Evolutionarily Conserved ELOVL4 Gene Expression in the Vertebrate Retina Pamela S. Lagali,1 Jiafan Liu,2 Rajesh Ambasudhan,2 Laura E. Kakuk,2 Steven L. Bernstein,3 Gail M. Seigel,4 Paul W. Wong,1,5 and Radha Ayyagari2,5 PURPOSE. The gene elongation of very long chain fatty acids-4 plays a critical role in retinal cell biology. (Invest Ophthalmol (ELOVL4) has been shown to underlie phenotypically hetero- Vis Sci. 2003;44:2841–2850) DOI:10.1167/iovs.02-0991 geneous forms of autosomal dominant macular degeneration. In this study, the extent of evolutionary conservation and the targardt macular dystrophies are inherited disorders of the existence and localization of retinal expression of this gene Sretina that are characterized by impaired central vision, was investigated across a wide variety of species. cellular atrophy of retinal and retinal pigment epithelial (RPE) METHODS. Southern blot analysis of genomic DNA and bioinfor- cells initiating in the macular region, and the presence of matic analysis using the human ELOVL4 cDNA and protein yellow fundus flecks surrounding the atrophic area resulting sequences, respectively, were performed to identify species in from the subretinal accumulation of lipofuscin-like material.1–4 which ELOVL4 orthologues and/or homologues are present. Both autosomal recessive and autosomal dominant forms of Retinal RNA and protein extracts derived from different spe- Stargardt’s disease (STGD) have been described.5,6 cies were assessed by Northern hybridization and immunoblot Classical or recessive Stargardt’s disease (STGD1; OMIM techniques to assess evolutionary conservation of gene expres- 248200; http://www.ncbi.nlm.nih.gov/omim, provided by the sion. Immunohistochemical analysis of tissue sections pre- National Institutes of Health, Bethesda, MD) is the most com- pared from various mammalian retinas was performed to de- mon form of hereditary macular dystrophy, occurring at an termine the distribution of ELOVL4 and homologous proteins incidence of approximately 1 in 10,000 live births, and ac- within specific retinal cell layers. counts for roughly 7% of all retinal dystrophies.7 A distinguish- RESULTS. The existence of ELOVL4 sequence orthologues and ing clinical feature of the disease is the dark or silent choroid homologues was confirmed by both Southern blot analysis and observed on fluorescein angiography of the fundus, resulting in silico searches of protein sequence databases. Phylogenetic from the accumulation of lipofuscin. Mutations in the ABCA4 (ABCR) gene on human chromosome 1p32 have been found to analysis places ELOVL4 among a large family of known and 8,9 putative fatty acid elongase proteins. Northern blot analysis underlie STGD1. This gene encodes a retina-specific adeno- revealed the presence of multiple transcripts corresponding to sine triphosphate–binding cassette transport protein found in ELOVL4 homologues expressed in the retina of several differ- photoreceptor cell outer segment disc membranes and func- ent mammalian species. Conserved proteins were also de- tions as an outwardly directed flippase to facilitate the shuttling tected among retinal extracts of different mammals and were of N-retinylidene-phosphatidylethanolamine from the interior to the exterior of the outer segments for subsequent retinoid found to localize predominantly to the photoreceptor cell layer 10,11 within retinal tissue preparations. recycling by RPE cells after phototransduction. ABCR mu- tations have also been implicated in the etiology of a variety of CONCLUSIONS. The ELOVL4 gene is highly conserved throughout other retinal diseases, including retinitis pigmentosa, cone–rod evolution and is expressed in the photoreceptor cells of the dystrophy, and age-related macular degeneration (AMD).9,12,13 retina in a variety of different species, which suggests that it Cases of autosomal dominant Stargardt’s macular dystrophy (adSTGD) are comparatively rare, and a number of genetic loci for diseases with typical adSTGD traits have been mapped, 14,15 1 including STGD3 and adMD (OMIM 600110) on 6q14 and From the Department of Biological Sciences, University of Al- 16 berta, Edmonton, Alberta, Canada; 2Department of Ophthalmology and STGD4 (OMIM 603786) on 4p. Forms of adSTGD are clini- Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, Ann cally quite similar to the phenotype characteristic of recessive Arbor, Michigan; the 3Department of Ophthalmology, University of Stargardt’s disease; however, there are a number of key differ- Maryland, Baltimore, Maryland; and the 4Department of Ophthalmol- entiating features. The most notable differences between ogy, State University of New York at Buffalo, Buffalo, New York. STGD1 and STGD3/adMD-associated phenotypes in particular 5Contributed equally to the work and therefore should be consid- are the later and more variable age of onset and the absence of ered equivalent authors. the dark choroid observed in fluorescein angiograms of pa- Supported by grants from the Foundation Fighting Blindness- tients with dominant STGD.15,17,18 Canada (PWW), the Foundation Fighting Blindness (RA), Research to Prevent Blindness (RA), research Grant EY13198 (RA) and core Grant Using a positional candidate approach, we and others re- EY07003 from the National Institutes of Health, a doctoral research cently identified the gene defect corresponding to the STGD3/ award from the Canadian Institutes of Health Research, and the E. A. adMD locus responsible for the disease phenotype in five 19 Baker Foundation of the Canadian National Institute for the Blind (PSL). related pedigrees throughout North America. The mutation, Submitted for publication September 26, 2002; revised January 10, a 5-bp deletion, lies in the elongation of very long chain fatty 2003; accepted February 5, 2003. acids-4 gene (ELOVL4), a novel member of a family of genes Disclosure: P.S. Lagali, None; J. Liu, None; R. Ambasudhan, that encode enzymatic components of the microsomal fatty None; L.E. Kakuk, None; S.L. Bernstein, None; G.M. Seigel, None; acid elongation system referred to as fatty acid elongases P.W. Wong, None; R. Ayyagari, None (ELOs). The ELOVL4 gene consists of six exons and encodes a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- 2.9-kb full-length mRNA transcript that in turn encodes a 314- amino-acid protein that contains the three typical features that ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 20,21 Corresponding author: Paul W. Wong, Department of Biological define a fatty acid elongase : five putative transmembrane Sciences, G-502 Biological Sciences Center, University of Alberta, Ed- segments, a single HXXHH histidine box redox center motif, monton, Alberta, T6G 2E9, Canada; [email protected]. and a carboxyl-terminal dilysine targeting signal thought to be Investigative Ophthalmology & Visual Science, July 2003, Vol. 44, No. 7 Copyright © Association for Research in Vision and Ophthalmology 2841 Downloaded from iovs.arvojournals.org on 09/28/2021 2842 Lagali et al. IOVS, July 2003, Vol. 44, No. 7 responsible for the retrieval of transmembrane proteins to the through 6. PCR cycling parameters used were as follows: 1 cycle of endoplasmic reticulum (ER) from the Golgi apparatus.22 The denaturation at 94°C for 3 minutes; 30 cycles of denaturation at 94°C 5-bp deletion in the gene identified in dominant STGD/adMD- for 1 minute, primer annealing at 45°C for 1 minute, primer extension affected pedigrees causes a reading frame shift that results in at 72°C for 3 minutes; and 1 cycle of extension at 72°C for 7 minutes. premature termination of the translation product, leading to Probes were purified with a gel extraction kit (QIAquick; Qiagen Inc., substitutions in amino acids 264-271 and the loss of the 51 Mississauga, Ontario, Canada) and radiolabeled with [␣32P]-dCTP (Am- carboxyl-terminal residues in the mutant protein. This muta- ersham Biosciences, Baie d’Urfe´, Quebec, Canada) by random primer tion has subsequently been verified in affected members labeling using a DNA labeling system (Multiprime; Amersham Bio- present in other branches of the same family.23 In addition, a sciences). Hybridization of each probe to the zooblot was performed in novel complex mutation in the ELOVL4 gene has been de- hybridization solution (Hybrisol II; Serologicals Corp., Norcross, GA) at scribed in an unrelated family that manifests diverse macular 42°C for 16 hours using 106 cpm of labeled, purified probes per dystrophy phenotypes.24 milliliter of hybridization solution. The blot was then washed twice for Thus, it is clearly established that the ELOVL4 gene plays a 15 minutes in 2ϫ sodium chloride/tri-sodium citrate buffer (SSC), once critical role in the pathogenesis of macular degeneration. How- for 30 minutes in 2ϫ SSC and 0.1% sodium dodecyl sulfate (SDS), and ever, the precise nature of this role is at present unknown. once for 10 minutes in 0.1% SSC with 0.1% SDS all at 42°C, followed by Clues to the importance of this gene in retinal cell biology have exposure to autoradiograph film (X-Omat; Eastman Kodak, Rochester, also come from the identification of both mouse and yeast NY) at Ϫ70°C for 24 hours. homologues, implying evolutionarily conserved gene expres- sion. This limited assessment of conservation, its implied role RNA Isolation and Northern Blot Analysis in fatty acid metabolism, and its association with retinal degen- Total RNA was isolated from primate ocular tissues and retinal tissue eration suggests that ELOVL4 is an essential retina-expressed derived from various mammalian species using extraction reagent gene. To further characterize this gene in normal retinal biol- (Trizol; Invitrogen), according to the manufacturer’s protocol. Five ogy, we used nucleic acid hybridization and bioinformatic and micrograms of each total RNA sample was electrophoresed on a 1% immunodetection analyses to examine the sequence conserva- agarose-formaldehyde gel and subsequently blotted onto a nylon mem- tion and expression of ELOVL4 gene products across a wide brane (Pall Corp., Mississauga, Ontario, Canada) by capillary transfer.