Unsupervised selleck chemicals hierarchical clustering split the cases into two large groups, Cluster A and Cluster B, resulting from differential expression of forty-seven miRNAs located on 14q32.2 and 32.31. Two prior studies [39], [40] showed similar differential miRNA expression patterns in adult mutant GIST based on 14q status, as well as other clinico-pathological variables. However neither of those studies addressed the methylation status of the retained 14q allele in their cases showing 14q loss. We found no direct relationship between 14q genomic status and 14q32 miRNA expression in this cohort. Eighty-two percent of adult mutant cases tested showed 14q loss, yet many of these in fact show relatively higher 14q miRNA expression than cases with the normal (diploid) FISH result as seen in all pediatric cases.

The 14q32 region is a known imprinted region in both mice (where the corresponding region is located on distal chromosome 12) and humans [37], [38], [41], containing maternally- and paternally-expressed genes. The miRNAs located within this cluster all map within a 40 kb interval and are controlled by a differentially methylated region (IG-DMR) 200 kb away [37], [38], [42]. miRNAs in this region are only expressed from the maternal allele [37], [38], as the paternal allele is silenced by methylation, and these miRNAs are thought to be transcribed as a large single poly-cistronic cluster (precursor transcript) rather than as individual primary transcripts [37]. Therefore, deletion of the active maternal allele is required for complete loss of expression of these miRNAs.

We hypothesised that the adult mutant cases showing 14q loss with relatively higher 14q miRNA expression (Cluster A) must retain the active maternal allele, while the cases with lower 14q miRNA expression (Cluster B) retain the silent paternal allele, resulting in down-regulation of these miRNAs. To investigate this, we applied the diagnostic assay used Drug_discovery for the detection of uniparental disomy (UPD) for chromosome 14q [30]. UPD is the inheritance of both homologues of a chromosome from one parent [43]. 14q32 contains the respectively maternally- and paternally- expressed MEG3 and DLK1 genes, which contribute to different phenotypes in maternal and paternal UPD14 [30], [41], [43] and are regulated by a differentially methylated region (DMR) that extends over the MEG3 promoter. This is the IG-DMR referred to above which controls the miRNA cluster. The assessment for UPD14 relies on a methylation-specific multiplex PCR to amplify methylated and unmethylated elements of the DMR and identify normal pattern methylation, maternal or paternal UPD14 [30].