Genetics and molecular pathology of Stargardt-like macular degeneration

Abstract

Stargardt-like macular degeneration (STGD3) is an early onset, autosomal dominant macular degeneration. STGD3 is characterized by a progressive pathology, the loss of central vision, atrophy of the retinal pigment epithelium, and accumulation of lipofuscin, clinical features that are also characteristic of age-related macular degeneration. The onset of clinical symptoms in STGD3, however, is typically observed within the second or third decade of life (i.e., starting in the teenage years). The clinical profile at any given age among STGD3 patients can be variable suggesting that, although STGD3 is a single gene defect, other genetic or environmental factors may play a role in moderating the final disease phenotype. Genetic studies localized the STGD3 disease locus to a small region on the short arm of human chromosome 6, and application of a positional candidate gene approach identified protein truncating mutations in the elongation of very long chain fatty acids-4 gene (ELOVL4) in patients with this disease. The ELOVL4 gene encodes a protein homologous to the ELO group of proteins that participate in fatty acid elongation in yeast. Pathogenic mutations found in the ELOVL4 gene result in altered trafficking of the protein and behave with a dominant negative effect. Mice carrying an Elovl4 mutation developed photoreceptor degeneration and depletion of very long chain fatty acids (VLCFA). ELOVL4 protein participates in the synthesis of fatty acids with chain length longer than 26 carbons. Studies on ELOVL4 indicate that VLCFA may be necessary for normal function of the retina, and the defective protein trafficking and/or altered VLCFA elongation underlies the pathology associated with STGD3. Determining the role of VLCFA in the retina and discerning the implications of abnormal trafficking of mutant ELOVL4 and depleted VLCFA content in the pathology of STGD3 will provide valuable insight in understanding the retinal structure, function, and pathology underlying STGD3 and may lead to a better understanding of the process of macular disease in general.

Figure 1. Summary of known MD and AMD loci

A) A table summarizing known MD and AMD loci generated from selective data mining of the Retnet database for the words macula and fundus (http://www.sph.uth.tmc.edu/retnet/). In some cases different mutations in the same gene define distinct disorders. MOI, mode of inheritance; AD, autosomal dominant; AR, autosomal recessive; XL, X-linked; MF, multifactorial. B) Profile of macular disease genes or related genes in the human genome. Loci summarized in A are distributed throughout the genome on 18 human chromosomes. There are apparent hot spot of MD and AMD loci on chromosomes 1 and 6. C) Distribution of MD and AMD loci according to mode of inheritance. Approximately 2/3 of the macular disease loci ascertained correspond to disorders inherited as a single gene defect by means of a standard Mendelian mode of inheritance (AD, AR, XL). One third of the loci correspond to AMD-related genes governed by a more complex mode of inheritance (MF).

Figure 2. MD and AMD interactome

Gene loci underlying MD (Gray) and/or AMD (Green) conditions were submitted to Ingenuity (http://www.ingenuity.com/) and String databases (http://string.embl.de/) to define potential functional interacting pathways. The generation of a single interactome suggests that the MD/AMD gene loci/gene products considered are functionally related. Ingenuity incorporated additional gene loci/proteins (yellow) as part of the pathway stemming from the initial input of the disease genes. Direct interactions are shown in solid lines (for example, protein to protein binding, phosphorylation, etc.); indirect interactions are shown in dashed lines (for example, effects through signaling pathways, expression); and undefined interactions are shown as red lines. Proteins are also functional coded:

Cytokine/growth factor		Ion channel
Chemical/toxicant		Peptidase
Enzyme		Transcription regulator
G-protein coupled receptor		Transmembrane receptor
Group/complex/		other transporter
Growth factor		Undefined

Figure 3. Fundus photographs of two individuals from a large Canadian STGD3 pedigree, an affected STGD3 male member and his affected nephew. (Photographs taken from Lagali et al., 2000 and used with permission from the Canadian Journal of Ophthalmology)

Fundus examination reveals a variety of progressive abnormalities, from RPE defects (A,C) to pattern dystrophy (B) and macular atrophy surrounded by flecks (D). Both individuals show a consistent progressive loss of visual acuity over time, but the rate at which this occurs and the degree of changes in the fundus is markedly different between the two affected individuals Fundus photograph of the right eye of the STGD3 affected Uncle at age 39 years with a visual acuity of 20/30 (A) and again at the age of 48 with a visual acuity of 20/80 (B). Fundus photographs of right eye of the nephew at 21 years of age with a visual acuity of 20/200 (C) and after a reexamination at age 43(D) with a visual acuity of 20/400.

Fig. 4. STGD3 maps to human chromosome 6

(A) Summary of linkage studies localizing the STGD3 disease locus to the long arm of human chromosome 6. (B) A list of microsatellite gene markers that defines the relative region to which STGD3 maps. Markers highlighted in yellow represent markers in which formal levels of accepting linkage were obtained in the linkage studies summarized.

Figure 5. Schematic representation of human wild type and different known mutations in ELOVL4 gene and their protein products

(A) ELOVL4 gene structure. Boxes depict exons and intervening lines depict introns. ELOVL4 consists of six exons and five introns and codes for a 3085-nucleotide transcript. All known STGD3-causing mutations (red lines depicting deletions or nucleotide changes) fall in exon 6 of the ELOLV4 gene. (B) Effect of STGD3-causing mutations on the protein sequence. Sequences of wild type and mutant proteins starting at amino acid 245 are shown. For the mutant ELOVL4 protein, changes in amino acid sequence as compared to wild type are indicated in red type. Amino acid regions that are deleted from the sequence are highlighted in yellow. For all three known STGD3 mutations the end effect is a premature stop to the protein sequence and a deletion of the endoplasmic reticulum retention signal (indicated in blue type in the wild type sequence).

Figure 6. Visual expression profiling based on Unigene data for identified ELOVL4 Expressed Sequence Tags (ESTs). (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi? uglist= Hs.101915)

Figure 7. Mutant ELOVL4 protein expression results in aggresome formation

An expression construct was created to express the wild type ELOVL4 allele fused to a green fluorescent protein marker (EGFP-Wt ELOVL4). A second expression construct was created to express the STGD3-causing 5-bp deletion ELOVL4 allele, also fused to a green fluorescent protein marker (EGFP- 5-bp del-ELOVL4). Cos-7 cells grown on dual chambers were transfected with EGFP-Wt ELOVL4 or EGFP-mut ELOVL4 using lipofectamine plus reagent. Post-transfected cells were fixed with methanol and processed for immunocytochemistry using antibodies specific to vimentin and visualized with Alexa fluor 555 probe–tagged secondary antibody. The Alexa fluor 555 probe fluoresces under a different wavelength than GFP. Fluorescence images were acquired using appropriate filters and lasers. GFP is imaged as green fluorescence, vimentin is detected as red fluorescence, and blue fluorescence results from 4′,6-diarnidino-2-phenylindole (DAPI) staining of the nucleus. GFP-ELOVL4 fluorescence is demonstrated in panels A D, and vimentin distribution is shown in panels B,E. Panels C,F are the merged image of panels A,B and D,E. Formation of aggresomes is associated with the reorganization of intermediary filament protein, vimentin. In Wt-ELOVL4– transfected cells, vimentin was found to be distributed in a reticulate pattern all over the cells, where as in cells expressing mutant-ELOVL4 protein, distribution of vimentin was found to be altered. Vimentin labeling in these cells was observed to be relocated to the perinuclear region where mutant ELOVL4 is accumulated as an aggregate. Formation of aggresome in mutant ELOVL4 transfected cells is visualized by this reorganization of vimentin. Scale bar is 5 μM.

Figure 8. Phenotype of homozygous ELOVL4 5-bp deletion knock-in pups (E_mut +/+)

A) Skin phenotype. Comparison of the skin abnormality in E_mut +/+ pups from birtth to 4 h postnatal (compared to wild type) demonstrates a progression towards a scaly, dry, wrinkled skin phenotype followed by death. B) At birth, the weight of control and E_mut+/+ pups is not significantly different from the wild type, but within the first 4 h of birth, the weight of E_mut +/+ pups decreases drastically. (C,D) Measurements of long chain FA in the epidermal free FA pool (C) and of the amide linked FA of epidermal ceramide/glucosylceramide. (D) show a similar pattern of elevated levels of C>26 related FA in E_mut+/+ samples as compared to wild type. Moreover there is a drastic decrease in levels of C>28 related FA in E_mut+/+ samples as compared to the wild type. (Modified after Vasireddy et al., 2007)

Figure 9. Fatty acid elongation in the cells and possible role of ELOVL4 in the elongation of long chain fatty acid

(Modified after Sprong et al., 2001; Hubbard, 2006; Agbaga et al., 2008; Katz and Minke, 2009; Oda et al., 2009). Enzymatic steps involving ELOVL4 are indicated (large yellow rectangle). Both steps are independent of the synthesis of DHA, the most abundant long chain FA in the retina. DHA is also known as C22:6n3 (“C:22” indicates that the molecule is 22 carbons long; the “:6” indicates that the structure has 6 cis double bonds; and “n3” indicates that the first double bond is located at the third carbon from the omega end of the structure). The function of ELOVL4 is to mediate the synthesis of very long chain FA (VLFA). A select number of polyunsaturated fatty acids (PUFA) that are mentioned in the text are shown. In general a PUFA is a FA that contains more than one double bond in its molecular structure. Abbreviations: ACP, acyl carrier protein; C, carbon chain; Co A, coenzyme A; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; FAS, fatty acid synthase; TCA cycle, tricarboxylic acid cycle; VLCFA, very long chain fatty acids.

Figure 10. Points of possible therapeutic strategies to treat retinal degeneration in STGD3 patients

Illustrated is a cell expressing ELOVL4 from a heterozygote STGD3-affected patient. The view is simplified because events are shown for the expression of the wild type (Wt) ELOVL4 allele (DNA, mRNA, and protein illustrated in black) separate from the STGD3-causing ELOVL4 allele (DNA, mRNA and protein illustrated in blue). In the affected heterozygous condition the aggresome consists of both mutant and wild type ELOVL4 protein and not just of mutant ELOVL4 protein as illustrated. Aggresome formation therefore represents an exodus of both mutant and Wt ELOVL4 out of the ER. The ideal treatment strategy would be targeted at the functional cause of the cellular dysfunction that underlies the clinical phenotype. At present the two most likely causes are the depletion of Wt ELOVL4 from the ER (and therefore no VLCFA synthesis) or the possible toxic effects of the aggresomes formed in the cytoplasm (leading to cell death). Putative therapies will either help promote (green arrow, +) an event that leads to an increased production of the ELVOL4 end product, VLCFA or inhibit (red arrow, -) functional points that lead to the negative effects of mutant ELOVL4 allele expression. Strategic points of intervention might include: 1) Enhanced expression of Wt ELOVL4; 2) VLCFA replacement; 3) removal of mutant ELOVL4 mRNA; 4) enhanced expression of chaperone expression and targeting of the mutant ELOVL4 to the proteosome. Triangles and squares indicate molecular chaperones.

Figure 11. A close-up of the ELOVL4/ABCA4 interactome

Where possible the gene symbol is given followed by the OMIM symbol for the disorders that the gene underlies. The mode of inheritance of the disorder has also been color coded.