DNA LINKAGE STUDIES OF DEGENERATIVE RETINAL DISEASES

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This proposal is for funds to continue our DNA linkage studies of families with degenerative retinal diseases such as autosomal dominant retinitis pigmentosa (ADRP) and Usher's syndrome. The objectives are to find tightly linked DNA markers for these diseases, to map the disease loci to specific chromosomal regions and to use these linked DNA markers in characterizing the disease loci. In 1986 the National Retinitis Pigmentosa Foundation established a collaborative program to facilitate DNA linkage studies of retinitis pigmentosa (RP). Dr. Daiger is Program Coordinator for this program. The aims of the program are to identify and characterize appropriate RP families, to send family blood samples to the Human Genetic Mutant Cell Repository for preparation and storage of transformed cell lines, and to provide these cell lines to Dr. Daiger's laboratory for preparation, testing and handling of DNAs. At present the RP collection contains 91 cell lines from one large ADRP family and 77 lines from an extended Usher's syndrome family. The collection should increase by 50 to 100 lines in 1988. We have prepared and tested DNAs from most of these samples and have acquired 91 additional DNAs from other RP families. This proposal is for the research component of the program, that is, DNA linkage testing and data analysis. In addition to the classical genetic markers we and others have tested in these families, we already have tested over 22 DNA probes in selected families. We propose to test 150 to 200 individuals with 20 new probes per year using conventional Southern gel analysis. We will also develop and apply a novel, high efficiency procedure for detecting "mini-VNTR" polymorphisms in human DNA, based on DNA amplification using the polymerase chain reaction (pcr) method. There are hundreds of polymorphic DNA probes currently available from the scientific community and we hypothesize that like numbers of mini VNTRs will be found. Methods are tissue culture, extraction of genomic DNAs, preparation of probe DNAs Southern gel analysis, detection of DNA polymorphisms by pcr amplification, and two point and multipoint linkage analysis. When linkage is established we will test additional families and will use molecular methods to improve the information content of the marker, develop closer markers and establish flanking markers. Linked markers for ADRP and Usher's syndrome will be of value in early diagnosis, in detection of genetic heterogeneity and eventually, in isolation and characterization of the mutant genes.