Figure 1. The association of miRNA tissue specificity and the number (Nd) of diseases it implicated in. Each blue circle represents a miRNA whose x,y coordinates are its Nd and its tissue specificity index value. MiRNAs are grouped into different groups according to the Nd, and then the average tissue specificity index value of each group was calculated, shown as pink triangle. The pink triangles are connected with green lines. doi:10.1371/journal.pone.0003420.g001 dysfunctions. Therefore, its dysfunction would result in diseases with a higher probability. However, it remains largely unknown whether this association exists for human miRNAs and diseases. To investigate this issue, we classified all the human disease- associated miRNAs into two groups: miRNAs that are conserved in other species (group 1) and miRNAs that are human specific (group 2). We assigned one human miRNA into the conserved miRNAs if its family members can be found in other species according to the family annotations of miRBase[19]. Otherwise, we assigned it into the human-specific group. We counted the number of miRNAs that are associated with at least one disease or zero disease for these two groups. Our results show that miRNAs in group 1 tend to be associated with diseases with a higher probability significantly (P = 3.9610233, Fisher’s Exact Test, Table 2). Single nucleotide polymorphisms (SNPs) are the most common genetic variants in the human genome[20] and SNP density can be used as a metric of the conservation of DNA sequences. We calculated the number of SNPs for miRNAs associated with at Table 2. The number of miRNAs implicated in one or more than one human diseases for two groups. Num1 Num2 P value Group1 191 213 3.9610233 Group2 128 1 Groups1 contains 404 human miRNAs conserved in other species. Group2 contains 129 human specific miRNAs. Num1 is the number of miRNAs that are not reported related to disease. Num2 is the number of miRNAs implicated in at least one disease. P value was calculated using Fisher’s Exact Test. doi:10.1371/journal.pone.0003420.t002 MicroRNAs and Human Disease least one disease and no diseases. For all 236 miRNAs associated with disease in our dataset, we identified 20 SNPs in 20 miRNAs precursor sequences. The SNP occurring probability is 0.0847 (20/236). The SNP occurring probability of miRNAs that are not associated with diseases is 0.2727 (81/297). Significantly, miRNAs associated with diseases show lower SNP occurring probability than miRNAs that are not associated with diseases (P = 1.361028, Fisher’s exact test, Figure 2). Similar result was found when searching SNPs in mature miRNA sequences (SNP occurring probability 0.0381 vs. 0.0707, P = 0.07). We also tested the significance by randomly picking up the same number of miRNAs as the number of miRNAs associated with diseases from the whole miRNAs and calculated SNP occurring probability in this group of miRNAs, which was then compared with the real SNP occurring probability. We repeat this process 5000 times and got the P value (P,2.061024, Figure 3). These findings suggested that miRNA conservation is associated with human disease susceptibility, which will help in the understanding of miRNAs’ roles in diseases. miRNA sets and miRNA diseases The dysfunction of genes in a gene set, such as a signaling pathway or a biological process often result in the same disease. However, it is unclear whether miRNAs implicated in the same disease tend to emerge as miRNA sets. In this study, we defined two types of miRNA sets: miRNA families that contain groups of homologous miRNAs (miRNA duplicates) and miRNA clusters that contain groups of neighboring miRNAs on the human genome. It has been reported that duplicates of protein coding genes are functionally interchangeable[21], suggesting that they may be involved in similar biological processes with similar roles, therefore, may be implicated in the same disease. However, it is of keen interests but remain unclear whether a family of miRNAs tend to play roles in the same disease. In this study, we revealed that miRNAs in 57% of the diseases have at least one family member in that disease associated miRNAs, which is significantly higher than the random (P = 2.061024, Randomization Test). For example, 50% (3/6) of the miRNAs in miR-8 family was related to the thyroid cancer. This finding suggested that the miRNA family members might have similar functions and play roles in similar biological processes, and therefore whose dysfunction would lead to similar phenotype. Bartel and his colleagues reported that neighboring miRNAs show significant coexpression by a microarray profiling analy- Figure 2. SNP occurring probability of miRNAs implicated in at least one disease (disease miRNA), and miRNAs that are not reported to be implicated in disease (no-disease miRNAs). doi:10.1371/journal.pone.0003420.g002 PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3420