Choroideremia is an X-linked retinal dystrophy that causes progressive visual loss and blindness in affected males. The biochemical and molecular bases for this disorder are unknown. Choroideremia was localized to the region Xq13-21 by tight linkage to five restriction fragment length polymorphisms. Two unusual families in which the retinal disease is being inherited concordantly with deafness, mental retardation and obesity in an X-linked manner have also been studied. Probands in one (M) family have an Xq21.3 interstitial deletion and are missing two tightly linked RFLP loci; probands in the other (B) family appear to have a much smaller deletion that is more difficult to document by cytogenetic or Southern analysis. We propose to isolate the gene for choroideremia to study its etiology and pathogenesis and improve genetic management. PLAN: A library enriched for B deletion DNA was made by reannealling single-stranded normal DNA with EcoR1 """"""""sticky"""""""" ends in the presence of excess blunt end denatured DNA from a B family proband using the phenol enhanced reassociation technique (PERT). 1) Probe normal, M and B patients' DNA with PERT library sequences to identify sequences deleted in the M or B probands. 2) Test DNA from within the B deletion by screening patients with uncomplicated choroideremia for Southern blot alterations. Find more B deletion sequences in a cosmid library made from 48XXXX DNA. 3) Use genomic sequences in the B deletion as starting points for isolating more overlapping DNA spanning the deletion. Test this DNA by probing the panel of choroideremia patients and examining mRNA from retina and retinal pigmented epithelium for expressed genes. 4) If 1) is unsuccessful, assess the distance between the junction point of the B deletion and the markers missing in the M probands by Southern blot analysis of 100-1000 kb restriction fragments resolved by pulsed field gel electrophoresis. If the junction point can be defined at a distance, begin at the markers to isolate overlapping cosmid sequences, move into the B deletion and proceed as in step 3) above.
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