Marcinkowska et al. BMC Genomics 2011, 12:183 http://www.biomedcentral.com/1471-2164/12/183 database [36] over half (14/26) of top-validated CNV- miRNAs (Table 1 and Table 2) were shown to be expressed in at least several tissues/cell lines (detailed expression profiles are shown in Additional file 3). MiRNA whose expression is not reported in mimiRNA were either not analyzed for expression or did not show expression in the analyzed tissues. Additionally, three out of ten (30%) top-validated CNV-miRNAs (Table 1 and Table 2) which expression in primary fibroblast cell lines was analyzed by the micro-fluidics-based TaqMan Human MiRNA Array show high level of expression [18]. Based on the currently available sequence data for miRNAs deposited in miRBase and blast searches of the vertebrate genomic sequences we also determined evolu- tionary conservation of the miRNAs found in top-vali- dated CNV regions. Most of these miRNAs seem to be specific only for primates. There are, however, 8 miR- NAs that are conserved across mammals or vertebrates (Table 1 and Table 2). The functional relevance of several of the CNV-miR- NAs identified in this survey was previously reported in the literature (manual screening; Table 1 and Table 2). CNV-miRNAs are involved in many processes and phe- notypes (diseases), including organ development [37], angiogenesis [38], male infertility [39], transplant rejec- tion [40], multiple sclerosis [41] and cancer. Many CNV-miRNAs are specifically deleted, amplified or expressed in different types of cancers [42-47] and can regulate the expression of important cancer-related genes [37,48]. The copy number variation of those func- tionally relevant miRNAs can modulate or predispose one to the aforementioned phenotypes. In the next step, we determined whether the overlap of CNVs and miRNA loci was random (null hypothesis) or whether the CNVs were underrepresented at these loci (alternative hypothesis). To test this hypothesis, we compared fractions of miRNA loci and fractions of the genome covered by differentially defined CNV regions. Figure 1A shows that the fraction of miRNA loci cov- ered by two sets of ‘polymorphic’ CNVs is approxi- mately two times lower than expected (fraction of the covered genome). Although this effect was only margin- ally significant (Figure 1A), it suggested that at least highly polymorphic CNVs are under negative (purifying) selection at miRNA genes. Conversely, the fraction of miRNAs (0.292) covered by ‘DGV-deposited’ CNVs cor- responded almost exactly to the fraction of the genome covered by those CNVs (0.299). The CNV purification effect was not observed, even after narrowing ‘DGV- deposited’ CNV regions by different validation factors defined above (Figure 1B and 1C). The fact that the pur- ifying effect did not apply to the ‘DGV-deposited’ CNVs suggested that a significant portion of these CNVs are very rare, private, or significantly oversized or represents Page 5 of 9 Figure 1 Comparison of observed and expected number (fraction) of miRNA loci located in different CNV regions. Expected values were estimated based on the fraction of the genome covered by CNVs. A) Graph showing the fractions of miRNA loci (observed number of CNV-miRNAs; green bars) and the genome (expected number of CNV-miRNAs; orange bars) covered by two sets of ‘polymorphic’ CNVs. Binomial probabilities of equal or lower than the observed number of miRNA loci covered by CNVs are indicated over the bars. B) and C) The fractions of miRNA loci and the genome covered by ‘DGV-deposited’ CNV regions gradually narrowed by the increasing number of overlapping CNVs (DGV records) (B) and the increasing number of reporting references (C). false positive artifacts. This observation is consistent with the conclusions from other recently published results [32,49]. Although copy number variation can influence gene expression through different mechanisms (e.g., position effect and deletion or duplication of regulatory elements