In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Structural defects of the heterotrimeric type I collagen molecule are well known to cause the dominant bone disorder osteogenesis imperfecta. Several years ago the BEMB 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 proteins that interact with collagen cause recessive 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, with white sclerae, normal head circumference, rhizomelia, and severe undertubulation of long bones. Biochemically, both groups have normal collagen sequences with absence of 3-hydroxylation of the Pro986 residue, but full overmodification of the helical prolines and lysines. The helical overmodification indicates that absence of the components of the 3-hydroxylation complex leads to delayed folding of the collagen helix. We have now shown that the basis of the phenotypic and collagen biochemical similarity of types VII and VIII OI is that CRTAP and P3H1 are mutually protective. Also, in LEPRE1-null cells, the secretion of CRTAP into conditioned media is increased and accounts for 15-20% of the decreased CRTP detected in cells. Recently, we have collaborated with an Italian team to study the CRTAP mutation found in a non-lethal proband with severe OI caused by homozygosity for a null insertion/deletion mutation. The levels of CRTAP transcripts and protein do not correlate with survival, which may be related to functions of the secreted CRTAP in matrix. Importantly, this study provided the first demonstration that absence of CRTAP results in a severe deficiency of collagen deposited into matrix (10-15% of control), with disorganization of the minimal fibrillar network. The BEMB also collaborated with a Japanese team to study a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval signal from P3H1.This mutation occurs in siblings with non-lethal OI. Our report showed that failure to retain P3H1 in the ER leads to a modest reduction in Pro986 3-hydroxylation but causes overmodification of the collagen helix. This study demonstrated that the KDEl signal is essential for P3H1 function. The BEMB identified two children with a mutation in the 3rd component of the collagen 3-hydroxylation complex, CyPB, which is incoded by PPIB. These siblings have recessive OI of moderate severity with white sclerae but without rhizomelia. They have a homozygous mutation in the start of codon of the peptidly prolyl isomerase gene, which results in a total absence of CyPB protein. Surprisingly, the 3-hydroxylation of collagen Pro986 and the hydroxylation of helical lysine and proline residues were both normal. First of all, this means that two component of the 3-hydroxylation complex, CRTAP and P3H1, can complete collagen modification in the absence of the 3rd component. Second, normal helical modification indicates that the folding rate of the collagen helix is normal. Since CyPB had been previously thought to be the unique collagen cis-trans prolyl isomerase, normal collagen folding in the absence of CyPB means that there must be redundancy for this important function in human cells. Among our LEPRE1-deficient patients, the BEMB identified a common mutant allele, IVS5+1G to T, which occurred in both African-Americans and West Africans. This so-called "West-African allele" has been found only in individuals of African descent. We determined a carrier frequency in Mid Atlantic USA of 1 in 200-300 African-Americans. In a collaboration which Charles Rotimi of NHGRI, we determined contemporary Ghanians and Nigerians had a carrier frequency for this lethal recessive mutations of 1.5%! This high carrier frequency makes the inheritance of severe OI in African distinct from the dominant form prevalent in North America and Europe, where recessive OI occurs in 5-7% of OI cases. The age of the mutation is calculated to be about 600 years old, consistent with a founder mutation that originated in West African and was introduced into North America by the Atlantic slave trade. Our studies have shown that the mutation is not found in a number of countries in Central and West Africa and hence is not pan-African SNP;the reasons for the limitation of this founder mutation to Ghana/Nigeria may reside in the use of languages with common roots spoken in this region. More recently, we have investigated the mechanism of type XI OI, a recessive form of OI caused by absence of the immunophilin FKBP65. A case of moderately severe type XI OI has total absence of FKBP65 protein. Collagen folding is normal in the cells with absence of FKBP65, showing that the chaperone activity of FKBP65 does not play a major role in collagen biochemistry. However, we demonstrated a dramatic decrease in the collagen deposited into matrix in culture despite normal collagen secretion. On mass spectrometry, the collagen telopeptide lysine involved in cross-linking is not hydroxylated in the absence of FKBP65, which would undermine collagen matrix incorporation. Immunofluorescence shows sparse, disorganized collagen fibrils in matrix. We contributed further to understanding of the FKBP10 mechanism and phenotype by identifying a small deletion in FKBP10 as the cause of a congenital contracture syndrome, Kuskokwim syndrome. Although the deletion destabilizes FKBP65, there is 5% residual protein. In Kuskokwim syndrome, hydroxylation of the collagen telopeptide is reduced to 2-10% but is not eliminated. This small amount of FKBP65 appears to be sufficient that affected individuals do not have significant bone disease. Thus, FKBP10 can cause OI alone, OI plus contractures (Bruck syndrome) or contractures along. Finally, we have proposed that pathways common to dominant and recessive OI are likely to provide key insights into disease mechanism. These commonalities include alterations in collagen post-translational modification and folding, abnormalities in both cartilage and bone (osteochondrodystrophy), ER stress, collagen-protein binding, cell-matrix effects, increased bone turnover and hypermineralization of bone tissue. For a gene that causes recessive OI, but is likely to affect osteoblast development more than it affects type I collagen per se, we collaborated with German colleagues on WNT1 mutations causing OI phenotypes ranging from severe bone fragility to early onset osteoporosis. Mutant WNT1 fails to activate the canonical LRP5-mediated b-catenin signaling pathway.

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