Abstract
Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.
Highlights
Researchers have been focusing their efforts on the study of the non coding part of the human DNA and, in particular, on its predicted role in the regulation of gene expression [1]
To verify whether the characterized copy-number variations (CNVs) overlap the VISTA enhancers, we compared the positions of their loci with three sets of CNVs: ‘Database of Genomic Variants (DGV)-deposited’ (N = 67,419; 30.37% genome coverage) [14], ‘‘polymorphic-Copy Number Polymorphism (CNP)’’ (N = 1,319; 1.06% genome coverage) [16] and ‘‘polymorphic-DC’’ (N = 5,037; 2.3% genome coverage)
[17] CNVs. ‘‘DGV-deposited’’ CNVs include all 67,419 CNVs deposited in the Database of Genomic Variants (DGV update Nov 02, 2010– variation.hg18.v10.nov.2010.txt, file available at http://projects.tcag.ca/variation). ‘‘PolymorphicCNP’’ [16] and ‘‘polymorphic-DC’’ [17] are two sets of highly polymorphic CNVs validated by high-quality genotyping in two CNV-discovery studies using highdensity arrays
Summary
Researchers have been focusing their efforts on the study of the non coding part of the human DNA and, in particular, on its predicted role in the regulation of gene expression [1]. Comparative sequence analysis has proved to be a valuable instrument to identify regulatory elements that have been highly conserved throughout evolution [2], many of these being noncoding sequences shown to act as enhancers in experimental models [3,4]. A database of human and mouse noncoding fragments with a gene enhancer activity has been developed [4]. To characterize CNVs overlapping VISTA enhancer loci, we have compared the coordinates of human VISTA enhancer loci with CNVs deposited to the Database of Genomic Variants (DGV) and Indels (small insertions and deletions of 100 bp-1 kb length) and with two highly polymorphic sets of deleted and duplicated regions. We have shown that highly polymorphic CNVs are under negative selection at VISTA enhancer loci, suggesting that copy number variable enhancers could represent functional variants. To confirm the functional relevance of enhancers and to verify whether dysmorphic features or mental retardation could be associated with rare or private duplications and deletions in these elements, we have designed the Enhancer Chip custom array that is described below
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