The Metabolic Risk
Complications of Obesity Genes (MRC-OB) Study: Genetic Basis of
the Metabolic Syndrome
The metabolic syndrome is a common chronic disorder in humans associated
with obesity, insulin resistance, and alterations in plasma lipid
profile. It results in decreased longevity and increased morbidity,
especially due to cardiovascular complications such as hypertension,
stroke, and coronary heart disease. Central to the cardiovascular
disease risk is the association of the metabolic syndrome with a
specific lipoprotein/lipid profile, expressed as elevation of fasting
plasma triglyceride levels, decreased HDL cholesterol levels, and
predominance of dense profiles of LDL and HDL particles.
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Figure 1:
Obesity in U.S. Adults. Percentages are listed for adults
with BMI? 30 which corresponds to a 5’4’ woman being 30 lbs
overweight. Source: Mokdad AH et al. (1999) J Am Med Assoc
282:16; (2001) 286:10. Graphics: Center for Disease Control,
Atlanta, GA. |
The overall etiology of the metabolic syndrome is complex, reflecting
extensive interaction of metabolic, neuroendocrine, and genetic
factors. Unraveling these complex interactions is a challenging
task, but may provide details for future intervention in the development
and prevention of the progression of the syndrome and its cardiovascular
complications.
For over 25 years, Dr. A.H. Kissebah and collaborators have been
funded to investigate the complex biology of the metabolic syndrome.
For the past eight years, efforts have focused on determining its
genetic etiology. The group phenotyped and genotyped 2207 individuals
distributed over 507 families of Northern European descent, and
identified numerous quantitative trait loci (QTL) linked to various
phenotypic manifestations of the disorder (Kissebah et al. 2000).
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Figure 2:
Results of MRC-OB Genome Scan for Human Chromosome 7. The
blue line depicts the multipoint LOD score for plasma triglyceride
levels, the red line the LOD score for plasma LDL cholesterol
levels along human chromosome 7. |
Our current project in the laboratory focuses on a QTL on human
chromosome 7q36 that is strongly linked to variation of plasma triglyceride
and plasma LDL levels. The long-term objective is to identify genetic
variants that are associated with the adverse lipid/lipoprotein
disorder characteristic of the metabolic syndrome. We hypothesize
that this QTL on human chromosome 7 contains genetic variants in
genes and/or regulatory elements that are responsible for the altered
lipid metabolism in subjects with the metabolic syndrome, and thus
contribute to the complex pathobiology and the cardiovascular complications
of this disorder. We are applying comprehensive analyses using single
nucleotide polymorphisms (SNPs) in linkage disequilibrium and haplotype
analyses to identify the gene(s) responsible for the linkage found
in our and other studies to this locus.
Collaborators: |
John Blangero (San Antonio, TX) |
|
Howard Jacob (MCW) |
|
Ahmed Kissebah (MCW) |
|
Anne Kwitek (MCW) |
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Lisa Martin (Cincinnatti, OH) |
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Gabriele Sonnenberg (MCW) |
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Xujing Wang (MCW) |
Program for Genomic Applications
(BerkeleyPGA): Genetic Variation in Apolipoprotein A5
Over the past two years, we have analyzed the genetic sequence
variation across the apolipoprotein gene cluster on human chromosome
11. This cluster includes three well-known apolipoprotein genes
(APOA4, APOC3, and APOA1). In addition to these genes, we discovered
a novel member of the gene family (called APOA5) by comparative
sequence analysis (Pennacchio et al. 2001). The new gene is located
approximately 27 kb distal to APOA4 and 37 kb from APOC3. The novel
gene plays an important role in the regulation of plasma lipid levels.
Mice over-expressing human APOA5 have decreased plasma triglyceride
levels, while mice lacking the protein apoA5 have increased plasma
levels of triglycerides. Subsequently, initial studies in humans
showed that three single nucleotide polymorphisms (SNPs 1-3) in
and around APOA5 were significantly associated with altered plasma
triglyceride levels in two human populations.
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Figure 3:(A)
APOA5 Genomic Structure and PolymorphismLocation.
The gene is transcribed from left to right as indicated by
the large horizontal arrow. Exons are depicted by boxes with
protein-encoding regions shaded black. The position and identity
of SNPs identified in APOA5 are shown below the schematic.
SNPs found within the open reading frame show the predicted
amino acid substitution in parentheses (synonymous changes
are underlined). SNPs previously identified are indicated
as SNPs1–4 in parentheses (25). For all the SNPs the
major allele basepair sequence is identical to that found
in chimpanzee, except for 12,238T>C where they are reversed.
(B) Common APOA5 Haplotypes and Their Relative Frequencies
in 419 Caucasian Samples (Berkeley Lipid Study Population).
The five SNPs used in this analysis are boxed in panel A.
The SNPs are depicted in the following order: 1131T>C,
c.3A>G, c.56C>G, IVS3 þ 476G>A, c.1259T>C.
Haplotype frequencies were predicted using the Expectation-Maximization
algorithm (5000 iterations, (31)). The three depicted haplotypes
account for 97.6% of all haplotypes, none of the other predicted
haplotypes had a frequency of greater than 1%. The minor alleles
that define the haplotypes are highlighted in bold. In an
independent Caucasian population, we find the APOA5*1, APOA5*2
and APOA5*3 haplotype frequencies are 83.4%, 8.0% and 8.4%,
respectively. These samples are from 367 unrelated Caucasian
individuals of Northern European descent, collected in the
Midwest.
Source: Pennacchio et al. (2002) Hum Mol Genet 11(24):3033.
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In our genetic analysis of multiple ethnic groups, we identified
additional sequence variants in and around APOA5, and described
two haplotypes that were independently associated with increased
plasma triglyceride concentrations in Caucasians, African-Americans,
and Hispanics (Pennacchio et al. 2002). Approximately 25-50% of
individuals in these populations carry at least one of the two risk
haplotypes, designated APOA5*2 and APOA5*3.
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Figure 4:
Pairwise Linkage Disequilibrium between SNPs in the APOA5
Gene Region. Pairwise LD was calculated according to Lewontin
(1988). |D’|, the normalized LD measure, is depicted
for all pairwise comparisons. |D’| values of 1 are indicated
by red squares, white squares indicate |D’| values of
less than 0.5. The approximate location of SNPs is shown by
the lines connecting individual rows and columns to the diagram
of the genomic region.the two genes in this region.
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Our ongoing work has focused on the analysis of sequence variation
across the entire gene cluster, and the resulting linkage disequilibrium
and haplotype patterns. The goal of these efforts is to test whether
the association seen with SNPs in APOA5 is really reflecting a direct
effect of this novel gene, or whether the effect is due to functional
variants in other neighboring apolipoprotein genes.
This project is part of the Program for Genomic Applications (PGA)
sponsored by the National Heart, Lung, and Blood Institute NHLBI).
For more information, see http://pga.lbl.gov
or http://www.nhlbi.nih.gov/resources/pga/index.htm.
Collaborators: |
Len Pennacchio (Berkeley, CA) |
|
Edward Rubin (Berkeley, CA) |
Analysis of Protein
Tyrosine Phosphatase 1 Beta
Protein Tyrosine Phosphatase 1 Beta (PTP-1B) is a gene located
on human chromosome 20. The phosphatase has two proposed specific
functions:
1.) It associates with the insulin receptor and dephosphorylates
it, thereby disrupting the ability of the receptor to respond to
insulin binding;
2.) It dephosphorylates JAK-2, an important mediator of the leptin
signaling cascade.
For these reasons, PTP-1B has been an important target for synthetic
inhibitors as potential treatment for diabetes. Numerous compounds
have been tested, however, the response has been highly variable
and not consistent.
Genetic studies have identified individual SNPs that were associated
with type 2 diabetes and obesity in human populations. However,
no comprehensive analysis of linkage disequilibrium or haplotype
structure of the gene region has been performed, nor is it clear
whether the SNPs shown to be associated with human disorders are
actually functional.
For these reasons, we have begun to analyze the linkage disequilibrium
structure across the genomic region around PTP-1B. In parallel,
we are examining whether cell lines homozygous for different haplotypes
in this gene exhibit different properties with regards to gene expression
or protein levels, or protein activity. The project combines sequencing
and SNP genotyping with extensive expression analyses and protein
biochemistry to understand the link between genome variation and
actual measurable phenotypes in vitro.
Analysis of the Cellular
Proteome During Angiogenesis
Angiogenesis, the formation of new blood vessels, is an essential
physiological mechanism and plays a crucial role in a number of
diseases. While the formation of new vessels is clearly beneficial
in exercise or after myocardial infarction, vascularization of cancerous
tumors leads to metastasis and thus is detrimental to the individual.
Genetic factors significantly impact on the ability of an organism
to initiate angiogenesis. For example, different strains of inbred
rats have varying potential to form new vessels upon stimulation
by exercise or hypoxia. Furthermore, in consomic rat lines where
one chromosome at a time has been introgressed from a normal strain
into a rat with a genetic background inhibiting angiogenesis, the
angiogenic ability is recovered at varying degrees in some strains
depending on the chromosome that has been transferred.
As part of the MCW Proteomics Center funded by the National Heart,
Lung, and Blood Institute, we utilize these consomic rat strains
to analyze the cellular proteome of microdissected vascular endothelial
cells that are undergoing angiogenesis in vivo or in vitro. The
Proteomics Center is divided into four major components: Component
1, under the direction of Dr. L. Smith at UW Madison, will focus
on the development of novel mass spectrometric instrumentation that
will permit the efficient analysis of samples as small as individual
cells. Component 2 (Dr. A. Greene, MCW) will provide microdissected
cells cultured and stimulated to undergo angiogenesis from consomic
animals with different genetic backgrounds. Component 3 (our lab)
will use the samples provided through component 2 and use the technology
developed as part of component 1 to identify the proteome involved
in angiogenesis. Finally, a bioinformatics component (Component
4, Dr. S Twigger, MCW) will facilitate our data analysis and release,
and develop new tools to aid in the mass-spectrometric identification
of proteins. Detailed information about the proteomics center can
be found at http://proteomics.mcw.edu.
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Figure 6:
MALDI-TOF Spectra for Total Protein Extracts from Cultured
Vascular Endothelial Cells from Different Strains of Rats.
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It is the goal of the protein analysis component (component 3)
to merge the technological advances in analytical mass spectrometry
from component 1 with existing analytical capabilities to identify
and quantify the proteome of cell lines, tissue samples, and ultimately
individual cells undergoing angiogenesis. We will use the angiogenic
process as a model and test system to develop a comprehensive proteomics
approach for disease-related proteome analysis.
Our approach requires the transfer of novel technology from component
1, thorough testing and comparison of results to results obtained
with previously established approaches of protein and proteome analysis,
and the adaptation of sample preparation procedures to fully utilize
the newly developed methodology for the successful analysis of the
entire cellular proteome during angiogenesis. Specifically, we will
1.) develop improved approaches to selectively isolate and analyze
subsets of proteins from cells and tissue samples undergoing angiogenesis,
initially focusing on membrane-bound proteins and phosphoproteins;
2.) develop miniaturized methods to directly compare protein quantities
from angiogenic and non-angiogenic cells by exploring novel protein
and peptide labeling methods in collaboration with Perbio Sciences
AB;
3.) develop experimental approaches for the analysis of posttranslational
modifications by mass spectrometry; and
4.) implement and test new mass spectrometry instrumentation with
our experimental system.
Over the entire period, we will gradually expand our analysis from
the initial study of selected individual proteins of vessels and
cells during angiogenesis to ultimately include the entire proteome
as new technologies from Component 1 become available.
We closely collaborate with Pierce Milwaukee (Perbio Sciences AB,
see http://www.piercenet.com/)
on the development of new approaches for protein sample preparation
and separation, protein quantification by mass spectrometry, and
protocol development.
Collaborators: |
Andrew Greene (MCW) |
|
Peter Newman (MCW) |
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Walid Qoronfleh (Pierce Milwaukee) |
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Mike Ramsey (Oak Ridge National Laboratory) |
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Lloyd Smith (UW Madison) |
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Simon Twigger (MCW) |
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Mike Westphall (UW Madison) |
Molecular Analysis
of the Genome of Cyclospora cayetanensis
In collaboration with Dr. Claudia Olivier and her group at UW Whitewater,
we are analyzing the genome of a coccidian human parasite, Cyclospora
cayetanensis. This parasite is endemic to underdeveloped countries
such as Guatemala or Nepal, and has recently caused several outbreaks
of cyclosporiasis, a severe intestinal infection with diarrhea,
dehydration, and weight loss, in the U.S. and Canada.
Very little is known about the parasite and its biology. It cannot
be grown in culture, so the only source of genetic material is from
oocysts isolated from human stool samples of individuals acutely
infected.
Initially, Dr. Olivier generated a partial genomic library, and
we are now attempting to generate a cDNA library from mRNA isolated
from partially sporulated oocysts. Our lab is assisting with some
of the required instrumentation and technology, as well as with
sequencing.
Collaborators: Claudia Olivier (UW Whitewater)
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