In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and then apply the knowledge gained from our studies to the treatment of children with these conditions. Structural defects of type I collagen molecule are well known to cause the dominant bone disorder OI. A dozen years ago, we identified defects in two components of the collagen prolyl 3-hydroxylation complex, CRTAP and P3H1 (encoded by LEPRE1) as the cause of recessive OI. Our work generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in proteins that interact with collagen cause the rare forms of OI. Recessive OI is now a major area of investigation for the BEMB. The phenotypes of types VII and VIII OI are distinct from classical dominant OI, but difficult to distinguish from each other. We showed that mutual stabilization of CRTAP and P3H1 underlies the phenotypic and biochemical similarity of types VII and VIII OI. With collaborators at the Boltzman Osteology Institute, we recently focused on the bone of the non-lethal subset of type VIII OI patients. Bone histology was similar to type VII OI, although it had the distinctive feature of extremely thin trabeculae and patches of increased osteoid, suggesting mineralization is slower in type VIII than VII OI. BMDD yielded increased mineralization of type VIII bone, as in classical OI and type VIII OI, but the proportion of bone with low mineralization was increased in type VIII bone vs type VII. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry. We generated a CyPB KO mouse, which has reduced bone density and strength, but increased brittleness. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports LH1 activity and its absence strongly modulates LH chaperone complexes. KO bone has significant reduction of hydroxylation of crosslinking residue K87, which alters fibril structure and reduces bone strength. With collaborators at the University of North Carolina we showed that CyPB interacted with all LH forms (LH1-3). In tendon, CyPB modulates collagen cross-linking by differentially affecting lysine hydroxylation in the helical and telopeptide domains of collagen. While lysine hydroxylation was decreased in the collagen helix, it was increased in the telopeptide cross-link sites. CyPB modulates crosslinking by affecting lysine hydroxylation in a site-specific manner. In skin, collagen lysine 87 is also underhydroxylated and underglycosylated, despite upregulation of LH1 and LH2 protein. In addition, lack of lysine hydroxylation leads to two unusual collagen crosslinks, derived from the telopeptides. The skin of KO mice has reduced resistance to nanoindentation, adding a role in skin mechanics to PPIB functions. We delineated a mutation in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. The BRIL S40L substitution results in minimal expression and secretion of PEDF by mutant FB and osteoblasts. In contrast to the gain-of-function BRIL mutation that causes type V OI, the BRIL S40L causes decreased mineralization and expression of bone markers. Only type I collagen shows similar expression pattern in both mutations, with decreased expression, secretion and matrix incorporation. We have generated a murine model for this mutation, in which both heterozygotes and homozygotes are viable, which is currently being characterized. We have also further characterized the bone tissue in type V OI, caused by a dominant gain-of-function mutation at the 5-end of the transcript, which adds 5 amino acid residues to the cytoplasmic amino terminus of BRIL, in collaboration with colleagues in Austria and Canada. The BMDD of type V patient bone was shown to have increased mineralization in both cortical and trabecular bone, comparable to the increased mineralization found in bone with structural defects of collagen, although collagen has normal primary structure and post-translational modification in type V OI. Bone in type V OI is immature and has a mesh-like structure with osteocyte lacunar density nearly double that of normal bone, pointing to elevated primary bone formation and altered bone remodeling. Type XIV OI is a moderately severe form of OI which was identified in 2013. It is caused by recessive defects in TMEM38B, which encodes TRIC-B, an ER cation channel. In probands with recessive null defects in TRIC-B, we demonstrated impaired ER calcium flux, resulting in ER-stress along the ATF4 pathway. TRIC-deficiency was shown to be collagen-related, impairing collagen synthesis and assembly at multiple steps. Collagen helical lysine hydroxylation was reduced, although the levels of LH1 protein were increased. We also investigated the unique aspects of TMEM38B deficiency in 8 patients with type XIV OI in collaboration with colleagues in the UK and Vienna. There is striking variability of OI severity even between siblings. Like other OI types, type XIV patients have low DXA BMD. However, unlike other OI types, bone mineralization is not increased and nanoporosity is low. Low bone turnover in type XIV OI is likely explained by an intrinsic defect in osteoclasts, which normally express TMEM38B. Recently, we delineated the first X-linked recessive form of OI, made more exciting by its novel bone mechanism. X-linked OI is moderately severe with pre- and post-natal fractures of ribs and long bones, dysplastic bone with bowing and crumpling. It is caused by missense mutations in MBTPS2, which encodes Site-2 protease (S2P). S2P is a critical component of Regulated Intramembrane Proteolysis (RIP), a process in which S1P and S2P, located in the Golgi Membrane, sequentially cleave regulatory proteins transported from the ER membrane in times of cell stress or sterol metabolite deficiency. The levels of mutant S2P transcripts and protein are normal, but RIP function on substrates OASIS, ATF6 and SREBP are impaired. At the bone tissue level, hydroxylation of type I collagen K87 residues is half normal, altering collagen crosslinking in bone. Osteoblasts with S2P defects also have a differentiation defect.

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Webb, Emma A; Balasubramanian, Meena; Fratzl-Zelman, Nadja et al. (2017) Phenotypic Spectrum in Osteogenesis Imperfecta Due to Mutations in TMEM38B: Unraveling a Complex Cellular Defect. J Clin Endocrinol Metab 102:2019-2028
Zou, Yaqun; Donkervoort, Sandra; Salo, Antti M et al. (2017) P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye. Hum Mol Genet 26:2207-2217
Kang, Heeseog; Aryal A C, Smriti; Marini, Joan C (2017) Osteogenesis imperfecta: new genes reveal novel mechanisms in bone dysplasia. Transl Res 181:27-48
Blouin, St├ęphane; Fratzl-Zelman, Nadja; Glorieux, Francis H et al. (2017) Hypermineralization and High Osteocyte Lacunar Density in Osteogenesis Imperfecta Type V Bone Indicate Exuberant Primary Bone Formation. J Bone Miner Res 32:1884-1892
Fratzl-Zelman, Nadja; Barnes, Aileen M; Weis, MaryAnn et al. (2016) Non-Lethal Type VIII Osteogenesis Imperfecta Has Elevated Bone Matrix Mineralization. J Clin Endocrinol Metab 101:3516-25
Dubnikov, Tatyana; Ben-Gedalya, Tziona; Reiner, Robert et al. (2016) PrP-containing aggresomes are cytosolic components of an endoplasmic reticulum quality control mechanism. J Cell Sci :
Terajima, Masahiko; Taga, Yuki; Chen, Yulong et al. (2016) Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen. J Biol Chem 291:9501-12
Lindert, Uschi; Cabral, Wayne A; Ausavarat, Surasawadee et al. (2016) MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta. Nat Commun 7:11920
Forlino, Antonella; Marini, Joan C (2016) Osteogenesis imperfecta. Lancet 387:1657-71
Ben-Gedalya, Tziona; Moll, Lorna; Bejerano-Sagie, Michal et al. (2015) Alzheimer's disease-causing proline substitutions lead to presenilin 1 aggregation and malfunction. EMBO J 34:2820-39

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