Osteoblastic differentiation of bone marrow mesenchymal stromal cells in Bruck Syndrome.

BACKGROUND: Osteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus. METHODS: We analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. RESULTS: No mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different. CONCLUSIONS: Our work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

COL1A1 and miR-29b show lower expression levels during osteoblast differentiation of bone marrow stromal cells from Osteogenesis Imperfecta patients.

BACKGROUND: The majority of Osteogenesis Imperfecta (OI) cases are caused by mutations in one of the two genes, COL1A1 and COL1A2 encoding for the two chains that trimerize to form the procollagen 1 molecule. However, alterations in gene expression and microRNAs (miRNAs) are responsible for the regulation of cell fate determination and may be evolved in OI phenotype. METHODS: In this work, we analyzed the coding region and intron/exon boundaries of COL1A1 and COL1A2 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. COL1A1 and miR-29b expression were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. RESULTS: We have identified eight novel mutations, where of four may be responsible for OI phenotype. COL1A1 and miR-29b showed lower expression values in OI type I and type III samples. Interestingly, one type III OI sample from a patient with Bruck Syndrome showed COL1A1 and miR-29b expressions alike those from normal samples. CONCLUSIONS: Results suggest that the miR-29b mechanism directed to regulate collagen protein accumulation during mineralization is dependent upon the amount of COL1A1 mRNA. Taken together, results indicate that the lower levels observed in OI samples were not sufficient for the induction of miR-29b.