Osteopetrosis is an inherited disease characterized by the absence of bone resorption, and is ascribed to defects in differentiation or function of osteoclasts which derived from hematopoietic lineage. Study of the molecular mechanism underlying osteopetrosis will most likely provide detailed knowledge on the control of osteoclast differentiation and signal transduction. The alternative approach to determine the molecular mechanisms implicated in osteoclast differentiation relies on genetic studies of osteopetrosis and consists in defining the gene(s) involved in this disease. We have taken advantage of the mouse, the primary experimental system for genetic analysis of mammalian cell differentiation and diseases, to study the molecular basis of this genetic defect. The murine grey-lethal (gl) mutant displays a defect in osteoclast differentiation and develops a severe osteopetrosis which closely resembles the human infantile disease, and which can be rescued by bone marrow transplantation, as in human.
The major objectives of our research project are the characterization of the gene responsible for the gl mutation and the definition of the role of gl in osteoclast differentiation. To this end, positional cloning is the only and most powerful approach to define the gene responsible for this defect. The generation of a contig covering the chromosome 10 region which includes the gl locus will allow the isolation of this osteopetrotic gene.
Three concurrent and complementing approaches will be undertaken.
1. Fine localization of the gl geneA backcross panel between the mouse gl (Mus domesticus) and Mus spretus is presently produced.
Presently, 460 animals have been analyzed. Each DNA will define the genetic origin for B-raf, c-fyn and c-ros genes which mapped on chromosome 10 and in the vicinity of the gl locus. Moreover, the same DNAs have been analyzed using specific microsatellite oligonucleotides (Mit45, Mit55, 108, 215) localized in the same chromosomal region, which are good markers because they are polymorphic between the mouse strains domesticus and spretus. By this approach, we have localized the grey-lethal gene in a 2,3 ± 1,3 centimorgans genetic interval. A second backcross panel using Mus m. molossinus is presently analyzed.
During this analysis, we have detected two polymorphisms specific for the gl chromosome 10. Using oligonucleotides specific for the microsatellites D10 Mit55 and D10 Mit108, we generated two different PCR products with gl and wild-type genomic DNAs. This result strongly suggested that we are in the close vicinity of the gl locus and will facilitate the cloning of the gene.
2. Generation of a chromosome 10 specific contigTwo mouse YAC (yeast artificial chromosome) libraries are screened by polymerase chain reaction. Using specific oligonucleotides for the c-fyn gene a 160kb clone has been isolated and used as an anchor point for our chromosomal 10 walk toward the gl locus. After isolation and sequencing of the two ends of this YAC, new oligonucleotides have been defined and used for the next walk. Hence each step defined specific chromosome 10 probes which will be used in the perspective of the mouse genome project. We have constructed a contig covering both sides from our anchor locus, c-fyn. We are presently pursuing our chromosome walk and have isolated several clones including the polymorphisms described previously.3. Characterization of the gl cellular defectSince the osteoclasts derive from the monocyte-macrophage hematopoietic lineage, we are characterizing these precursors in the gl mutant. Preliminary in vitro cell cultures demonstrated an increase of these populations in the gl mutant in comparison with wild-type littermates. In addition, a decrease of mature B cells in the mutant gl spleen has been observed. Long term repopulation experiments are presently initiated.