Large-scale in silico mapping of complex quantitative traits in inbred mice

Understanding the genetic basis of common disease and disease-related quantitative traits will aid in the development of diagnostics and therapeutics. The processs of gene discovery can be sped up by rapid and effective integration of well-defined mouse genome and phenome data resources. We describe here an in silico gene-discovery strategy through genome-wide association (GWA) scans in inbred mice with a wide range of genetic variation. We identified 937 quantitative trait loci (QTLs) from a survey of 173 mouse phenotypes, which include models of human disease (atherosclerosis, cardiovascular disease, cancer and obesity) as well as behavioral, hematological, immunological, metabolic, and neurological traits. 67% of QTLs were refined into genomic regions <0.5 Mb with ∼40-fold increase in mapping precision as compared with classical linkage analysis. This makes for more efficient identification of the genes that underlie disease. We have identified two QTL genes, Adam12 and Cdh2, as causal genetic variants for atherogenic diet-induced obesity. Our findings demonstrate that GWA analysis in mice has the potential to resolve multiple tightly linked QTLs and achieve single-gene resolution. These high-resolution QTL data can serve as a primary resource for positional cloning and gene identification in the research community

Principles for the post-GWAS functional characterisation of risk loci

Several challenges lie ahead in assigning functionality to susceptibility SNPs. For example, most effect sizes are small relative to effects seen in monogenic diseases, with per allele odds ratios usually ranging from 1.15 to 1.3. It is unclear whether current molecular biology methods have enough resolution to differentiate such small effects. Our objective here is therefore to provide a set of recommendations to optimize the allocation of effort and resources in order maximize the chances of elucidating the functional contribution of specific loci to the disease phenotype. It has been estimated that 88% of currently identified disease-associated SNP are intronic or intergenic. Thus, in this paper we will focus our attention on the analysis of non-coding variants and outline a hierarchical approach for post-GWAS functional studies

The role of neutrophil myeloperoxidase in models of lung tumor development

Chronic inflammation plays a key tumor-promoting role in lung cancer. Our previous studies in mice demonstrated that neutrophils are critical mediators of tumor promotion in methylcholanthrene (MCA)-initiated, butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated the role of neutrophil myeloperoxidase (MPO) activity in this inflammation promoted model. Increased levels of MPO protein and activity were present in the lungs of mice administered BHT. Treatment of mice with N-acetyl lysyltyrosylcysteine amide (KYC), a novel tripeptide inhibitor of MPO, during the inflammatory stage reduced tumor burden. In a separate tumor model, KYC treatment of a Lewis Lung Carcinoma (LLC) tumor graft in mice had no effect on tumor growth, however, mice genetically deficient in MPO had significantly reduced LLC tumor growth. Our observations suggest that MPO catalytic activity is critical during the early stages of tumor development. However, during the later stages of tumor progression, MPO expression independent of catalytic activity appears to be required. Our studies advocate for the use of MPO inhibitors in a lung cancer prevention setting

Signaling mechanisms of the semaphorin receptor plexin -B1.

Semaphorins are chemorepulsive molecules that signal via plexin-containing receptor complexes to effect changes in the cytoskeleton required for growth cone turning or collapse. Semaphorin 4D is one such member which directly interacts with plexin-B1. The biochemical signaling mechanisms downstream of this receptor have not been well characterized, however, they appear to involve regulation of members of the Rho family of small GTPases which is a method common to other guidance receptor signaling systems. We observed that the intracellular domain of plexin-B1 is able to directly interact with the Rae GTPase. Rho and Cde42 do not interact with plexin-B1 and the interaction is specific to plexin-B1 and not plexin-A or -C family members. The interaction is dependent on the effector domain of Rae and was found to be, enhanced upon Sema4D co-expression. We also observed that plexin-B1 can compete with PAK for Rae and inhibit Rae-induced PAK activation. This effectively sequesters Rae activity. This is consistent with events downstream of other repulsive guidance receptors where Rae and Cdc42 are inhibited and Rho is activated. We also observed that expression of active Rae can enhance the ability of plexin-B1 to interact with Sema4D. Active Rae is able to stimulate the number of receptors at the cell surface without affecting plexin-B1 expression. To further direct signaling events at the level of the plexin-B1 receptor, we purified a plexin-B1 interacting protein from mouse brain called LARG. LARG is a known exchange factor for Rho and our analysis show that the PDZ domain of LARG directly interacts with the C-terminal type I PDZ-binding site of plexin-B1. Furthermore, Sema-4D stimulated activation of Rho is dependent on the interaction of plexin-B1 with LARG. All together our observations provide molecular descriptions for the role of Rae and Rho GTPases downstream of the plexin-B1 receptor.Ph.D.BiochemistryBiological SciencesNeurosciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123490/2/3079543.pd

The Plexin-B1/Rac interaction inhibits PAK activation and enhances Sema4D ligand binding

The small GTPase Rac has been implicated in growth cone guidance mediated by semaphorins and their receptors. Here we demonstrate that plexin-B1, a receptor for Semaphorin4D (Sema4D), and p21-activated kinase (PAK) can compete for the interaction with active Rac and plexin-B1 can inhibit Rac-induced PAK activation. We have also demonstrated that expression of active Rac enhances the ability of plexin-B1 to interact with Sema4D. Active Rac stimulates the localization of plexin-B1 to the cell surface. The enhancement in Sema4D binding depends on the ability of Rac to bind plexin-B1. These observations support a model where signaling between Rac and plexin-B1 is bidirectional; Rac modulates plexin-B1 activity and plexin-B1 modulates Rac function