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. A severe recessive form of OI was first postulated in 1979. More recently, investigators have noted that some patients with clinical OI do not have defects detected in the type I collagen genes during sequencing. These patients without mutations in collagen can be divided into those who have abnormal collagen biochemistry and those with normal electrophoretic migration of the collagen chains. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interacted with type I collagen. Seven 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 has generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in the components of a complex in the endoplasmic reticulum that modifies collagen cause recessive OI. In the expanded nosology for OI, defects in CRTAP and LEPRE1 are designated as types VII (OMIM #610682) and VIII (OMIM #610915) OI, respectively. Among our LEPRE1-deficient patients, we identified a common mutant allele, IVS5+1G to T, which occurred in both African-Americans and West African families. This so-called """"""""West-African allele"""""""" accounts for a third of the known LEPRE1 mutations, and has been found only in individuals of African descent. To our surprise, contemporary West Africans have a carrier frequency for this lethal recessive mutation of 1.5%! 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. Both groups of children have severe/lethal OI with white sclerae, normal or small head circumference, rhizomelia, metacarpal shortening 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 by prolyl 4-hydroxylase and lysly hydroxylase. This overmodification of the helix was unexpected and 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 protectively in the complex. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry compared to types VII and VIII. We have generated a CyPB KO mouse to explore these distinctions further. Knock-out mice are small, with reduced bone density and strength, but increased brittleness. Only 1-2% 3-hydroxyltion is detected in KO cells, showing the importance of CyPB to complex function. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports collagen lysyl hydroxylase (LH1) activity and its absence allows site-specific alterations in helical lysine hydroxylation, in particular significant reduction of hydroxylation of crosslinking residue K87. The decreased crosslink ratio alters fibril structure and reduces bone strength. The effects of CyPB on collagen glycosylation crosslinking and fibrillogenesis are novel findings. Recessive mutations in FKBP10, which encodes FKBP65, cause type XI OI. Mutations in this gene also cause Bruck Syndrome, which is OI plus congenital contractures. These mutations are allelic, since siblings with the same mutation my have OI or Bruck Syndrome. Thus, contractures are shown to be a variable manifestation of FKBP10 mutations. We also identified an FKBP10 mutation in Kuskokwim syndrome (KS), a recessive congenital contracture disorder found among Yupil Eskinos in Alaska. The causative mutation is an in-frame deletion which removes the highly conserved p.Tyr293 residue in FKBP65s third PPIase domain. This mutation destabilizes the protein but leaves residual 5%. FKBP65 supports LH2 function, so it absence substantially decreases hydroxylation of the telopeptide lysine important for collagen crosslinking. Thus FKBP65 mutations affects collagen indirectly through loss of LH2 function. Most recently, we have delineated a muttion in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. We identified a patient with severe OI whose fibroblasts and osteoblasts secreted minimal amounts of PEDF and whose bone histology was typical of type VI OI, but whose serum PEDF was in the normal range. Whole exome sequencing revealed a de novo mutation in IFITM5 in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL. Both IFITM5 transcripts and BRTIL protein levels were normal in proband cells. However, SERPINF1 expression was minimal. Expression of type I collagen was similarly decreased in proband osteoblasts, and the pattern of osteoblast markers was consistent with a primary PEDF defect. Since this mutation in IFITM5 was causing bone-specific type VI OI, we compared these osteoblasts to osteoblasts with the type V OI-causing IFITM5 mutation at the 5:-end of the gene. In these cells we demonstrated increased SERPINF1 expression and PEDF secretion during osteoblast differentiation, connecting the two OI-causing genes in an important pathway under delineation.
