Identification of novel genes involved in Paget's disease by Differential Display PCR
In order to identify novel genes which were expressed in Paget's disease, the Differential Display PCR technique was used to compare gene expression in samples of Pagetic and normal bone, and in human bone long term marrow cultures. Several genes appeared to be differentially expressed in Pagetic bone in comparison to controls, however the display patterns were difficult to interpret and consistent differences were not observed. Pagetic marrow cultures were studied and were found to be abnormal in that osteoclast-like cell formation was 20-fold higher in Pagetic marrow than in age-matched controls. This study also examined Pagetic marrow samples aspirated from both affected and unaffected sites, and it was found that large numbers of oseteoclast-like cells formed in Pagetic cultures regardless of the site of marrow aspiration, suggesting that there may be an abnormality in osteoclast precursors in Paget's disease. Differential display of these marrow samples revealed consistent differences in gene expression between Pagetic and normal marrow, and of four differentially expressed mRNAs which were cloned and sequenced, one was selected for further study owing to its significant homology to murine Centrosomin A, a gene thought to be involved in cell cycle division and microtubule formation. The centrosomin-like DD clone was confirmed to be significantly over-expressed in Pagetic marrow by semi-quantiative PCR, and using 232bp DD product as a probe, a ZAP Express osteoclastoma cDNA library was screened by hybridisation to identify cross-hybridising clones. Thirty four positive clones were identified, and two of these clones, CLC 1 and CLC 2, were isolated and sequenced. Database searches identified CLC 2 (4kb) as being a partial clone of human eukaryotic translation initiation factor-3 (eIF-3) - p180 subunit. Further characterisation of both CLC-1 and 2 may provide important information on their functions, both within the cell and their possible role in Paget's disease.