that encodes the alpha-1-subunit (Cav1.1) of the voltage-gated skeletal muscle L-type calcium channel in a patient with malignant hyperthermia. CLINICAL FINDINGS: An otherwise healthy 34-yr-old female developed fulminant malignant hyperthermia (MH) under sevoflurane anesthesia during laparoscopic donor nephrectomy. The first sign was an increase in end-tidal CO(2). Malignant hyperthermia was suspected early, and resuscitative measures, including supportive and specific treatment, were successfully implemented. The patient rejected the open muscle biopsy for the Caffeine-Halothane Contracture Test (CHCT); therefore, only molecular genetic testing was performed. Sequencing of the entire ryanodine receptor type 1 transcript did not reveal any MH causative mutations. However, a novel homozygous mutation, p.Arg1086Ser, was identified in the CACNA1S gene that encoded for the alpha-1-subunit of the skeletal muscle L-type calcium channel (Cav1.1). A CACNA1S mutation, p.Arg1086His, involving the same Arg1086 residue that is mutated in our patient has previously been reported in association with MH in three independent families. CONCLUSION: The homozygous p.Arg1086Ser mutation of CACNA1S, the gene that encodes the alpha-1-subunit of the voltage-gated skeletal muscle L-type calcium channel, is a novel mutation associated with malignant hyperthermia.

Oscillatory dynamics in rock-paper-scissors games with mutations.

We study the oscillatory dynamics in the generic three-species rock-paper-scissors games with mutations. In the mean-field limit, different behaviors are found: (a) for high mutation rate, there is a stable interior fixed point with coexistence of all species; (b) for low mutation rates, there is a region of the parameter space characterized by a limit cycle resulting from a Hopf bifurcation; (c) in the absence of mutations, there is a region where heteroclinic cycles yield oscillations of large amplitude (not robust against noise). After a discussion on the main properties of the mean-field dynamics, we investigate the stochastic version of the model within an individual-based formulation. Demographic fluctuations are therefore naturally accounted and their effects are studied using a diffusion theory complemented by numerical simulations. It is thus shown that persistent erratic oscillations (quasi-cycles) of large amplitude emerge from a noise-induced resonance phenomenon. We also analytically and numerically compute the average escape time necessary to reach a (quasi-)cycle on which the system oscillates at a given amplitude.

Beta-adrenergic modulation of arrhythmogenesis and identification of targeted sites of antiarrhythmic therapy in Timothy (LQT8) syndrome: a theoretical study.

Timothy syndrome (TS) is a malignant form of congenital long QT syndrome with a mode of arrhythmia onset often triggered by enhanced sympathetic tone. We sought to explore mechanisms by which beta-adrenergic stimulation (BAS) modulates arrhythmogenesis and to identify potential targeted sites of antiarrhythmic therapy in TS. Using a dynamic Luo-Rudy ventricular myocyte model incorporated with detailed intracellular Ca(2+) cycling, along with its one-dimensional multicellular strand, we simulated various clinical scenarios of TS, with stepwise increase in the percentage of G406R Ca(v)1.2 channels from 0 to 11.5 and 23%, and to 38.5 and 77%, respectively, for heterozygous and homozygous states of TS1 and TS2. Progressive prolongation of action potential duration (APD) and QT interval, accompanied by amplification of transmural dispersion of repolarization, steepening of APD restitution, induction of delayed afterdepolariztions (DADs), and both DAD and phase 3 early afterdepolariztion-mediated triggered activities, correlated well with the extent of G406R Ca(v)1.2 channel mutation. BAS amplified transmural dispersion of repolarization, steepened APD restitution, and facilitated inducibility of DAD-mediated triggered activity. Systematic analysis of intracellular Ca(2+) cycling revealed that sarcoplasmic reticulum Ca(2+) ATPase (uptake current) played an essential role in BAS-induced facilitation of DAD-mediated triggered activity and, in addition to L-type calcium current, it could be an effective site of antiarrhythmic therapy under the influence of BAS. Thus G406R Ca(v)1.2 channel mutation confers not only a trigger, but also a substrate for lethal ventricular arrhythmias, which can be exaggerated by BAS. It is suggested that, besides beta-adrenergic blockers and L-type calcium current channel blockers, an agent aimed at reduction of sarcoplasmic reticulum Ca(2+) ATPase uptake current may provide additional antiarrhythmic effect in patients with TS.

Ancestral population genomics: the coalescent hidden Markov model approach.

With incomplete lineage sorting (ILS), the genealogy of closely related species differs along their genomes. The amount of ILS depends on population parameters such as the ancestral effective population sizes and the recombination rate, but also on the number of generations between speciation events. We use a hidden Markov model parameterized according to coalescent theory to infer the genealogy along a four-species genome alignment of closely related species and estimate population parameters. We analyze a basic, panmictic demographic model and study its properties using an extensive set of coalescent simulations. We assess the effect of the model assumptions and demonstrate that the Markov property provides a good approximation to the ancestral recombination graph. Using a too restricted set of possible genealogies, necessary to reduce the computational load, can bias parameter estimates. We propose a simple correction for this bias and suggest directions for future extensions of the model. We show that the patterns of ILS along a sequence alignment can be recovered efficiently together with the ancestral recombination rate. Finally, we introduce an extension of the basic model that allows for mutation rate heterogeneity and reanalyze human-chimpanzee-gorilla-orangutan alignments, using the new models. We expect that this framework will prove useful for population genomics and provide exciting insights into genome evolution.

Grand canonical free-energy calculations of protein-ligand binding.

The principles behind the computation of protein-ligand binding free energies by Monte Carlo simulation in the Grand Canonical Ensemble are described in detail, and two variations of the calculation are presented. The computation can be performed by bathing a protein binding site either with ligand images that interact with each other or with ligand images that pass through each other. The second method is theoretically more rigorous, but we show that both methods lead to the same result, and there are distinct numeric advantages to using ligand images that interact with each other. The Grand Canonical simulation provides gas-phase binding free energies that can be converted to aqueous energies by generalized Born-surface area (GB/SA) solvation calculations to provide values that agree with experiment within +/-1.5 kcal/mol. However, the accuracy of these simple solvation calculations is a major limiting factor in the accuracy of the overall binding free-energy computation. The Grand Canonical simulation has several characteristics beneficial to free-energy calculations. One is that the number of parameters that must be set for the simulation is small and can be determined objectively, making the outcome more deterministic, with respect to choice of input conditions, as compared to perturbation methods. Second, the simulation is free from assumptions about the starting pose or nature of the binding site. A final benefit is that binding free energies are a direct outcome of the simulation, and little processing is required to determine them.

MDR1 gene polymorphism: therapeutic response to paroxetine among patients with major depression.

The multidrug resistance transporter, P-glycoprotein (P-gp), encoded by polymorphic MDR1 (ABCB1) gene, is involved in efflux transport of several antidepressants and acts as a barrier to different exogenous noxa in the blood-brain barrier. MDR1 gene belongs to the best understood mediators of drug resistance. Different polymorphisms in MDR1 have been found to be connected with P-gp expression and function. The aims of the study were to investigate the potential influence of MDR1 polymorphisms, exon 26 C3435T and exon 21 G2677T/A, on treatment response to paroxetine (20 mg/day) in patients with major depression. To assess and evaluate therapeutic response to paroxetine, all patients were rated weekly using the HAMD-17 scale. Responders were defined as subjects with a decrease in HAMD scale by >or=50% at week 6 of treatment. The study population included 127 patients with major depression (diagnosed by Structured Clinical Interview for DSM-IV disorders). Our results indicated that MDR1 variants G2677T and C3435T are not associated with therapeutic response to paroxetine in patients with major depressive disorder. The associations between paroxetine and P-glycoprotein still need to be clarified.

Human evolution and its relevance for genetic epidemiology.

The invitation to write the prefatory article to this volume of the Annual Review of Genomics and Human Genetics inspired me to collect some thoughts, a few involving ideas that are not new, but perhaps worth resurrecting in light of recent observations made with the data emerging from the Human Genome Diversity Project (HGDP). Data from the many relevant studies based on the HGDP have been made public, as was originally the hope and plan of the project. Here I try to give a short summary of the evolution of modern humans, a unique species in many respects but of special interest to readers of this volume, and a few thoughts on the general rates of evolution that might be relevant to medical genetics and genetic epidemiology. I have made no attempt to give a general bibliography, not even of results from the HGDP, since most authors' conclusions are still unpublished. Citations are limited to very few general concepts and articles discussed in this preface.

Genetic improvement of processes yielding microbial products.

Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benefit; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal. Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening/selection for higher producing microbial strains and the application of recombinant DNA technology.

Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery.

The concept of constitutively active G-protein-coupled receptors is now firmly rooted in receptor pharmacology. Many independent research groups have contributed to its acceptance since its introduction by Costa and Herz in 1989. This concept necessitated a revised ligand classification, and a new category of inverse agonists was introduced alongside existing agonist and antagonist ligands. Initially, it was hoped that new therapeutic modalities would become available. However, the drug industry has not adopted inverse agonism as a design criterion and instead accepted that some compounds emerge as (neutral) antagonists in compound screening, whereas other compounds possess inverse agonistic activity. In this article, we summarize aspects of the impact of constitutive activity on the drug-discovery process: for example, its use in orphan receptor assays, its link with pharmacogenetics and genomics, and its relevance for currently marketed drugs.