Understanding the molecular basis of human genetic diseases described to date has rapidly advanced in recent years due to the advent of recombinant DNA technology. Traditionally, defective gene products associated with a genetic disorder would be isolated and characterized based on a previous knowledge of the normal gene products. This process, known as """"""""forward genetics,"""""""" has limitations in that the protein associated with the disease must first be identified. This major drawback has recently been overcome with the establishment of """"""""reverse genetics,"""""""" which relies on the use of genetic linkage studies to define the chromosomal localization of the disease followed by cloning of chromosomal structural abnormalities at defined chromosomal regions. Although several diseases, which affect the human dentition, have been clinically defined and their patterns of inheritance determined, no conclusive protein defects have been established for any of these diseases. Defects of the dentin have been classified into two major subgroups: dentin dysplasia and dentinogenesis imperfecta (DGI). Of these two groups, dentinogenesis imperfecta is the most common with an incidence of 1:8000. Dentinogenesis imperfecta is an autosomal dominant disorder which affects both dentitions. Linkage analysis has established this disease to be located on the long arm (q) of human chromosome 4 in the region ql3-q2l. The goal of this proposal is to delineate the primary genetic defects which are responsible for dentinogenesis imperfecta. Our hypothesis is that mutations within a single gene localized within human chromosome 4 q 13-q21, whose gene product, is solely expressed by odontoblast cells during dentinogenesis, is responsible for DGI. We plan to use DNA, isolated from a patient with a hemizygous interstitial deletion of the chromosome 4 ql3-q2l, to construct a regional genomic library using the phenol-enhanced differential reassociation (PERT) technique. This regional chromosome 4 library will be screened for sequences which are expressed during active formation of the dentin extracellular matrix. These sequences will be characterized by their temporal-spatial expression pattcrns using in situ hybridization thus demonstrating their relevance to dentin formation and DGI. The mutations which occur in available kindreds with DGI types II and III will be mapped once the putative 'DGI' gene has be identified. Lastly, the """"""""DGI"""""""" protein will be isolated from dentin matrix and characterized by production of synthetic polypeptide antibodies. These studies will provide a better understanding of genetic diseases associated to mineralizing tissues.
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