A Novel Gene for Autosomal Dominant Stargardt-like Macular Dystrophy with Homology to the SUR4 Protein Family Albert O. Edwards,1 Larry A. Donoso,2 and Robert Ritter, III1 PURPOSE. To describe a novel gene causing a Stargardt-like fatty acid biosynthesis in the pathogenesis of macular degen- phenotype in a family with dominant macular dystrophy and eration. The PCR-based assay for the 5-bp deletion will facilitate the exclusion of all known genes within the disease locus. more accurate genetic counseling and identification of other METHODS. Meiotic breakpoint mapping in a family of 2314 branches of the family. (Invest Ophthalmol Vis Sci. 2001;42: individuals enabled refinement of the location of the disease 2652–2663) gene. The genomic organization and expression profile of known and putative genes within the critical region were determined using bioinformatics, cDNA cloning, and RT-PCR. acular dystrophies with subretinal flecks may arise from The coding sequence of genes expressed within the retina was Mmutations at multiple disease loci,1,2 the most common scanned for mutations, by using DNA sequencing. of which is the ABCA4 gene on chromosome 1 that gives rise 3,4 RESULTS. The disease-causing gene (STGD3) was further local- to Stargardt disease (STGD1) or fundus flavimaculatus. We 2,5 ized to 562 kb on chromosome 6 between D6S460 and a new previously reported a founder effect for a dominant condi- polymorphic marker centromeric to D6S1707. Of the four tion (STGD3) phenotypically similar to Stargardt disease local- genes identified within this region, all were expressed in the ized to chromosome 6q.2,6 A genealogical and molecular inves- retina or retinal pigment epithelium. The only coding DNA tigation of several families with autosomal dominant Stargardt- sequence variant identified in these four genes was a 5-bp like macular dystrophy led to the recognition that all studied deletion in exon 6 of ELOVL4. The deletion is predicted to lead families were related through a common founder that immi- to a truncated protein with a net loss of 44 amino acids, grated to North America in 1730.2,5–8 We also showed that the including a dilysine endoplasmic reticulum retention motif. Stargardt-like disease (STGD2), previously reported to be The ELOVL4 gene is the fourth known example of a predicted linked to chromosome 13,9 was actually a branch of the family human protein with homology to mammalian and yeast en- we had been studying on chromosome 6.5 zymes involved in the membrane-bound fatty acid chain elon- Disease-causing mutations segregating within single families gation system. The genomic organization of ELOVL4 and can be difficult to identify with certainty, because all affected primer sets for exon amplification are presented. patients share the identical genomic DNA segment on which CONCLUSIONS. ELOVL4 causes macular dystrophy in this large the founding mutation arose. Thus, all DNA sequence variation family distributed throughout North America and implicates within the disease locus or critical region is shared by all affected patients and segregates with the trait. In addition to the customary criteria for distinguishing between polymor- From the 1Department of Ophthalmology, University of Texas phisms and mutations, exclusion of other potential disease- Southwestern Medical Center, Dallas; and the 2Henry and Corinne causing sequence variation within the disease locus can con- 10 Bower Laboratory, Wills Eye Hospital, Philadelphia, Pennsylvania. firm the mutation. Therefore, it is critical to exclude Supported in part by Grant EY12699 from the National Institutes sequence variation in all genes within the disease locus. of Health (AOE, LAD), Career Development Awards from Research to The disease locus on chromosome 6q14 for this autosomal Prevent Blindness and the Foundation Fighting Blindness (AOE), and dominant Stargardt-like macular dystrophy family has been unrestricted departmental funds from Research to Prevent Blindness (University of Texas Southwestern and Wills Eye Hospital). The Scholl- progressively refined to between novel markers within the maier Foundation, Fort Worth, Texas; the Anne Marie and Thomas B. region defined by D6S1625 and D6S1707 by us and between 11,12 Walker, Jr. Fund for Age-Related Macular Degeneration, Dallas, Texas; D6S460 and D6S391 by other groups. While this manu- and the Walter Center for Macular Degeneration, Dallas, Texas pro- script was in preparation, a 5-bp deletion in the ELOVL4 gene vided additional support at University of Texas Southwestern. The in three branches of this family was reported by Zhang et al.11 Henry and Corinne Bower Laboratory for Macular Degeneration; the In addition to ELOVL4, they reported exclusion of two other Elizabeth C. King Trust; the estates of Margaret Mercer, Harry B. genes within their 3100-kb critical region between D6S460 and Wright, and Martha W. S. Rogers; the Association for Macular Diseases; 11 and Macular Degeneration International, all of Philadelphia, Pennsyl- D6S391. We are aware of eight additional genes within their vania, provided additional support at Wills Eye Hospital. LAD is the reported critical region that were not excluded and thus could Thomas D. Duane Professor of Ophthalmology, Wills Eye Hospital and harbor a disease-causing sequence variation. Herein, we report Jefferson Medical College, Thomas Jefferson University, Philadelphia, our independent identification and characterization of the Pennsylvania. gene for autosomal dominant Stargardt-like dominant macular Submitted for publication March 26, 2001; revised May 25, 2001; accepted June 5, 2001. dystrophy and the exclusion by DNA sequencing of the com- Commercial relationships policy: N. plete coding region of all candidate genes within our 562-kb The publication costs of this article were defrayed in part by page critical region, defined by D6S460 and a novel marker charge payment. This article must therefore be marked “advertise- (260P22.A) centromeric to D6S1707. The expression profile in ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. human retina, genomic organization, and known functional Corresponding author: Albert O. Edwards, Department of Oph- information are presented for 15 genes in or near the critical thalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9057. region for STGD3 that are candidates for other chromosome 6q [email protected] retinopathies. Investigative Ophthalmology & Visual Science, October 2001, Vol. 42, No. 11 2652 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/24/2021 IOVS, October 2001, Vol. 42, No. 11 Gene for Dominant Stargardt-like Dystrophy 2653 FIGURE 1. Color photograph of the retina of a 52-year-old family member with autosomal dominant Stargardt-like macular dystrophy, showing RPE atrophy, subretinal flecks, and temporal pallor of the optic nerve. MATERIALS AND METHODS 74°C for 5 minutes. Amplified products were denatured for 4 minutes at 94°C in the presence of formamide stop dye (6.5 ␮l), snap cooled on DNA Extraction ice, resolved at room temperature on 8% denaturing polyacrylamide White blood cells were isolated by centrifugation of whole blood at gels at 80 W, and transferred to nylon membranes (Roche Molecular 1000g and DNA purified with a kit (Masterpure Genomic DNA; Epi- Biochemicals, Indianapolis, IN) overnight. DNA was cross-linked to the centre Technologies, Madison, WI), according to the manufacturer’s membranes for 5 minutes (Photoprep I; Photodyne, Newberlin, WI). instructions. This research was approved by the institutional review The immobilized products were denatured for 10 minutes in 0.4 M boards and followed the tenets of the Declaration of Helsinki. NaOH and neutralized in 2ϫ SSC. An oligonucleotide containing an AC15 repetitive sequence was Polymorphic Marker Identification, 3Ј-tailed with digoxigenin (DIG)-11-dUTP and terminal transferase ac- Amplification, and Analysis cording to the manufacturer’s instructions (Roche Molecular Biochemi- cals) and hybridized to the resolved PCR products at 42°C in 5ϫ SSC, Previously reported primers for short tandem repeat (STR) amplifica- 1% blocking reagent, 0.1% N-laurosarkosine, 0.02% SDS. Hybridized tion were purchased from Research Genetics (Huntsville, AL). Novel membranes were washed in 6ϫ SSC and 0.1% SDS three times for 5 dinucleotide (AC)n repeat motifs were identified using a BLAST 2 Sequences search (available publicly from the National Center for minutes, followed by a single wash in 0.1 M maleic acid, 0.15 NaCl, and Biotechnology Information [NCBI] at http://www.ncbi.nlm.nih.gov/ 0.3% Tween-20 at pH 7.5 for 1 minute. The products were visualized BLAST/) of finished and unfinished genomic sequence within the by incubating membranes with alkaline phosphatase-conjugated anti- STDG3 critical region produced by the Human Chromosome 6 Se- DIG antibody for 60 minutes, washing twice in 0.1 M maleic acid and quencing Group at the Sanger Centre (Cambridgeshire, UK) with an 0.15 M NaCl at pH 7.5 for 15 minutes, followed by a single equilibra- tion in 100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCL at pH 9.5 (AC)60 sequence motif. Primers were designed in flanking sequence for 2 amplification. Polymerase chain reactions (PCRs) were performed on for 2 minutes. The membranes were incubated with the chromogenic genomic DNA (12.5 ng) in the presence of 1.5 mM MgCl, 1.25 nM each substrate (nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indoyl dNTP, 2.5 pM each primer, and 0.11 U Taq polymerase in a final phosphate 4-toludine sale [NBT/BCIP]) until the desired band intensity volume of 6.5 ␮l under the following conditions: initial denaturation at was achieved. All results were analyzed by at least two independent 95°C for 4 minutes, followed by 30 cycles at 94°C for 10 seconds, 55°C investigators and compared with a control sample (CEPH 1331-01) for for 10 seconds, and 74°C for 10 seconds, and then a final extension at registering genotypes between assays.