[Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells].

OBJECTIVE: To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells. METHODS: Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot. RESULTS: By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells. CONCLUSION: WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.

[Proliferation, differentiation, and gene expression profile of osteoblast of patient with spondyloepiphyseal dysplasia tarda with progressive arthropathy].

OBJECTIVE: To explore the proliferation, differentiation, and gene expression profile of the osteoblasts (OBs) of patient with spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA). METHODS: OBs from the head of femur of a SEDT-PA patient resected during operation were cultured. ELISA was used to examine the bone-specific alkaline phosphatase (BAP), osteocalcin (OC), and osteoprotegrin (OPC) in the lysate of OBs. The proliferation and survival of the OBs were evaluated with MTT method and (3)H-TDR incorporation. Flow cytometry was used to observe the cell cycle. Northern blotting was used to evaluate the mRNA expression of WISP3, a member of the CCN family, mRNA in the OBs. Gene differential expression microarray was used to determine the cDNA expression. Osteoblasts from the head of femur of a patient about the same age with fracture of femur neck was used as control. RESULTS: The cultured OBs from the SEDT-PA patient showed a higher survival capacity (0.86 +/- 0.04 vs 0.71 +/- 0.10) and more (3)H-TDR incorporation (1363 +/- 350 vs 867 +/- 128). The expressions of OC and OPG were down-regulated to the 1/4.3 and 1/4.69 of the control respectively. There was no significant difference in the expression of BAP. WISP3 was very lowly expressed in the OBs of the SEDT-PA patient. In comparison with the normal control, there were up-regulation of 22 genes, including those coding chemotactic factors and inflammatory factors, and down-regulation of 16 genes in the OBs of the SEDT-PA patient with a synthetic effect of more rapid proliferation of OBs and reduction of synthesis of extracellular matrix, resulting in osteopenia. CONCLUSION: Not significantly different between SEDT-PA patient and normal person, the WISP3 expression may not be a direct factor of SEDT-PA.

[Pathology and molecular pathogenesis of spondyloepiphyseal dysplasia tarda with progressive arthropathy caused by compound CCN6 heterogeneous gene mutations].

OBJECTIVE: To characterize the clinical manifestations, features of roentgenography and MR imaging, and the pathology of articular cartilage and matrix of spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA), to screen the mutations of the disease-causing CCN6 gene, and try to elucidate the molecular pathogenesis of SEDT-PA. METHODS: A questionnaire survey on the clinical manifestations and history was conducted among a pedigree of SEDT-PA with 57 persons (53 living members) in tolal, including 2 probands, a 19-year old female and a 9-year old male. Physical examination and roentgenography and MR imaging were used on the 2 probands to characterize the features of their joints and articular cartilage. The femoral head extracted during replacement of hip of the proband 1 underwent hematoxylin-eosin staining and toludine blue (TB) staining to observe the pathological changes and ultra-microstructure of the articular chondrocytes and cartilage matrix using electron microscopy. Peripheral blood samples were collected from these 53 living members and 100 healthy controls. PCR was used to examine and sequence the exons of CCN6. 3D-conformational illustration of mutant CCN6 proteins were predicted using the Prospect Software. RESULTS: The clinical manifestations, radiology, and MR imaging established the diagnosis of SEDT-PA. Pathologic examination demonstrated that the articular cartilage chondrocytes became hyper-proliferative and immature, while the density and diameter of matrix collagens were dramatically decreased. Mutation studies showed the two probands carried a deletion (840delT) mutation in maternal allele, that caused the truncated CCN6 protein to miss 43 residues in C-terminus; and a substitution mutation (1000T-->C, Ser334Pro) in paternal allele, which was also inherited down to other 4 members in the SEDT-PA kindred. The predicted 3D-conformational changes of the truncated mutant and the Ser334Pro mutant CCN6 proteins demonstrated that in comparison with the wild CCN6 protein, the single long peptide loop in the region from signal peptide to the beginning 24 amino acid residues in the first domain (IGFBP) was subjected to folding into two smaller cross-loops accompanied with a much shorter C-terminus in 840 delT truncated mutant CCN6 protein, and no substantial 3D-conformational change of Ser334Pro mutant CCN6 protein was detected except for the C-terminal peptide towards the opposite direction. CONCLUSION: Novel 840delT mutation of CCN6 gene is the leading cause of SEDT-PA though coexistence of T1000C substitution is necessary for the clinical onset of SEDT-PA, in which marked abnormalities of cartilage chondrocytes and matrix are morphologically and functionally presented.