Gyrate atrophy of the choroid and retina (GA) is an autosomal recessive chorioretinal degeneration, characterized by night blindness in childhood and progressive loss of peripheral vision leading to blindness in the 4th or 5th decades. Affected patients have marked hyperornithinemia due to deficient activity of ornithine amino-transferase (OAT or OKT). OAT is a major catabolic enzyme for ornithine and requires pyrodoxine (Vitamin B6) as a cofactor. Based on the results of vitamin B6 therapeutic trials and in vitro OAT assays, the majority of GA patients are classified as B6-non-responsive and a few patients as B6-responsive. The recent cloning of rat cDNA complementary to OAT mRNA offers the opportunity to analyze the molecular defects in GA. We propose to use this clone (a generous gift of Dr. Henry C. Pitot) to screen a human liver cDNA library and clone the corresponding human OAT cDNA which will then be used as the hybridization probe to study the genetic defect in GA. We have skin fibroblasts from 6 families with the B6-nonresponsive variant and 1 family with the B6-responsive variant. Genomic DNA will be isolated and analyzed by Southern blot hybridization. Major deletions of the OAT gene could be detected by this technique. Restriction fragment length polymorphism (RFLP) at the OAT gene locus, once established in control individuals, will be examined in GA patients, obligate heterozygotes and other family members to identify any polymorphism unique to the mutant. To analyze the size and the level of mRNA in GA, Northern blot hybridization of cellular mRNA will be done. We should find hybridizable mRNA in some cell lines, especially those with residual OAT activity. S1 nuclease mapping will be used to detect any mRNA splicing defects as a result of small deletion or base substitution of the DNA. Depending on the experimental results, representative GA lines will be selected for cloning cDNA complementary to the mRNA. These clones will be further analyzed by restriction mapping, and sequencing. Human liver OAT will be purified using an affinity chromatography technique we recently developed. Rabbit antiserum to human OAT will be prepared to detect the presence of any immunologically cross-reactive material (CRM) in GA fibroblasts. The proposed experiments are expected to yield important information in understanding the genetic defect and heterogeneity in GA at the molecular level which should have clinical application in the diagnosis of GA, heterozygote identification and family counseling.
