Genetic Linkage Analysis by Mitotic Recombination

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DESCRIPTION (provided by applicant): Great advancements in developing therapeutics and cures for inherited disorders will result from identification of the gene mutations that cause these disorders. For rare genetic disorders, family pedigrees are scarce and genetic linkage and positional cloning is difficult, inefficient and/or impossible. Fibrodysplasia ossificans progressiva (FOP), the most disabling form of heterotopic ossification known to mankind, is a very rare autosomal dominant genetic disorder with low reproductive fitness and its inheritance from parent to child in families is rarely seen. FOP is characterized by skeletal malformations of the great toes and by progressive induction of bone formation at ectopic sites. BMP4 mRNA and protein are over-expressed in lymphocytes and lesional cells from patients who have FOP and BMP4 over-expression serves as a reliable molecular marker for the condition. The BMP4 gene is not mutated in FOP, and the BMP4 locus has been excluded from linkage to the condition. Although FOP has been linked to chromosome locus 4q27-31 by genetic linkage analysis in four small families, the reliability of these data in predicting the genetic locus and the identification of the mutated gene within the linked locus is uncertain. The paucity of multi -generational families also renders doubt about the fidelity of the localization, as genetic heterogeneity may exist. Our FOP research group coordinates an integrated global network of physicians who are responsible for the care of FOP patients worldwide. Even with this resource, it is unlikely that additional multigenerational families will be identified for use in further linkage analyses (meiotic recombination). We therefore have devised a novel and innovative alternate approach using mitotic recombination in somatic cells. By enhancing the production of mitotic ally-generated recombinant progeny cells, this method will create a large cellular pseudo-family from a single BMP4 over-expressing "parent" FOP cell line. Somatic cell recombination will create a loss of the mutant chromosomal locus in a subset of the cellular "progeny", which will be identified by the loss of BMP4 over-expression (the marker for the FOP phenotype). The chromosomal site of recombination will be identified by the loss of heterozygosity (LOH) of chromosomal microsatellite markers within these "progeny" cells, and will facilitate a highly focused and targeted positional cloning approach to L identify the mutated gene locus in FOP patients.