GENETICS OF OBESITY, THE METABOLIC SYNDROME, AND RELATED ABNORMALITIES
In collaboration with Dr. Ahmed Kissebah (MCW), Dr. Lisa Martin (Children's Hospital Cincinnati, OH) and Dr. John Blangero (Southwest Foundation for Biomedical Research, San Antonio, TX), our work has focused on the lipid abnormalities associated with the metabolic syndrome in humans. As part of these efforts, Dr. Ed Smith, a postdoctoral fellow in the laboratory, has analyzed a QTL on human chromosome 7 linked to plasma triglyceride levels. We have comprehensively examined the linkage disequilibrium and haplotype structure of the entire QTL region using over 1000 SNPs. The statistical analysis of the genotype data has identified several regions that contribute to the overall linkage. Two novel genes not previously described to be involved in lipid metabolism account for over 40% of the total linkage, and at least two other intergenic regions of unknown function also contribute significantly to the observed effect (Cover of Genomics, issue 88(4), October 2006). We are currently finishing the haplotype analysis to assess the additive and interactive effects of SNPs to confirm the association effects and identify functional variants in genes of interest. The project will be continued by Dr. Yi (Sherry) Zhang, a new postdoctoral fellow in the laboratory. Recently, we have begun a new collaboration with Dr. Scarlett Shi (MCW) to use proteomics to profile the composition of lipoprotein particles in human serum. Another Graduate student, Lisamarie Collins, has developed a chromatographic approach to separate LDL, IDL, and HDL subclasses. Utilizing membrane extraction and isotopic labeling approaches developed by Dr. Shama Mirza, a postdoctoral fellow in the laboratory, we are currently quantifying the assocaiated proteins. The work is being supported by an Inducement Grant from the Biotechnology and Bioengineering Cener to Dr. Shi. The approach, if successful, will aid our studies examining the lipid abnormalities in individuals with the Metabolic Syndrome and may reveal candidate pathways and proteins that could direct our genetic studies. In another project, a graduate student in the laboratory, Jeff Eckert, has shown that haplotypes in PTPN1, a gene involved in signaling of the insulin receptor and the leptin receptor, are associated with plasma leptin levels and obesity in our TOPS cohort. We have also shown that the risk of haplotype increases gene expression in vitro and in adipose tissue biopsy samples. While it is currently unclear how this effect is mediated, this analysis shows for the first time a direct correlation of genetic risk and a cellular functional effect in obesity. Finally, we have begun to examine the association of SNPs in selected candidate genes with fatty liver and non-alcoholic steatohepatitis (NASH), a liver disorder leading to liver dysfunction. This work in collaboration with Dr. Samer Gawrieh (MCW) is utilizing DNA isolated from archived liver biopsy samples. To date, we have isolated DNA from over 250 samples, and have begun to examine the role of SNP variants on fatty liver, NASH, and liver inflammation. We have shown association to some variants in PTPN1 and PPARG. Also, we have begun to collect blood samples and biopsy samples from patients undergoing bariatric sugery at Froedert Hospital for expression profiling. The first few samples have been profiled using Illumina technology, and have revealed gene networks altered in gene expression in both affected liver tissue and peripheral circulating blood cells. This work is supported by an Inducement Grant from the Biotechnology and Bioengineering Center to Dr. Gawrieh, and the initial data suggest that we may be able to identify expression signatures characteristic of NASH and fatty liver that can unambiguously be identified in blood samples for clinical diagnosis. GENETICS OF LEUKEMIA IN DOWN SYNDROME Individuals with Down syndrome usually have an additional copy of the entire chromosome 21. On average, this leads to an over–expression of all genes on the chromosome by ~50%, and is believed to result in the phenotypic abnormalities commonly seen in affected individuals. One of the striking features of individuals with Down syndrome is their significantly increased risk of developing a specific form of leukemia, acute megakaryoblastic leukemia (AMLK). One gene on chromosome 21, RUNX1, codes for a transcription factor that is essential in the normal development of megakaryocytes from the bone marrow. The project of one of the graduate students in the laboratory, Kelly Duffy, is to explore whether genetic sequence variation in and around this gene may lead to an altered expression of the resulting transcription factor. In our initial work, we have developed a SNP genotyping method that allows the successful genotyping of trisomic cell lines. Preliminary results suggest that RUNX1 is up–regulated ~1.5 fold in trisomy 21. Our analysis of haplotype–specific RUNX1 gene expression has revealed two SNP haplotypes that increase RUNX1 gene expression in chromosomally normal lymphoblast cell lines. In collaboration with Dr. Stephanie Sherman (Emory University, Atlanta, GA), we are currenty examining whether this up–regulation of RUNX1 gene expression is maintained and enhanced in individuals with trisomy 21. We are testing trisomy cell lines to identify samples homozygous for the different haplotypes, and test the levels of RUNX1 gene expression using real–time PCR. THE GENETICS OF PROTEUS SYNDROME
A. Proteomics We are applying this approach to the analysis of vascular endothelial cells isolated from different rat strains and cultured under normoxic or hypoxic conditions to identify proteins involved in angiogenesis. For these studies, cells are lysed, and separated into a mitochondrial, a soluble (cytosolic), and an insoluble (membrane) fraction. In addition, we are analyzing the culture media samples. All protein samples are further fractionated using isoelectric focusing. The resulting subfractions are analyzed using standard capillary LC–MS–MS analysis after tryptic digest of the protein samples. (Method pictured left; the image at the bottom shows the analysis of relative peptide ratios using the software package ZoomQuant.) Overall, we routinely identify over 1500 proteins from the total cell lysate. Analyses are under way to determine the proteins differentially expressed under hypoxic (i.e. angiogenesis stimulating) conditions. In another research effort, we have begun to use surface chemistry approaches to develop arrays suitable for proteomics analysis. Here we identify proteins that are bound to specific ligands attached to a gold surface by mass spectrometry. This project in collaboration with the University of Texas Southwestern Medical School utilizes peptoid molecules as baits for protein binding. We have shown that we can use both MALDI–TOF and LC–MS–MS analysis to identify proteins bound to peptoids, and have adapted the approach to high–throughput screening. B. SNP Genotyping We have developed a novel approach using the Invader technology to genotype trisomic DNA samples from individuals with Down syndrome. In individuals with three chromosomal copies, a biallelic SNP can result in four potential genotypes: AAA, AAa, Aaa, aaa. Traditional SNP genotyping platforms have difficulties to distinguish the AAa and Aaa genotypes. We have adapted the Invader technology to use genomic DNA as starting material for our analysis, and can reliably distinguish the heterozygotes. In collaboration with Dr. Stephanie Sherman at Emory University we have compared our genotyping results with results obtained from the samples by pyrosequencing, an established method for genotyping Down syndrome, and have shown that the results are identical. C. Molecular Diagnosis of Chromosomal Abnormalities In collaboration with Dr. Aoy Tomita-Mitchell (BBC) and Dr. Michael Mitchell (MCW), we have begun to develop a combinatorial approach to test for chromosomal abnormalities in fetal DNA. Fetal DNA can be found in small amounts in samples of maternal blood. Using a combination of biallelic SNPs and quantitative copy number probes, we are testing whether our approach allows the detection of trisomy 21 using maternal blood samples without amniocentesis or chorionic villi sampling as a non–invasive low–risk alternative to currently available technologies for clinical diagnostics. |
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Our major research effort is geared towards the analysis of the Metabolic Syndrome in humans, a disorder characterized by obesity, insulin resistance, dyslipidemia, and hypertension. Several projects are trying to address different aspects of this complex disorder and its genetic basis. We are using both family–based genome–wide analysis and candidate gene studies to elucidate the genetic alterations affecting lipid metabolism, plasma lipid levels, and disease manifestations of these abnormailities, such as abdominal obesity and non–alcoholic fatty liver disease.
In collaboration with Dr. David Bick (MCW), Kelly Duffy has used Affymetrix 100K arrays to identify copy number variants in a sample from a patient with Proteus Syndrome. Proteus Syndrome is a sporadic multi system disorder characterized by progressive asymmetric overgrowth of body parts, including cerebriform connective tissue nevi, epidermal nevi, capillary, venous, and lymphatic vascular malformations and dysregulated adipose tissue. It is thought that Joseph Merrick (the Elephant Man, pictured right, 
As part of the NHLBI–funded National Center for Proteomics Research, we have continued to analyze cellular sub–proteomes quantitatively by mass spectrometry. Dr. Mirza and co–workers have developed methods to separate cellular subfractions, and to quantify all proteins in these subfractions by an isotopic labeling approach using 18O as a substrate. The relative mass shift induced by the isotope in tryptic peptides can be detected using standard mass spectrometry technology.