Michael Olivier

Linkage and Association Between Distinct Variants of the APOA1/C3/A4/A5 Gene Cluster and Familial Combined Hyperlipidemia

Objective—Combined hyperlipidemia is a common disorder, characterized by a highly atherogenic lipoprotein profile and a substantially increased risk of coronary heart disease. The purpose of this study was to establish whether variations of apolipoprotein A5 (APOA5), a newly discovered gene of lipid metabolism located 30 kbp downstream of the APOA1/C3/A4 gene cluster, contributes to the transmission of familial combined hyperlipidemia (FCHL). Methods and Results—We performed linkage and association tests on 128 families. Two independent alleles, APOA5c.56G and APOC3c.386G, of the APOA1/C3/A4/A5 gene cluster were overtransmitted in FCHL (P =0.004 and 0.007, respectively). This was paired with reduced transmission of the common APOA1/C3/A4/A5 haplotype (frequency 0.4461) to affected subjects (P =0.012). The APOA5c.56G genotype accounted for 7.3% to 13.8% of the variance in plasma triglyceride levels in probands (P <0.004). The APOC3c.386G genotypes accounted for 4.4% to 5.1% of the variance in triglyceride levels in FCHL spouses (P <0.007), suggesting that this allele marks a FCHL quantitative trait as well as representing a susceptibility locus for the condition. Conclusions—A combined linkage and association analysis establishes that variation at the APOA1/C3/A4/A5 gene cluster contributes to FCHL transmission in a substantial proportion of northern European families.

Genetic variation in aldosterone synthase predicts plasma glucose levels.

The mineralocorticoid hormone, aldosterone, is known to play a role in sodium homeostasis. We serendipitously found, however, highly significant association between single-nucleotide polymorphisms in the aldosterone synthase gene and plasma glucose levels in a large population of Chinese and Japanese origin. Two polymorphisms—one in the putative promoter (T-344C) and another resulting in a lysine/arginine substitution at amino acid 173, which are in complete linkage disequilibrium in this population—were associated with fasting plasma glucose levels (P = 0.000017) and those 60 (P = 0.017) and 120 (P = 0.0019) min after an oral glucose challenge. A C/T variant in intron 1, between these polymorphisms, was not associated with glucose levels. Arg-173 and -344C homozygotes were most likely to be diabetic [odds ratio 2.51; 95% confidence interval (C.I.) 1.39–3.92; P = 0.0015] and have impaired fasting glucose levels (odds ratio 3.53; 95% C.I. 2.02–5.5; P = 0.0000036). These results suggest a new role for aldosterone in glucose homeostasis.

Current analysis platforms and methods for detecting copy number variation

Copy number variation (CNV), generated through duplication or deletion events that affect one or more loci, is widespread in the human genomes and is often associated with functional consequences that may include changes in gene expression levels or fusion of genes. Genome-wide association studies indicate that some disease phenotypes and physiological pathways might be impacted by CNV in a small number of characterized genomic regions. However, the pervasiveness and full impact of such variation remains unclear. Suitable analytic methods are needed to thoroughly mine human genomes for genomic structural variation, and to explore the interplay between observed CNV and disease phenotypes, but many medical researchers are unfamiliar with the features and nuances of recently developed technologies for detecting CNV. In this article, we evaluate a suite of commonly used and recently developed approaches to uncovering genome-wide CNVs and discuss the relative merits of each.

A haplotype map of the human genome

Inherited genetic variation has a critical but as yet largely uncharacterized role in human disease. Here we report a public database of common variation in the human genome: more than one million single nucleotide polymorphisms (SNPs) for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations, including ten 500-kilobase regions in which essentially all information about common DNA variation has been extracted. These data document the generality of recombination hotspots, a block-like structure of linkage disequilibrium and low haplotype diversity, leading to substantial correlations of SNPs with many of their neighbours. We show how the HapMap resource can guide the design and analysis of genetic association studies, shed light on structural variation and recombination, and identify loci that may have been subject to natural selection during human evolution.

Fatty acid binding protein 3 (fabp3) is associated with insulin, lipids and cardiovascular phenotypes of the metabolic syndrome through epigenetic modifications in a northern european family population

BackgroundFatty acid-binding proteins (FABPs) play regulatory roles at the nexus of lipid metabolism and signaling. Dyslipidemia in clinical manifestation frequently co-occurs with obesity, insulin resistance and hypertension in the Metabolic Syndrome (MetS). Animal studies have suggested FABPs play regulatory roles in expressing MetS phenotypes. In our family cohort of Northern European descent, transcript levels in peripheral white blood cells (PWBCs) of a key FABPs, FABP3, is correlated with the MetS leading components. However, evidence supporting the functions of FABPs in humans using genetic approaches has been scarce, suggesting FABPs may be under epigenetic regulation. The objective of this study was to test the hypothesis that CpG methylation status of a key regulator of lipid homeostasis, FABP3, is a quantitative trait associated with status of MetS phenotypes in humans.MethodsWe used a mass-spec based quantitative method, EpiTYPER®, to profile a CpG island that extends from the promoter to the first exon of the FABP3 gene in our family-based cohort of Northern European descent (n=517). We then conducted statistical analysis of the quantitative relationship of CpG methylation and MetS measures following the variance-component association model. Heritability of each methylation and the effect of age and sex on CpG methylation were also assessed in our families.ResultsWe find that methylation levels of individual CpG units and the regional average are heritable and significantly influenced by age and sex. Regional methylation was strongly associated with plasma total cholesterol (p=0.00028) and suggestively associated with LDL-cholesterol (p=0.00495). Methylation at individual units was significantly associated with insulin sensitivity, lipid particle sizing and diastolic blood pressure (p<0.0028, corrected for multiple testing for each trait). Peripheral white blood cell (PWBC) expression of FABP3 in a separate group of subjects (n=128) negatively correlated with adverse profiles of metabolism (βWHR = −0.72; βLDL-c = −0.53) while positively correlated with plasma adiponectin (β=0.24). Further, we show that differential methylation of FABP3 affects binding activity with nuclear proteins from heart tissue. This region that we found under methylation regulation overlaps with a region actively modified by histone codes in the newly available ENCODE data.ConclusionsOur findings suggest that DNA methylation of FABP3 strongly influences MetS, and this may have important implications for cardiovascular disease.

Genetic Variation in the Peroxisome Proliferator Activated Receptor-Gamma Gene Is Associated with Histologically Advanced NAFLD

BackgroundThe peroxisome proliferator activated receptor-gamma (PPARG) is a nuclear receptor that regulates adipocyte differentiation, insulin sensitivity and lipid metabolism, thus, it represents a good candidate gene for non-alcoholic fatty liver disease (NAFLD). Purpose and MethodWe investigated the association of two PPARG variants (Pro12Ala and C1431T) with NAFLD and its histological features. DNA was extracted from 274 archived, formalin-fixed liver biopsy specimens from 212 patients with NAFLD and 62 controls with normal liver histology.Results Individual SNPs did not show significant association with NAFLD or its histological features. A haplotype comprised of both minor alleles (GT) was less enriched whereas a haplotype comprised of the two major alleles (CC) was more enriched in subjects with NAFLD compared to controls [9.3% vs. 28.1% for GT (P = 0.001, OR 0.26 (range 0.14–0.48) and 80.4% vs. 64.8% for CC (P = 0.037, OR 2.23 (range 1.30–3.81)]. Both haplotypes were significantly associated with steatosis and fibrosis. The GT haplotype was also associated with lobular inflammation.Conclusions Genetic variation in PPARG is associated with NAFLD, and the GT haplotype is associated with inflammatory and fibrotic changes that denote histologically advanced NAFLD.

Multiethnic genome-wide meta-analysis of ectopic fat depots identifies loci associated with adipocyte development and differentiation

Variation in body fat distribution contributes to the metabolic sequelae of obesity. The genetic determinants of body fat distribution are poorly understood. The goal of this study was to gain new insights into the underlying genetics of body fat distribution by conducting sample-size-weighted fixed-effects genome-wide association meta-analyses in up to 9,594 women and 8,738 men of European, African, Hispanic and Chinese ancestry, with and without sex stratification, for six traits associated with ectopic fat (hereinafter referred to as ectopic-fat traits). In total, we identified seven new loci associated with ectopic-fat traits (ATXN1, UBE2E2, EBF1, RREB1, GSDMB, GRAMD3 and ENSA; P < 5 × 10−8; false discovery rate < 1%). Functional analysis of these genes showed that loss of function of either Atxn1 or Ube2e2 in primary mouse adipose progenitor cells impaired adipocyte differentiation, suggesting physiological roles for ATXN1 and UBE2E2 in adipogenesis. Future studies are necessary to further explore the mechanisms by which these genes affect adipocyte biology and how their perturbations contribute to systemic metabolic disease.

18O labeling over a coffee break: a rapid strategy for quantitative proteomics.

Proteomics-based quantification methods for differential protein expression measurements are among the most important and challenging techniques in the field of mass spectrometry. Though numerous quantification methods have been established, no method meets all the demands for measuring accurate protein expression levels. Of the various relative quantification methods by isotopic labeling, (18)O labeling method has been shown to be simple, specific, cost-effective and applicable to a wide range of analyses. However, some researchers refrain from using the method due to long incubation periods required during the labeling process. To address this problem, we demonstrate a method by which the labeling procedure can be completed in 15 min. We digested and labeled samples using immobilized trypsin on micro-spin columns to speed up the enzyme-mediated oxygen substitution, thereby completing the labeling process within 15 min with high labeling efficiency. We demonstrate the efficiency and accuracy of the method using a four protein mixture and whole cell lysate from rat vascular endothelial cells.

Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification

The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS) analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1) promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO) subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

Integrated approach for the comprehensive characterization of lipoproteins from human plasma using FPLC and nano-HPLC-tandem mass spectrometry

The implication of the various lipoprotein classes in the development of atherosclerotic cardiovascular disease has served to focus a great deal of attention on these particles over the past half-century. Using knowledge gained by the sequencing of the human genome, recent research efforts have been directed toward the elucidation of the proteomes of several lipoprotein subclasses. One of the challenges of such proteomic experimentation is the ability to initially isolate plasma lipoproteins subsequent to their analysis by mass spectrometry. Although several methods for the isolation of plasma lipoproteins are available, the most commonly utilized techniques require large sample volumes and may cause destruction and dissociation of lipoprotein particle-associated proteins. Fast protein liquid chromatography (FPLC) is a nondenaturing technique that has been validated for the isolation of plasma lipoproteins from relatively small sample volumes. In this study, we present the use of FPLC in conjunction with nano-HPLC-ESI-tandem mass spectrometry as a new integrated methodology suitable for the proteomic analysis of human lipoprotein fractions. Results from our analysis show that only 200 microl of human plasma suffices for the isolation of whole high density lipoprotein (HDL) and the identification of the majority of all known HDL-associated proteins using mass spectrometry of the resulting fractions.