Cell-based assays to probe the ERK MAP kinase pathway in endothelial cells.

To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activated protein (MAP) kinase pathways. MAP kinases are principal regulators of fundamental processes in mammalian cells, including growth, cell division, differentiation, stress responses, and neoplastic transformation. Here we describe two cell-based assays querying the function of ERK (extracellular signal regulated kinase), one of the three principal MAP kinases in mammalian cells. We selected human umbilical vein endothelial cells (HUVECs), a primary cell type, because they show a very dynamic response to various activators. Both assays are phenotypic assays and use well-established phosphorylation-specific primary antibodies to study activation. Fluorochrome-coupled secondary antibodies were used to label phosphorylated target proteins; images were captured with the INCell Analyzer 3000 and analyzed with the INCell Analyzer 3000 software. The first of these two assays monitors phosphorylation of ERK1/2, while the second assay monitors activation of the transcription factor CREB (cAMP response element-binding protein). The assays described in this chapter cover major checkpoints of the ERK signaling pathway: (1) MAP kinase activation and (2) subsequent transcription factor activation. Both assays exhibit robust performance and can easily be used for high-throughput screening.

Relative contributions of genes, environment, and interactions to blood lipid concentrations in a general adult population.

The authors evaluated the contributions of nine genetic (G) variants (selected from 275 single nucleotide polymorphisms in 11 reverse cholesterol transport pathway genes), five environmental (E) factors (selected from 10), and G x G, E x E, and G x E interactions in explaining population variance of blood lipid concentrations. Total cholesterol, triglycerides, and high density lipoprotein (HDL) cholesterol were measured, and low density lipoprotein (LDL) cholesterol and HDL cholesterol/LDL cholesterol ratio were calculated in a population-based random sample of 1,543 men and women in Geneva, Switzerland, aged 35-74 years in 1999-2001. Explained variances (R2) for HDL cholesterol/LDL cholesterol ratio, HDL cholesterol, and LDL cholesterol, respectively, were 34%, 33%, and 19%, decomposed into main effects of G (6%, 4%, and 5%) and E (25%, 28%, and 11%), with just 3%, 2%, and 3% due to G x G, E x E, and G x E interactions, respectively. Risk factor clustering was only moderate: 70% of study subjects had < or =3 variants, 75% had < or =2 environmental exposures, and 69% had < or =5 of both types of factors. Multiple genes with weak associations, together with more dominating environmental factors, are involved in determining blood lipid concentrations. Interactions added little explained variance. Increasing trends in hypercholesterolemia are attributable to environmental changes affecting populations as a whole. Reducing obesity and smoking and moderating alcohol intake in entire populations should remain the primary strategies for lipid control.

Malic enzyme 2 may underlie susceptibility to adolescent-onset idiopathic generalized epilepsy.

Idiopathic generalized epilepsy (IGE) is a class of genetically determined, phenotypically related epilepsy syndromes. Linkage analysis identified a chromosome 18 locus predisposing to a number of adolescent-onset IGEs. We report a single-nucleotide polymorphism (SNP) association analysis of the region around the marker locus with the high LOD score. This analysis, which used both case-control and family-based association methods, yielded strong evidence that malic enzyme 2 (ME2) is the gene predisposing to IGE. We also observed association among subgroups of IGE syndromes. An ME2-centered nine-SNP haplotype, when present homozygously, increases the risk for IGE (odds ratio 6.1; 95% confidence interval 2.9-12.7) compared with any other genotype. Both the linkage analysis and the association analysis support recessive inheritance for the locus, which is compatible with the fact that ME2 is an enzyme. ME2 is a genome-coded mitochondrial enzyme that converts malate to pyruvate and is involved in neuronal synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). The results suggest that GABA synthesis disruption predisposes to common IGE and that clinical seizures are triggered when mutations at other genes, or perhaps other insults, are present.

The genomic sequence of the accidental pathogen Legionella pneumophila.

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

Population-based study of SR-BI genetic variation and lipid profile.

The variability of the Class B Type I Scavenger Receptor (SR-BI) gene in human populations and the relation of its variants to blood lipids was investigated in a random sample of 1756 untreated adult residents of Geneva, Switzerland, during 1999-2000. A three-step study approach yielded the following results: (1) resequencing the gene's exons and flanking regions in 95 subjects identified four common single nucleotide polymorphisms (SNPs with rare allele frequency >3%); (2) association study of the four common SNPs in subjects with extreme HDL-cholesterol (HDL-C) and LDL-C phenotypes (186 "atherogenic cases" and 185 "non-atherogenic controls") showed that the synonymous exon 8 C-T (allelic frequency 48%) polymorphism, A350A, was associated with atheroprotection in men (odds ratios (OR) = 0.36, 95% confidence intervals (CI) = 0.15-0.90, P < 0.03), but not in women (2.09, 0.79-5.49, P = 0.14); and (3) population clinical effects of A350A genotypes assessed in all 1756 subjects, showed that the case-control study findings reflected a protective HDL-C effect in men (CC: 1.17 mmol/L, CT: 1.22 mmol/L, and TT: 1.24 mmol/L, trend P = 0.0062) and a deleterious LDL-C effect in women (CC: 3.58 mmol/L, CT: 3.72 mmol/L, and TT: 3.79 mmol/L, trend P = 0.014). The allelic frequencies of the common SR-BI variants appear to be very similar in European and North American populations. The HDL-C effect increased with age. SR-BI A350A appears to have gender-specific and age-related effects on cholesterol transport lipoproteins.

Association of extreme blood lipid profile phenotypic variation with 11 reverse cholesterol transport genes and 10 non-genetic cardiovascular disease risk factors.

This study explored the genetic basis of the combination of extreme blood levels of HDL-C and LDL-C, a well-studied endophenotype for CVD, which has several attractive features as a target for genetic analysis: (1) the trait is moderately heritable; (2) non-genetic risk factors account for a significant but still limited portion of the phenotypic variance; (3) it is known to be moderated by a number of gene products. We exhaustively surveyed 11 candidate genes for allelic variation in a random population-based sample characterized for known CVD risk factors and blood lipid profiles. With the goal of generating specific etiological hypotheses, we compared two groups of subjects with extreme lipid phenotypes, from the same source population, using a case-control design. Cases (n=186) were subjects, within the total sample of 1708 people, who scored in the upper tertile of LDL-C and the lowest tertile of HDL-C, while controls (n=185) scored in the lowest tertile of LDL-C and the upper tertile of HDL-C. We used logistic regression and a four-tiered, systematic model building strategy with internal cross-validation and bootstrapping to investigate the relationships between the trait and 275 genetic variants in the presence of 10 non-genetic risk factors. Our results implicate a subset of nine genetic variants, spanning seven candidate genes, together with five environmental risk factors, in the etiology of extreme lipoprotein phenotypes. We propose a model involving these 14 genetic and non-genetic risk factors for evaluation in future independent studies.

Association between lipoprotein lipase (LPL) gene and blood lipids: a common variant for a common trait?

S447X, a serine substitution by a stop codon on base 99 of exon 9 of the lipoprotein lipase (LPL) gene, has beneficial effects on blood lipids. Other LPL alleles are associated with lipid levels, but whether one of these variants predominates remains elusive. We performed a systematic survey to identify single-nucleotide polymorphisms (SNPs) in all 10 LPL exons and flanking regions by resequencing the gene in 95 subjects. Of 24 variants, 14 were common (> or = 3%). We assayed the common SNPs in 186 cases with atherogenic lipid profiles (low HDL, high LDL) and 185 nonatherogenic controls (high HDL, low LDL). Only S447X and exons 6 (base +73) and 10 (base -11) were individually associated with case-control status (P<0.05, adjusted for major nongenetic covariates with known lipid effects). There were no significant SNP x gender interactions. In adjusted multi-SNP and haplotypic analyses, S447X was interpretable as the sole predictor, with a 2-3-fold reduction in the odds of being atherogenic vs. nonatherogenic (adjusted OR, 0.39; 95% CI, 0.21-0.73). S447X and base -11 of exon 10 were statistically interchangeable because they are strongly associated (r=0.92, P<0.0001), but we posit that the LPL association with lipid profile is more likely attributable to the functional S447X rather than the nonfunctional exon 10 SNP. It appears that the S447X variant of LPL may be another rare example (like APOE4, factor V-Leiden, and PPAR gamma Pro12Ala) of a common variant predisposing to a common disorder.

Mutations in LGI1 cause autosomal-dominant partial epilepsy with auditory features.

The epilepsies are a common, clinically heterogeneous group of disorders defined by recurrent unprovoked seizures. Here we describe identification of the causative gene in autosomal-dominant partial epilepsy with auditory features (ADPEAF, MIM 600512), a rare form of idiopathic lateral temporal lobe epilepsy characterized by partial seizures with auditory disturbances. We constructed a complete, 4.2-Mb physical map across the genetically implicated disease-gene region, identified 28 putative genes (Fig. 1) and resequenced all or part of 21 genes before identifying presumptive mutations in one copy of the leucine-rich, glioma-inactivated 1 gene (LGI1) in each of five families with ADPEAF. Previous studies have indicated that loss of both copies of LGI1 promotes glial tumor progression. We show that the expression pattern of mouse Lgi1 is predominantly neuronal and is consistent with the anatomic regions involved in temporal lobe epilepsy. Discovery of LGI1 as a cause of ADPEAF suggests new avenues for research on pathogenic mechanisms of idiopathic epilepsies.