Genome-Wide Association Studies and the Problem of Relatedness Among Advanced Intercross Lines and Other Highly Recombinant Populations

Model organisms offer many advantages for the genetic analysis of complex traits. However, identification of specific genes is often hampered by a lack of recombination between the genomes of inbred progenitors. Recently, genome-wide association studies (GWAS) in humans have offered gene-level mapping resolution that is possible because of the large number of accumulated recombinations among unrelated human subjects. To obtain analogous improvements in mapping resolution in mice, we used a 34th generation advanced intercross line (AIL) derived from two inbred strains (SM/J and LG/J). We used simulations to show that familial relationships among subjects must be accounted for when analyzing these data; we then used a mixed model that included polygenic effects to address this problem in our own analysis. Using a combination of F₂ and AIL mice derived from the same inbred progenitors, we identified genome-wide significant, subcentimorgan loci that were associated with methamphetamine sensitivity, (e.g., chromosome 18; LOD = 10.5) and non-drug-induced locomotor activity (e.g., chromosome 8; LOD = 18.9). The 2-LOD support interval for the former locus contains no known genes while the latter contains only one gene (Csmd1). This approach is broadly applicable in terms of phenotypes and model organisms and allows GWAS to be performed in multigenerational crosses between and among inbred strains where familial relatedness is often unavoidable.SUSCEPTIBILITY to diseases such as drug abuse is partially determined by genetic factors. The identification of the alleles that underlie disease susceptibility is an immensely important goal that promises to revolutionize both the diagnosis and the treatment of human disease. Genome-wide association studies (GWAS) in humans can locate common alleles with great precision. However, GWAS may be unable to identify the bulk of the heritable variability for common genetic diseases; some of this “missing heritability” is thought to be due to rare alleles (Manolio et al. 2009). Model organisms are complementary to human genetic studies and offer unique advantages including the ability to control the environment, perform dangerous or invasive procedures, and test hypotheses by manipulating genes via genetic engineering; a final advantage is that crosses between two inbred strains avoid many of the difficulties associated with rare alleles.Studies in model organisms have frequently employed intercrosses (F₂''s) to identify quantitative trait loci (QTL) that underlie phenotypic variability. F₂ crosses are easy to produce and easy to analyze; however, due to a lack of recombination they can identify only larger genomic regions and are thus unsuitable for identifying the genes that cause QTL (Flint et al. 2005; Peters et al. 2007). This is a serious limitation that can be addressed by using populations with greater numbers of accumulated recombinations. Darvasi and Soller (1995) suggested the creation of advanced intercross lines (AILs) by successive generations of random mating after the F₂ generation to produce additional recombinations. An AIL offers vastly improved mapping resolution while maintaining the desirable property that all polymorphic alleles are common.We used an AIL to study sensitivity to methamphetamine, which is a genetically complex trait that may be useful for identifying genetic factors influencing the subjectively euphoric response to stimulant drugs and susceptibility to drug abuse (Palmer et al. 2005; Phillips et al. 2008; Bryant et al. 2009). For example, a prior study suggested that the gene Casein Kinase 1 Epsilon (Csnk1e) might influence sensitivity to the acute locomotor response to methamphetamine in mice (Palmer et al. 2005). This conclusion has been bolstered by additional pharmacological (Bryant et al. 2009) and genetic studies. In addition, we have shown that polymorphisms in this gene are associated with sensitivity to the euphoric effects of amphetamine in humans (Veenstra-Vanderweele et al. 2006). Another group has subsequently reported that this same gene is associated with heroine addiction (Levran et al. 2008). Thus, genes that modulate the acute locomotor response to a drug in mice may also be important for sensitivity to similar drugs in humans as well as the risk for developing drug abuse.The purpose of this study was to develop a framework for rapid identification of high precision QTL and ideally specific genes that influence sensitivity to methamphetamine in mice by employing an AIL. We produced an F₂ cross (n = 490) and a corresponding 34th generation AIL (n = 688) derived from the inbred strains SM/J and LG/J. This allowed us to compare and integrate the results from F₂ and AIL mice. We examined the locomotor stimulant response to a 2-mg/kg dose of methamphetamine, which is extremely disparate in the two progenitor strains. We performed a GWAS using either simple regression, which ignored relatedness, or a mixed model that accounted for relatedness by using identity coefficients that were calculated from the pedigree. We also explored two methods to estimate significance: simple permutation and gene dropping. We discuss the performance of a mixed model that includes polygenic effects vs. simple regression and the performance of permutation vs. gene dropping. The methods used in this study are applicable to a variety of other phenotypes and populations.     

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.     

Patterns of Natural and Human-Caused Mortality Factors of a Rare Forest Carnivore,the Fisher (Pekania pennanti) in California

Wildlife populations of conservation concern are limited in distribution, population size and persistence by various factors, including mortality. The fisher (Pekania pennanti), a North American mid-sized carnivore whose range in the western Pacific United States has retracted considerably in the past century, was proposed for threatened status protection in late 2014 under the United States Endangered Species Act by the United States Fish and Wildlife Service in its West Coast Distinct Population Segment. We investigated mortality in 167 fishers from two genetically and geographically distinct sub-populations in California within this West Coast Distinct Population Segment using a combination of gross necropsy, histology, toxicology and molecular methods. Overall, predation (70%), natural disease (16%), toxicant poisoning (10%) and, less commonly, vehicular strike (2%) and other anthropogenic causes (2%) were causes of mortality observed. We documented both an increase in mortality to (57% increase) and exposure (6%) from pesticides in fishers in just the past three years, highlighting further that toxicants from marijuana cultivation still pose a threat. Additionally, exposure to multiple rodenticides significantly increased the likelihood of mortality from rodenticide poisoning. Poisoning was significantly more common in male than female fishers and was 7 times more likely than disease to kill males. Based on necropsy findings, suspected causes of mortality based on field evidence alone tended to underestimate the frequency of disease-related mortalities. This study is the first comprehensive investigation of mortality causes of fishers and provides essential information to assist in the conservation of this species.     

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and β clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.     

Influence of oral contraceptive use on endothelial t-PA release in healthy premenopausal women

We determined the influence of oral contraceptives (OC) on the capacity of the endothelium to release tissue-type plasminogen activator (t-PA). Twenty-three healthy premenopausal women were studied: 12 nonusers and 11 users of OC. Net endothelial release rates of t-PA were calculated as the product of the arteriovenous concentration gradient and forearm plasma flow in response to intra-arterial bradykinin (BK: 12.5-50 ng. 100 ml tissue(-1) x min(-1)) and sodium nitroprusside (SNP: 1.0-4.0 microg x 100 ml tissue(-1) x min(-1)). Net release of t-PA antigen and increment in t-PA activity across the forearm to BK increased (P < 0.01) in a dose-dependent fashion and to similar extents in the nonusers and users of OC. At the highest BK dose, net release of t-PA antigen was 64.5 +/- 8.2 and 66.2 +/- 15.4 ng x 100 ml tissue(-1) x min(-1) in the nonusers and users of OC, whereas the net increment in t-PA activity was 18.6 +/- 3.0 and 16.0 +/- 2.0 IU. 100 ml tissue(-1) x min(-1), respectively. There was no effect of SNP on t-PA release in either group. These results indicate that endothelial t-PA release is not altered in premenopausal women who use oral contraception.     

Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.     

Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L⁻¹ production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.     

Kinetic model of enzymatic hydrolysis of steam-exploded wheat straw

The hydrolysis kinetics of steam-exploded wheat straw treated with cellulase NS 50013 enzyme complex in combination with β-glucosidase NS 50010 is studied. The time dependence of the reducing sugars amount is followed at varying the temperature value and the amount of the enzyme introduced. The activation energy determined on the ground of the rate temperature dependence stays unchanged in the course of the process. The preexponential factor decreases with the increase of the degree of hydrolysis and is responsible for the process rate decrease. A new expression for the dependence of degree of hydrolysis of one of carbohydrate polymers (cellulose) in wheat straw on the time, the enzyme concentration and the temperature is obtained. It is of practical importance as well because it provides estimation of the degree of hydrolysis required at predetermined values of the temperature, the enzyme concentration and the time used. The expression can be used for control of the enzyme hydrolysis of cellulose in the wheat straw.