Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.

Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple-helical domains of type I collagen α1(I) and type II collagen α1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, CRTAP, or cartilage-associated protein, and prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis-trans isomerase cyclophilin-B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and sixteen with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, “popcorn” epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations.

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Type I procollagen is a heterotrimer composed of two pro␣1(I) chains and one pro␣2(I) chain, encoded by the COL1A1 and COL1A2 genes, respectively. Mutations in these genes usually lead to dominantly inherited forms of osteogenesis imperfecta (OI) by altering the triple helical domains, but a few affect sequences in the pro␣1(I) C-terminal propeptide (C-propeptide), and one, which has a phenotype only in homozygotes, alters the pro␣2(I) C-propeptide. Here we describe four dominant mutations in the COL1A2 gene that alter sequences of the pro␣2(I) C-propeptide in individuals with clinical features of a milder form of the disease, OI type IV. Three of the four appear to interfere with disulfide bonds that stabilize the C-propeptide conformation and its interaction with other chains in the trimer. Cultured cells synthesized pro␣2(I) chains that were slow to assemble with pro␣1(I) chains to form heterotrimers and that were retained intracellularly. Some alterations led to the uncharacteristic formation of pro␣1(I) homotrimers. These findings show that the C-propeptide of pro␣2(I), like that of the pro␣1(I) C-propeptide, is essential for efficient assembly of type I procollagen heterotrimers. The milder OI phenotypes likely reflect a diminished amount of normal type I procollagen, small populations of overmodified heterotrimers, and pro␣1(I) homotrimers that are compatible with normal skeletal growth. Osteogenesis imperfecta (OI)4 (1, 2), commonly known as brittle bone disease, is usually caused by mutations in the COL1A1 and COL1A2 genes that encode the pro␣1(I) and pro␣2(I) chains, respectively, of type I procollagen. This heterotrimeric collagen is composed of two pro␣1(I) chains and one pro␣2(I) chain. Molecular assembly of the trimer is a multistep process that occurs following synthesis of the full-length chains and release from the ribosome. The C-terminal propeptide (C-propeptide) of each chain folds into a structure that is stabilized by intra-chain disulfide bonds and exposes a chain selectivity domain (3) that directs the interaction of the correct three chains into trimers. Following stabilization of the trimer by inter-chain disulfide bonds, the collagen triple helical domains are nucleated by sequences at their C-terminal end and the helix then propagates in an N-terminal direction.The vast majority of OI-causing mutations (4, 5) 5 affect the triple helical domains and perturb either the nucleation or the propagation of the triple helix N-terminal to the altered sequence but do not affect initial assembly of the pro␣ chains (6). The delay in helix propagation permits prolonged access of modifying enzymes to the chains N-terminal of the alteration (7), whereas the sequence alterations themselves alter the helical structure (8, 9) and reduce molecular thermal stability (10, 11). The phenotypic outcome of these mutations is, in part, due to the synthesis of structurally altered triple helices, and reflects both the location and nature of the change. These mutations result in the full spectrum of OI severity,...