Marcinkowska et al. BMC Genomics 2011, 12:183 http://www.biomedcentral.com/1471-2164/12/183 miRNA-target recognition) is the single nucleotide poly- morphism (SNP). Computational and experimental stu- dies have revealed many SNPs located in different parts of pre-miRNA sequences [14-16]. The occurrence of SNPs (including INDELs) in pre-miRNA regions is sig- nificantly lower than that in the surrounding reference sequences [16]. While sequences of mature miRNAs are the most conserved, the sequences of anti-miRNAs and the stems (outside miRNA and anti-miRNA) and loops of pre-miRNAs are somewhat less conserved [16]. SNPs naturally occurring within pre-miRNA sequences may affect miRNA biogenesis and impair miRNA-mediated gene silencing, as demonstrated by functional assays [15,17]. Recently, large genome-wide association study has demonstrated that also SNPs located outside (>14 kb) of pre-miRNA sequences can modulate miRNA expression both as cis- and trans-regulators (miRNA- eQTLs). One of identified miRNA-eQTLs (rs1522653) was shown to correlate with expression of 5 different miRNAs [18]. MiRNA target sites are also conserved genetic ele- ments. Bioinformatic analyses show that SNPs are underrepresented in both experimentally validated and computationally predicted miRNA target sites, [16,19] and SNPs have the potential to either disrupt or create new miRNA target sites [19]. It has also been proposed that target site polymorphisms may play a role in evolu- tion by altering miRNA specificity and function. However, little is known about copy number variation (CNV) of miRNA genes. CNVs are segments of genomic DNA (roughly 1 kb to 1 Mb in length) that show vari- able numbers of copies in the genome due to deletions or duplications. CNVs recurrently occurring in a popu- lation are often called copy number polymorphisms (CNPs). Only a few CNV discovery studies report the presence of miRNAs in detected CNV regions and recognize their potential consequences [20-22]. Indeed, it was suggested that a comprehensive analysis of the co-localization of miRNAs and CNVs is needed [12]. Numerous studies show that CNVs can influence the expression of protein-coding genes in a copy number- dependent manner [23-25]. Recent results of genome- wide association study has confirmed such association for dozens of protein-coding genes and showed that CNVs capture at least 18% of the total detected genetic variation in gene expression [26]. It seems obvious that the expression of miRNA genes can also be modified by CNVs. This notion is supported by results from cancer genetics studies. For instance, there is a correla- tion between somatic copy number variation and the expression of miRNA genes, and miRNA genes recur- rently amplified or lost in cancer genomes can serve as oncogenes or cancer suppressor genes, respectively [27-31]. Page 2 of 9 In this study, by comparing the coordinates of human miRNAs with different sets of CNV regions (DGV- deposited and highly polymorphic), we identified over 200 human copy number variable miRNA loci. By com- paring fractions of miRNAs and the genome that are covered by differentially validated CNV regions, we showed that miRNA loci are underrepresented in highly polymorphic CNVs, but not in CNVs deposited in the DGV database. We discuss the potential functional rele- vance of identified copy number variable miRNAs and propose models of how different types of CNVs can affect miRNA dosage. Results and Discussion Prior to bioinformatic identification of copy number variable miRNA genes (CNV-miRNAs), we compared the frequency of SNPs in annotated pre-miRNA sequences (3.7 SNPs/1,000 bp) and in reference human genome (4.8 SNPs/1,000 bp). Significantly lower number of SNPs in the pre-miRNA sequences (Fisher’s exact test; p < 0.0001) most likely results from SNP purifica- tion effect and confirms general conservation of the ana- lyzed pre-miRNA sequences. These analyses confirmed a SNP purification effect in pre-miRNA sequences reported previously [16]. The much higher number of SNPs identified in annotated pre-miRNA sequences in our study (N = 229; Additional file 1) versus N = 65 reported previously [16] results from the increased num- ber of both SNPs (dbSNP - build 130; Apr 30, 2009; only annotated as ‘single’; ~14 million SNPs) and miR- NAs (miRBase - v 13.0), available in versions of data- bases used in this study. To identify CNV-miRNAs, we compared the positions of miRNA loci with three sets of CNVs: ‘DGV-deposited’ (N = 29133; 30% genome coverage), ‘polymorphic-SMC’ (N = 1319; 1.2% genome coverage) [32] and ‘poly- morphic-DC’ (N = 5037; 2.3% genome coverage) [22] CNVs. ‘DGV-deposited’ CNVs include all 29133 CNVs deposited in the Database of Genomic Variants (DGV update Aug 05, 2009 - http://projects.tcag.ca/variation). Two sets of ‘polymorphic’ CNVs (’polymorphic-SMC’ [32] and ‘polymorphic-DC’ [22]) include highly poly- morphic CNVs (minor allele frequency >0.01) validated by high-quality genotyping in two recent CNV-discovery studies using CNV-dedicated high-density hybrid arrays (combining traditional SNP probes and probes targeting CNVs) [22,32]. In both of these studies, precise break- points and unambiguous copy numbers were deter- mined for each analyzed sample. All ‘DGV-deposited’ CNV-miRNA regions were further characterized by the following validation factors: (i) number of publications reporting CNVs (references), (ii) number of overlapping CNVs (DGV records) and (iii) number of observations in discovery studies (frequency) (Additional file 2). Since