Kingsley: You mentioned that achondrogenesis 1B is the clinical name for a condition that results from homozygosity for null mutations in the sulfate transport. What does that look like? Is there any cartilage formed?

D. Cohn: These mice are extremely small, and they die because their chest doesn't grow properly. The cartilage looks very similar to that in diastrophic dysplasia: histologically we see rings of collapsed collagen around the chondrocytes. The growth plates are extremely disorganized, there are zones of degenerating cartilage, and the chondrocytes have very little ability to align at the growth plate. Having said that, if you measure the level of sulfation of aggrecan, for example, in tissue, there is still a level of sulfate on aggrecan that remains. So there are other ways — perhaps only salvage pathways — of getting sulfate in. Transport of sulfated amino acids may be up-regulated, or there could be secondary accessory pathways for the transport of sulfate into those cells. We don't know the precise level of the defect in post-translational sulfation that produces achondrogenesis 1B: is it that the level of sulfation of the extracellular matrix molecules is 5% of normal or 10% of normal? It is not known what level of decreased sulfation results in each of these phenotypes.

Russell: With regard to the COMP story, Dick Heinegard makes a big point about the distribution through cartilage being non-homogeneous. I was wondering whether in the syndromes you were describing anyone has looked at that.

D. Cohn: His data suggest that there is a higher concentration of COMP in the immediate pericellular region around the chondrocyte, and that there is then a diffuse distribution of COMP throughout the rest of the matrix. We have repeated some of these experiments with lower concentrations of antibody, and as we reduce the concentration of antibody we get a much more homogeneous picture. I wonder to what extent there really is more COMP immediately adjacent to the chondrocytes. In pseudoachondroplasia chondrocytes, whether there is a clearing of COMP around the chondrocyte is not known. We haven't had much cartilage from patients with which to test that idea. But we are doing direct experiments looking at which specific amino acid residues of COMP might be involved in binding chondrocytes.

Bard: One of your electron micrographs caught my eye. It was in COMP. It showed type II collagen fibrils showing normal 640 A periodicity. Is this correct?

D. Cohn: It seems that type II collagen is able to assemble in a proper way, but the fibril diameter is just a little bit bigger.

Bard: Normally it's very thin.

D. Cohn: Yes, so the abnormality is in the lateral assembly of the type II collagen.

Bard: Do you have any idea why this was happening?

D. Cohn: Not really. This is speculation, but perhaps it is because of a disrupted interaction with type IX collagen.

Chen: I'm struck by the concentration of the mutations in COMP within the calmodulin domains. It is known that the extracellular growth factor (EGF) domain also binds calcium, and thus may be important for the function of COMP. Why are there no mutations identified in EGF domains?

D. Cohn: It surprised us as well. We certainly expect that there are phenotypes that will result from mutations in the EGF domain ofCOMP. We have looked at a number of other spondyloepiphyseal dysplasias and spondyloepimetaphyseal dysplasias to try to identify them. As yet, no mutations in that domain have been identified in any phenotype. Having said that, another way you can look for them is to ask whether in osteochondrodysplasias in which there are inclusions in the RER, you can identify any others in which the inclusions stain with COMP antibody. We have now looked at a couple of dozen different phenotypes with inclusions in the rough endoplasmic reticulum and we don't see any others that stain with antibodies to COMP. Perhaps the best way to address that problem is to construct a transgenic mouse with a mutation in that domain to explore the possibility that a skeletal phenotype would result. This may then bring into play the ability to detect things that we wouldn't detect by just clinically ascertaining patients. This would include everything from lethal phenotypes to those that are really very mild.

"Newman: Some of these chondrocytes making the aberrant proteins look pretty sick. Can you attribute some ofthe symptomology to altered chondrocyte function apart from matrix occlusion? In other words, perhaps it is not so much that the matrix is compromised as that the growth of the chondrocytes is.

D. Cohn: One of the questions would be, is the accumulation of so much of this abnormal protein within the rough endoplasmic reticulum affecting cellular metabolism in some more general way? A number of people have looked to see whether there is increased apoptosis in chondrocytes from these patients. There are some limited data on pseudoachondroplasia, which suggests that there may be, but in general that's not what we see. The distinct clinical outcomes of mutations in each of the different proteins, despite the fact that they all accumulate a tremendous amount of abnormal protein within the rough endoplasmic reticulum, I think argues a little bit against that.

"Hall: Presumably, you're also getting disrupted feedback from the extracellular matrix to the cells. Thus you are not maintaining differentiation in the normal way.

D. Cohn: That makes a lot of sense and is likely to be true, although there are very few data which say that it is. Now knowing the underlying biology of some of these conditions, we have the ability to look at this, with our eyes more open.

Novartis 232: The Molecular Basis of Skeletogenesis. Copyright © 2001 John Wiley & Sons Ltd Print ISBN 0-471-49433-X elSBN 0-470-84665-8

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