BONE AND EXTRACELLULAR MATRIX BRANCH
This past year, the Bone and Extracellular Matrix Branch (BEMB) made a series of discoveries that have shifted the paradigm for collagen-related bone disorders. The group identified the genes, which have been postulated to exist since 1979, that cause recessive osteogenesis imperfecta (OI). Recessive OI is a relatively rare form of OI that constitutes 5 to 7 percent of OI cases but has a phenotype at the most severe end of the clinical spectrum, overlapping types II (lethal) and III (progressive deforming) OI. The BEMB investigators took a candidate gene approach to the question of recessive OI, postulating that the subset of children with OI who did not have a collagen mutation (the cause of dominant OI, which is the prevalent form) but who did have abnormal collagen biochemistry would have a defect in a molecule that interacted with collagen. The BEMB researchers demonstrated that all cases with collagen overmodification but with no collagen mutation were deficient in either of two proteins that are part of a complex responsible for the 3-hydroxylation of alpha1(I) Pro986, CRTAP (Cartilage Associated Protein), or P3H1 (prolyl 3-hydroxylase 1). They showed that all cases have null mutations in the respective genes, with a consequent reduction or absence of mRNA by nonsense-mediated decay, absence of the corresponding protein in cell lysates, and the absence of hydroxylation of Pro986 on patient collagen. The secondary effect of the absence of the components of the endoplasmic reticulum (ER) hydroxylation complex included delayed secretion of collagen from cells but an increase in the total amount of secreted collagen, abnormal fibril structure with irregular circumference. Interestingly, absence of complex components results in excess modification of the collagen helix, suggesting a delay in the folding of the helix.
The group generated the Brtl mouse, a non-lethal knockin model for OI, and, in recent investigations, demonstrated selective secretion of mutant collagen by Brtl cells. The cells selectively retain collagens with one mutant chain as compared to collagens with two or no mutant chains. Engorgement of the ER suggests that such retention may stress the Brtl cells. ER stress may also play a role in the phenotypic variability of Brtl mice, which have two distinct phenotypes. Pups with the lethal phenotype evidence elevated Gadd153, which promotes cellular apoptosis. Conversely, pups with a moderate skeletal phenotype evidence elevated levels of alpha B-crystallin, which is protective against apoptosis.
The BEMB continues to lead the Consortium for Mutations in Osteogenesis Imperfecta, which comprises eight international laboratories. Genotype-phenotype analyses have identified different patterns for the alpha1 and alpha2 chains and have provided testable hypotheses. Two regions in the carboxyl half of the a1(I) chain, which contain exclusively lethal mutations, align with the Major Ligand-Binding Regions (MLBR) II and III. The Consortium database supports the Regional Model previously proposed by the BEMB for lethal mutations in the a2(I) chain, in which eight regularly spaced clusters of lethal mutations coincide with the binding sites of matrix proteoglycans to the collagen fibril.
The BEMB’s clinical investigations continue to play an important role in the treatment of OI. Previous controlled trials with bisphosphonate pamidronate have distinguished the beneficial and detrimental aspects of such compounds in Brtl mice and in children with OI. In children with types III and IV OI, treated patients experienced a significant increase in vertebral BMD z-scores and increased mid-vertebral height and vertebral area. However, the increases in BMD tapered off after one to two years of treatment. Furthermore, treated patients did not experience positive functional effects in ambulation level, lower-extremity strength, or amelioration of pain. The changes previously reported in these parameters in uncontrolled trials appear to have been placebo effects. The BEBM is currently engaged in a dose comparison trial, testing the hypothesis that a reduced cumulative dose of bisphosphonate can result in the same improvements as the higher dose but with decreased detrimental effects.
