Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 19
Текст из файла (страница 19)
The transition from compensated cardiachypertrophy to decompensated heart failure is accompanied by marked changesin the expression of an array of genes in the heart. What is the marker for earlymyocardial decompensation and can we intervene in its progression? This discovery is the target for genomics-based studies (Fig. 3.2) [9].Traditionally, the geneticist’s search for disease causing gene mutations was alengthy and resource-consuming process.
Genetics investigators searched for single mutated nucleotide(s) in the DNA sequence. This methodology, though highlyfruitful for locating single gene mutations, is not adequate for elucidating thecomplexities of multi-gene based diseases such as heart failure in which multiplegenes and gene clusters may be involved. As described above, multiple pathwaysare involved at different clinical stages and in different types of heart failure andseveral gene mutations or protein defects have been identified in cardiomyopathy.The Human Genome Project and related projects have paved the way for futurestudies in genomics.
In February 2001, completion was announced of the firstmap of the whole human genome, comprising more than 90% of the approximately 3 billion bases of the human genome [17, 18]. The Human Genome Project database will serve as an invaluable resource for identifying the completestructure of each gene, interindividual variations of gene structure related to biodiversity, the clustering of genes in each chromosome, and gene polymorphisms related to structure or function.
Furthermore, we can exploit gene expression profiles and genome-wide linkage analyses to identify genes contributing to individual variations in disease risk, to develop more efficacious treatments by targetingnovel genes or unidentified pathways, and to tailor individual responses to treatment on the basis of genetic constitution.The technology of the Human Genome Project and the genomics revolution arebeginning to transform our approach to such complex disorders as heart failureand cardiomyopathy by enabling investigators to look at the differential expressionof hundreds of thousands of genes simultaneously, and to compare gene expression during disease development and over the course of disease progression [19].The genomics approach by contrast to the single gene approach is a “holistic”technology that attempts to investigate the relationship between clusters of candidate genes expressed or active during the complex processes involved in disease[20, 21].An important development of the Human Genome Project has been the application of EST methods of gene discovery.
ESTs (Expressed Sequence Tags) are sequences derived from complementary DNA libraries, representing particular tissues or organs [22, 23]. Although ESTs are relatively short in sequence (hundreds3.3 Genomic Approach to Heart Failurea)A 10,000 element cDNA array containing:over 3000 known, characterized genesover 7000 uncharacterized EST clusterse.g. Profiled DCM and HCM heart tissueb)DCM (dilated cardiomyopathy) versusnormal (non-failing) heart samplesc)Fig. 3.2a) Microarray analysis; b) hierarchical clustering; c) verification of individual genes.51523 Heart Failure: A Genomics Approachof base pairs), they contain enough information to identify the transcript corresponding to the cDNA clone using simple nucleotide and protein database searching algorithms (e.g., BLAST).
EST technology is convenient for large-scale transcript profiling through the generation of partial DNA sequences (ESTs) from randomly selected cDNA clones. Using these clones, we can generate an expressionprofile useful for performing genome-wide comparisons between two or moretranscript populations and allow the identification of expression differences at thelevel of the single gene [24].
Moreover, identifying cDNA clones through EST generation provides a valuable genomic resource that is useful for the identificationof protein homologs, chromosome mapping, exon identification in genomic sequences, single nucleotide polymorphism identification and as a substrate forcDNA microarrays [25].With the publication of the complete sequence of the human genome, the number of genes encoded by the genome is estimated to be 32,000–38,000 [17, 18].The next step is to determine how many genes are involved in specific cells, tissues, organs. In a recent publication, the first to describe the number of genes expressed in a single organ system, our team took a global approach to address theissue.
Using three approaches (a current EST database, the complete nucleotidesequences of chromosomes 21 and 22 and cDNA microarray hybridization) we estimated that the number of genes expressed in the cardiovascular system ranges between 21,000 and 27,000 [26]. The high end of the estimate is a number close tothe total number of genes in the human genome, suggesting that the majority ofgenes in the human genome function in the normal maintenance and function ofan organ regardless of its specific function, while only a small proportion are allocated to cell-specific functions.With access to the human genome map and with the aid of ESTs, we can investigate simultaneously the genes involved in physiologic or pathologic states andbegin to develop a modern approach to puzzles of genomics.
Our laboratory hasapplied the EST approach in cardiovascular disease and heart failure. UtilizingEST technology, our laboratory has generated a compendium of genes expressedin the human cardiovascular system, with the ultimate goal of assembling the intricacies of development and of disease, particularly the pathways leading to heartfailure [9]. Through a computer-based in silico strategy, we have been able to identify on a large scale both known and previously-unsuspected genetic modulatorscontributing to the growth of the myocardium from fetal through adult, and fromnormal to a perturbed hypertrophic phenotype.
Our laboratory employed a wholegenome approach using ESTs to characterize gene transcription and to identifynew genes overexpressed in cardiac hypertrophy [24]. A detailed comparison of individual gene expression identified 64 genes potentially overexpressed in hypertrophy, and analysis of general transcription patterns revealed a proportional increasein transcripts related to cell/organism defense and a decrease in transcripts related to cell structure and motility in the hypertrophic heart. This approach is instriking contrast to the earlier time consuming and cumbersome gene-by-geneapproach in elucidating the genes and mechanisms involved in complexities of development and disease.3.3 Genomic Approach to Heart FailureAnother genomic advance is the DNA microarray chip.
DNA microarray chiptechnology makes use of EST data and represents a major advance in genomicsresearch. In microarray analysis, individual cDNA clones are amplified by polymerase chain reaction (PCR). A micro-sample of each cDNA clone is then chemically bonded onto a glass surface or nylon membrane in an array format. Thechip can also be made from oligonucleotides synthesized in situ on the surface ofthe array. Probes labeled with different fluorophores can be used to identify differential gene expression.
The strength and specificity of the interaction can be increased or decreased by altering the length of the oligonucleotides or by varyingthe conditions of the hybridization reactions. Fluorescence signals representinghybridization to each arrayed gene are then analyzed to determine the relativeabundance in the two studied samples of mRNA corresponding to each gene.Currently, there are two other techniques available for large-scale monitoring ofgene expression, or transcript profiling: differential display and serial analysis ofgene expression which are discussed elsewhere [21].Microarray is increasingly being used to investigate patterns of gene expression.Recently, microarray technology has been employed as a means of large-scalescreening of vast numbers of genes – if not whole genomes – that possess differential expression in two distinct conditions.
While new and exciting developmentshave arisen in such fields as cancer [27] and yeast, [28] very few cardiovascularbased microarray studies have been published [29]. In animal studies of myocardial infarction, Friddle et al. [30] used microarray technology to identify gene expression patterns altered during induction and regression of cardiac hypertrophyinduced by administration of angiotensin II and isoproterenol in a mouse model.A total of 55 genes were identified during induction or regression of cardiac hypertrophy.
They confirmed 25 genes or pathways previously shown to be alteredby hypertrophy, and further identified a larger set of 30 genes whose expressionhad not previously been associated with cardiac hypertrophy or regression.Among the 55 genes, 32 genes were altered only during induction, and 8 were altered only during regression. This study, using a genome-wide approach, demonstrated that a set of known and novel genes was involved in cardiac remodelingduring regression and that these genes were distinct from those expressed duringinduction of hypertrophy.In other studies, to examine the host gene expression involved during differentphases of viral myocarditis in a coxsackievirus B-infected mouse model, Taylor etal. [31] showed that 169 known genes of about 7,000 clones initially screened hada level of expression significantly different at one or more postinfection timepoints (days 3, 9 and 30) as compared with baseline.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
