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 acyltransferase (LRAT) gene, map its chromosomal localization, and screen for mutations in humans with various hereditary retinal degenerations. METHODS. Using DNA probes specific for LRAT, a bacterial artificial chromosome (BAC) clone containing the LRAT gene was isolated, subcloned into DNA fragments and relevant subclones characterized by sequencing. Exon‐intron junctions were determined by comparison with the cDNA sequence previously published. Southern blot analysis was performed on human genomic DNA samples digested with several restriction enzymes. Fluorescence in situ hybridization (FISH) analysis of normal metaphase chromosomes derived from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes and radiation hybrid mapping were used for localization of the LRAT gene. Single-strand conformation polymorphism analysis (SSCP) was used to screen for potential mutations in patients with age-related macular degeneration, Leber congenital amaurosis, 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 digested genomic DNA revealed a single band, suggesting a single copy of the LRAT gene. The human LRAT gene was localized to chromosome 4q31.2, a locus having no previous association with human eye disease. Additionally, the bovine LRAT homologue sequence was deduced and a general LRAT protein topology is suggested. No polymorphisms that segregated with retinal disease phenotypes were identified in 374 unrelated probands. CONCLUSIONS. The organization of the LRAT gene, based on cDNA clones derived from the retinal pigment epithelium (RPE) has been determined. Its structure is less complex than other acyltransferases such as lecithin cholesterol acyltransferase (LCAT) and acyl CoA acyltransferase (ACAT). The absence of polymorphisms in the probands examined suggests a very low mutation level in the LRAT gene from the diseases analyzed. (Invest Ophthalmol Vis Sci. 2001;42:31‐37) R ecently, we reported the characterization of cDNA clones encoding for lecithin retinol acyltransferase (LRAT), the enzyme responsible for the transformation of all-trans-retinol into all-trans-retinyl esters, in the retinal pigment epithelium (RPE) of the eye. 1 In the present study, we have isolated and characterized genomic DNA clones that contain the LRAT gene and by virtue of these genomic clones, have also determined its chromosomal locus. LRAT activity is fundamental in the continuation of the visual cycle. This important process takes place in the photoreceptor cells, and the RPE and is responsible for the generation of 11-cis-retinaldehyde, which is the chromophore for rhodopsin and the cone photopigments. 2,3 The mechanism of action of LRAT suggests that an acyl group, specifically the acyl group at position sn-1 of a membrane phosphotidylcholine, is removed and transferred to all-trans retinol during the formation of all-trans retinyl esters. 4 These retinyl esters are not only the storage form of all-trans-retinol (Vitamin A) but are also the substrate for the isomerohydrolase that generates 11-cis-retinol, another important intermediate in the visual cycle. 5,6 In addition to RPE, LRAT is expressed in other tissues involved in processing and transport of retinol such as intestine, testis, and liver. 7,8,9 Because of the relative instability of LRAT in the detergent solubilized form, its purification has represented a challenge. Nonetheless, by affinity labeling studies using LRAT specific inhibitors, its kinetic properties and substrate specific