Current Genomics - Volume 6, Issue 7, 2005

The term epigenetics defines the heritable changes in gene expression that occur through changes in the chromatin structure, rather than changes in the DNA sequence. The methylation of cytosines (m5C) in CpG dinucleotides (DNA methylation) and the modification of histones are fundamental epigenetic mechanisms that regulate eukaryotic gene expression. In general, increases in DNA methylation are associated with gene silencing whereas decreases in DNA methylation are often associated with gene expression. As a result, DNA modification provides an additional level of genetic information by incorporating a fifth nucleotide into the genetic code. Epigenetic regulation has been shown to be important in cellular differentiation, embryonic development, and when abnormal - carcinogenesis and disease. Until recently studies investigating epigenetic modifications of DNA were restricted to looking at specific gene loci. High throughput genomic methods such large scale bisulphate sequencing, restriction landmark genome scanning (RLGS), CpG island microarrays and mass spectroscopy methods have now been developed that allow researchers to assay levels of DNA methylation and chromatin structure over wide areas of the genome or the entire genome - the epigenome or methylome. This new area of research has been termed epigenomics, and its aim is to describe the complete set of DNA methyation modifications within a cell. Comparative studies are presently being conducted in order to understand epigenomic differences in a number of systems. These included epigenome comparisons between undifferentiated and differentiated embryonic cells, cancerous and non-cancerous cell types, and cells exposed to varying environmental parameters. Studying global changes in DNA methylation has the potential to identify early indicators of carcinogenesis or biomarkers for conditions that alter the normal epigenomic status within an organism.

The availability of complete genomes and global gene expression profiling has greatly facilitated analysis of complex genetic regulatory systems. We describe the use of a bioinformatics strategy for analyzing the cis-regulatory design of genes diferentially regulated during viral infection of a target cell. The large-scale transcriptional activity of human embryonic kidney (HEK293) cells to reovirus (serotype 3 Abney) infection was measured using the Affymetrix HU- 95Av2 gene array. Comparing the 2000 base pairs of 5' upstream sequence for the most differentially expressed genes revealed highly preserved sequence regions, which we call "modules". Higher-order patterns of modules, called "supermodules", were significantly over-represented in the 5' upstream regions of transcriptionally responsive genes. These supermodules contain binding sites for multiple transcription factors and tend to define the role of genes in processes associated with reovirus infection. The supermodular design encodes a cis-regulatory logic for transducing upstream signaling for the control of expression of genes involved in similar biological processes. In the case of reovirus infection, these processes recapitulate the integrated response of cells including signal transduction, transcriptional regulation, cell cycle control, and apoptosis. The computational strategies described for analyzing gene expression data to discover cisregulatory features and associating them with pathological processes represents a novel approach to studying the interaction of a pathogen with its target cells.

Fragile X syndrome is the most common inherited form of mental retardation and is due to the silencing of FMR1 gene coding for the FMRP protein. FMRP is an RNA binding protein endowed with Nuclear Localization and Nuclear Export Signals and is associated with actively translating polysomes as part of mRNP complexes. During the past years, efforts from many laboratories to unravel the function of this protein, resulted in the identification of several proteins (mostly RNA-binding) and few hundred of mRNAs that are targeted by FMRP. The puzzle illustrating the FMRP role depicts a protein implicated in different steps of mRNA metabolism. However, its precise mechanism of action is not still understood and the specificity of its function is probably dependent on RNA and/or proteins that interact and associate with it.

Many genes important during early development in vertebrates are regulated by sequences located at large distances from the protein coding region. Clues to the location of these long-range gene-regulatory elements can be obtained from comparing genomic sequences of evolutionarily distant species such as the mouse and human. However, identifying them functionally remains a major challenge. Analysis of distal regulatory sequences is important not only for a complete understanding of regulation of the gene in specific tissues, but also for exploring mechanisms and possible therapeutic strategies for diseases linked to variations in those sequences. Polymorphisms existing in far away regulatory sequences that are linked statistically to a disease can be associated with a gene only when such sequences are functionally implicated in regulating the expression of that gene. The mechanistic pathway that connects the disease to the malfunction of the gene can then be identified, and possible therapeutic interventions explored. A strategy that uses transgenic mice developed with a GFP-reporter gene tagged BAC clone to functionally identify such long-range regulatory sequences in the cardiac specific Nkx2-5 gene is illustrated. A combinatorial approach using the full length Nkx2-5 GFP-BAC and several of its truncations, chosen on the basis of cross-species genomic sequence alignment of highly conserved non-coding DNA, as transgenes helped delimit the boundaries of transcriptional regulation to sequences 27 kb upstream of the Nkx2- 5 gene. Identifying sequences involved in the regulation of genes distant to them are discussed in view of their potential for exploring mechanistic pathways for disease and possible therapeutic interventions.

First named Narc-1 (Neural apoptosis regulated convertase 1), PC9 is the ninth member of the family of proprotein convertases. This newly identified human subtilase contributes to cholesterol homeostasis and mutations in its gene, PCSK9 (Proprotein Convertase Subtilisin/Kexin type 9), are responsible for Autosomal Dominant Hypercholesterolemia. This is the first example of a dominant disease associated with a defect in a member of the convertase family. Hypercholesterolemia is a main risk factor of atherosclerosis and its vascular complications. In the general population, about 1 person out of 20 presents high plasma LDL-cholesterol. In particular, familial forms with autosomal dominant transmission affect about 1 person out of 500. Until recently, mutations in only two genes were associated with the disease: the LDLR gene encoding a transmembrane receptor implicated in endocytosis and degradation of circulating LDL, and the APOB gene encoding the main ligand of this receptor present at LDL surface. Pathophysiology of these two main forms of the disease has been extensively studied and is well understood. In 1999, two teams simultaneously published hypercholesterolemic families presenting neither LDLR nor APOB defects and, in 2003, a third major gene involved in Autosomal Dominant Hypercholesterolemia, PCSK9, was identified. To date, no substrate of PC9 has been found except itself. The purpose of the present review is to compile all reported data and current knowledge on PC9 and hypotheses of its role in cholesterol homeostasis and in pathophysiology of hypercholesterolemia.

We review current approaches that can extend our understanding of monogenic disease towards complex disease. Recent studies showed that currently established disease genes differ in their protein size, tissue specificity and the phylogenetic distribution of homologs. These characteristics can be explained by the fact that monogenic disease mutations must be sufficiently deleterious to produce a clearly recognizable phenotype, but also must not be lethal in an early embryonic stage. On the other hand, deletion of each gene in the human genome must be evolutionarily disadvantageous. For most genes, this disadvantage might manifest as an increased susceptibility to complex disease. Accordingly, mildly deleterious variants can be observed in a wide spectrum of genes. The phenotypic manifestation of these mildly deleterious variants might depend on somatic mutations, which cause the breakdown of compensating mechanisms in individual cells. At present, association studies are the most promising strategy for mapping complex disease phenotypes. However, these are restricted to the identification of common disease variants and often provide only marginally convincing statistical evidence. Novel computational strategies, which take prior biological knowledge into account, therefore might play a major role in the design and interpretation of large-scale association studies.

Dissecting the genetic basis of complex diseases remains one of great challenges in human genetics, because these diseases have polygenic determinations and involve multiple gene-gene and gene-environmental interactions. Definite conclusions about finding genes underlying complex diseases need substantial evidence from three levels of gene function. The traditional strategy of gene identification is to determine putative susceptibility genes on the DNA level, and then to find related association between susceptibility genes and complex diseases on the RNA and protein levels. However, with rapid development of technologies of proteomics and microarrays, a new high-throughput strategy backward from protein to RNA and further to DNA becomes available for gene discovery. This strategy can systemically test gene expression, analyze co-expressed genes or regulatory network, and detect the effects of environmental factors on the onset and development of complex diseases. Here we attempt to outline these two strategies using obesity as an example of a complex disease, and to compare their advantages and disadvantages. In conclusion, we suggest that these two strategies may complement each other and thus help to uncover a more comprehensively and more completely multifaceted spectrum of genetic determination for complex diseases.

Unstable expansions of trinucleotide repeats (TNRs) are associated with a growing number of neurological disorders (at least 14), including HD (Huntington's disease), fragile X-syndrome, MD (Myotonic dystrophy) and Freidreich's ataxia. These disorders are often characterized by a tendency of certain pathological alleles to further expand due to biases in the parental origin of mutations, at times, leading to the most severe forms. TNR expression involves changes in the repeat tract length, threshold value, secondary structure formation, interruptions, mismatch repair mechanism, genes and their involved sequences, the product thereof and anticipation. The interactions of each of these factors with the others influence manifestations of the disease. The exact cellular events and/or mechanism of varied expression in all the neurological diseases with similar repeats have not yet been clearly understood. A correlation between trinucleotide expansion, chromosomal fragile sites and neurological diseases has, however, been established. This review deals with the involvement of TNRs in neurological disorders with respect to the type of repeat, level of normal, premutation and expansion of repeats, chromosomal locus and the involved genes along with clinical implications. Possible answers to two basic questions regarding the mechanisms of involvement of repeat expansions and interrelationships between such expansions and fragile sites have been provided based on the available experimental data. Detection of trinucleotide repeat expansion and fragile genetic sites could be among the excellent parameters for screening and diagnosis of human neurodegenerative disorders. An insight into the mechanisms involved may help the clinicians to develop suitable treatments.