Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 11
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Host gene regulation during coxsackievirus B3 infection in mice: assessment by microarrays. Circ. Res. 2000;87(4):328–334.Johnatty SE, Dyck JR, Michael LH,Olson EN, Abdellatif M. Identificationof genes regulated during mechanicalload-induced cardiac hypertrophy. J.
Mol.Cell. Cardiol. 2000; 32(5):805–815.Shimkets RA, Lowe DG, Tai JT, Sehl P,et al. Gene expression analysis by transcript profiling coupled to a gene database query. Nat. Biotechnol. 1999;17(8):798–803.Schulze A, Downward J. Navigatinggene expression using microarrays – atechnology review. Nat. Cell. Biol. 2001;3(8):E190–195.272Global Genomic Analyses of Cardiovascular Disease:A Potential Map or Blind Alley?Maike Krenz, Gary Schwartzbauer, and Jeffrey Robbins2.1Blindly Searching for Structure-FunctionSome of the first successes in the new age of molecular biomedicine involved identifying the gene or genes responsible for a particular disease. These initial studiesrelied heavily upon extensive information about the gene product, or what is nowcalled today biological annotation.
Thus, a wealth of information about the protein(s) or biochemical pathways underlying such diseases as sickle cell anemia,Tay-Sachs disease and phenylketonuria existed, and these largely biochemical datadrove the nucleic acid studies, in which specific mRNAs were isolated and usedas probes to isolate the respective genomic sequences. This general approach,known as functional cloning, presupposes and depends upon extensive data at theprotein level, often laboriously gathered over a long period of time. Starting approximately 25 years ago, this methodology was complemented by and then supplantedusing the so-called “reverse genetics” or what is more accurately termed positionalcloning, in which pedigrees are constructed, linkage analyses carried out and the investigator then hones in on the (presumably) affected locus.
Importantly for the purposes of the present topic, unlike functional cloning, positional cloning presupposesno information about the gene product, and the genes for Duchenne muscular dystrophy [1], Fragile X Syndrome [2] and Huntington’s disease [3] are only a few of themany disease-causing genes identified using this technology.Identification of the candidate gene does not necessarily translate into an understanding of the pathological processes that actually underlie disease development.Thus, investigators have had to struggle for 15 years to understand how dystrophin actually causes the pathology of Duchenne Muscular Dystrophy, or how theprotein huntingtin leads to Huntington’s disease. For the cardiovascular system,these issues are illustrated by focusing on the disease, familial hypertrophic cardiomyopathy (FHC).
The genetic etiology and molecular pathology of FHC has beenthe subject of intense investigation since the seminal observation by Seidman andher colleagues, linking mutations in the major cardiac contractile protein, the bmyosin heavy chain, with the development of a genetically-based hypertrophic cardiomyopathy [4]. It has become clear over the ensuing years that a variety of mutations in multiple sarcomeric proteins can lead to cardiac disease and failure [5].Proteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E. van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co.
KGaA, WeinheimISBN: 3-527-30596-3282 Cardiac Disease and The TranscriptomeFHC is characterized by unexplained cardiac hypertrophy in the absence of increased cardiac load or other systemic abnormalities. Clinically, the autosomaldominant diseases show variable penetrance with hypertrophy occurring in eitherventricle; almost always (>95%), there is involvement of the intraventricular septum [6]. FHC is a major cause of sudden death in otherwise healthy appearingyoung adults [7].
However, even within a family in which the disease is due to asingle genetic defect, the severity, onset and penetrance of the pathology is highlyvariable, presumably due to the existence of modifier loci, although this has notyet been formally demonstrated. Early on, it was recognized that FHC mapped tomultiple loci and soon after the first reports linking the disease to b-MyHC, genelinkage/positional cloning approaches zeroed in on other disease loci and identified mutations in the other sarcomeric protein genes.
These studies and “candidate gene” studies, in which mutations in closely related proteins are looked for,identified mutations in cardiac myosin binding protein-C, components of the troponin complex, actin, titin, a-tropomyosin and others. The association of FHCwith mutations in this set of functionally related proteins led to the syndromebeing called “a disease of the sarcomere [8].”These studies illustrate the logical progression of a positional cloning approachand its strengths and weaknesses. The strengths include the relatively rapid progress that can be made once the pedigrees are established, the locus or locimapped, and the linkage validated by statistical analyses.
Identification of a geneor group of genes gives one the necessary biological annotation to begin “blind” candidate gene studies in which structurally related or functionally related sequencesare scanned for mutations. Collecting linkage data for these candidates is time consuming and the data are often incomplete, precluding the use of linkage analysis.Thus, the candidate gene approach, in which closely related genes (such as actinor troponin or myosin light chain in the case of FHC), or genes that can be logicallyplaced as playing a possible role in the characterized pathology are chosen, has beenquite useful.
However, often the resultant biological validation, that is showing thatthe particular mutation is causative, is lacking or the results ambiguous [9, 10].Unfortunately, identifying the primary genetic etiology has led to only limitedinsight into understanding the resultant pathology, which is the result of and reflects the downstream changes in the transcriptome and proteome. The progression from functional cloning ? positional cloning ? candidate gene is all intellectually founded in a reductionist approach: understanding the cause(s) of the disease by focusing on the primary genetic lesion.
For cardiovascular disease, eventhose that are monogenically based, we now realize that reductionism has its limitations, particularly when it comes to understanding a disease pathology in termsof choosing potential therapeutic targets. In a paradigm shift it is now appreciatedthat, in order to understand the molecular pathologies at work, the overall regulatory networks and interplay of the different organ systems, cell types and evensubcellular compartments and organelles need to be described.It is this explicit recognition of the value of a global approach for understanding cardiovascular disease that has generated the excitement associated with exploring the transcriptome. The advent of multiple methodologies for defining a2.2 The Starting Line: Garbage In – Garbage Out?cell’s total transcriptional output and how it might change during disease progression offers heretofore unrealized potential for truly understanding the pathologyof cardiovascular disease.
The different methods currently in use for profilinggene expression include differential display [11], expression sequence tag (EST)identification and sequencing [12, 13], serial analysis of gene expression (SAGE)[14] and microarray analyses with either cDNA or oligonucleotide based chips.However, the commercialization of the microarray technology through Affymetrix’sTM development of comprehensive chip sets for the human, mouse and othermodel organisms, has brought global analysis out of the boutique into the generalcardiovascular research community in a surprisingly short period of time.
A largenumber of cardiovascular laboratories have now adopted microarrays for analyzing the genetic output of both normal and diseased cardiovascular tissue at different developmental times. Instead of measuring the output of only a few genes,we can now quantitate and compare tens of thousands of transcripts in a more orless reproducible manner across different conditions.2.2The Starting Line: Garbage In – Garbage Out?The field of cardiovascular biology appears, at first glance, to be particularly wellsuited for the types of comprehensive analyses needed for the technology’s productive application to discovery-based research.
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