Systematic gene analysis for bone metabolism associated to TGF-β1

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We analyzed 13 Camurati-Engelmann disease families for mutation in TGF-β1. We found four kinds of mutations, R218C, R218H, C223R, and C223G. These four mutations could occur nine times independently by judging from haplotype analysis around TGFB1 gene in Camurati-Engelmann disease families. This fact indicates mutations in Camurati-Engelmann disease are not transmitted from founder, but are fresh mutations occurred independently (paper submitted). We characterized the biochemical alterations of mutated TGF-β1 protein by transfection-expresssion assay system. In mutated proteins LAP (latency associated polypeptides) cannot associate to active TGF-β1, nor fold TGF-β1 to inactivate. This LAP dysfunction could cause the constitutive activation of TGF-b1 signal transduction in vivo. To search the bone metabolism in detail in vivo, we started to make knock-in mouse having R213C found in CED patient. Nine chimera mice were generated, but all of them were infertile because of immobility of sperm collected from epididymis. The cause of sperm immobility is not clear. There are two possibilities, ES cell itself might be damaged and TGF-b1 alteration might cause spermatogenesis. We screened 3 new homologous recombinant ES clones to make new knock-in mice.Our mutation search in Camurati-Engelmann disease indicates that TGF-β1 regulate the bone metabolism. We search the SNPs (single nucleotide polymorphisms) in the TGF-β1, TGFB receptors, SMADs, LRP, and c-Fos genes. We performed association study using these SNPs and bone density in 500 Japanese. Association result shows that one polymorphism in LRP gene is the determinant for bone density (p-value < 0.03) (paper in preparation).