Genomic organization and mutation analysis of the gene encoding lecithin retinol acyltransferase in human retinal pigment epithelium

PURPOSE. TO determine the structure of the human lecithin retinol acyltrans ferase (LRAT) gene, map its chromosomal localization, and screen for mutati ons in humans with various hereditary retinal degenerations. METHODS. Using DNA probes specific for LRAT, a bacterial artificial chromos ome (BAC) clone containing the LRAT gene was isolated, subcloned into DNA f ragments and relevant subclones characterized by sequencing. Exon-intron ju nctions were determined by comparison with the cDNA sequence previously pub lished. Southern blot analysis was performed on human genomic DNA samples d igested with several restriction enzymes. Fluorescence in situ hybridizatio n (FISH) analysis of normal metaphase chromosomes derived from phytohemaggl utinin (PHA) stimulated peripheral brood lymphocytes and radiation hybrid m apping were used for localization of the LRAT gene. Single-strand conformat ion polymorphism analysis (SSCP) was used to screen for potential mutations in patients with age-related macular degeneration, Leber congenital amauro sis, retinitis pigmentosa, and cone-rod dystrophy. RESULTS. The human LRAT gene is organized into three exons of 219, 541, and 2058 bp and two introns of 103 and 4117 bp. Southern blot analysis of dige sted genomic DNA revealed a single band, suggesting a single copy of the LR AT gene. The human LRAT gene was localized to chromosome 4q31.2, a locus ha ving no previous association with human ep disease. Additionally, the bovin e LRAT homologue sequence was deduced and a general LRAT protein topology i s suggested. No polymorphisms that segregated with retinal disease phenotyp es were identified in 374 unrelated probands. CONCLUSIONS. The organization of the LRAT gene, based on cDNA clones derive d from the retinal pigment epithelium (RPE) has been determined. Its struct ure is less complex than other acyltransferases such as lecithin cholestero l acyltransferase (LCAT) and acyl CoA acyltransferase (ACAT). The absence o f polymorphisms in the probands examined suggests a very low mutation level in the LRAT gene from the diseases analyzed.