Genetic Disorders of Bone and Extracellular Matrix

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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 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 SHDBEM. 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. We are now focused on patients with type VII OI. This type is usually lethal by 1 year of age, but we have identified siblings who are full null for CRTAP but have severe non-lethal OI. Cultured osteoblasts from surgical discard from these patients is being investigated for differentiation and pathways related to collagen synthesis and modification, 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. We are currently utilizing the PPIB KO mouse to investigate the differentiation and functioning of bone cells, both osteoblasts and osteoclasts, and the impact on bone histomorphometry and mineralization. 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. 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 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. We are currently investigating the differentiation of type XIV human osteoblasts for comparison with those in a murine model generated by collaborators. 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 have a differentiation defect. We have generated a murine model that copies the amino acid substitution present in two independent patients. The impact of this mutation on various arms of the RIP pathway and the distinction of the OI mutations from the S2P mutations causing cholesterol defects is being investigated.