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1.  ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bull's‐eye maculopathy 
The British Journal of Ophthalmology  2007;91(12):1650-1655.
To determine the frequency and nature of mutations in the gene ABCA4 in a cohort of patients with bull's‐eye maculopathy (BEM).
A panel of 49 subjects (comprising 40 probands/families, 7 sibling pairs and a set of three sibs) with BEM, not attributable to toxic causes, was ascertained. Blood samples from each patient were used to extract genomic DNA, with subsequent mutation screening of the entire coding sequence of ABCA4, using single‐strand conformational polymorphism (SSCP) analysis and direct sequencing.
Fourteen probands (35%) were found to have a potentially disease‐causing ABCA4 sequence variant on at least one allele. Three patients had a Gly1961Glu missense mutation, the most common variant in Stargardt disease (STGD), with 2 of these subjects having a macular dystrophy (MD) phenotype and a second ABCA4 variant previously associated with STGD. The second most common STGD mutation, Ala1038Val, was seen in one patient with cone–rod dystrophy (CORD). Five novel ABCA4 variants were detected. Two sibships were identified with a similar intra‐familial phenotype but discordant ABCA4 variants.
Variations in the ABCA4 gene are common in BEM. Two sibships showed discordant ABCA4 variants. One of these sibships illustrates that ABCA4 variants can be identified in families that have another molecular cause for their disease, due to the high prevalence of ABCA4 disease alleles in the population. The discordance evident in the second sibship may yet also be a chance finding in families with macular disease of another genetic cause, or it may represent a complex mode of inheritance determined/modified by the combination of ABCA4 alleles.
PMCID: PMC2095527  PMID: 18024811
2.  Partial paternal uniparental disomy (UPD) of chromosome 1 in a patient with Stargardt disease 
Molecular Vision  2007;13:96-101.
Stargardt disease (STGD) is the most common juvenile macular dystrophy, characterized by central visual impairment. All recessively inherited cases are thought to be due to mutations in the ABCA4 gene, mapped to 1p21-p13.
To describe a form of non-mendelian inheritance in a patient with STGD identified through the course of a conventional mutational screening performed on 77 STGD families. DNA from the patient and relatives was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray; results were confirmed by direct sequencing. Haplotype analyses, standard and high-resolution (HR) karyotypes, and multiplex ligation-dependent probe amplification (MLPA) were also performed.
A patient with STGD caused by the homozygous p.Arg1129Leu mutation in the ABCA4 gene was found to be the daughter of a noncarrier mother and a father who was heterozygous for this change. Haplotype analysis suggested that no maternal ABCA4 allele was transmitted to the patient. Microsatellite markers spanning the entire chromosome 1 identified a homozygous region of at least 4.4 Mb, involving the ABCA4 gene. The cytogenetic study revealed normal female karyotype. Further evaluation with MLPA showed the patient had a normal dosage for both copies of the ABCA4 gene, thus suggesting partial paternal isodisomy but not a maternal microdeletion.
We report that recessive STGD can rarely be inherited from only one unaffected carrier parent in a non-mendelian manner. This study also demonstrates that genomic alterations contribute to only a small fraction of disease-associated alleles for ABCA4.
PMCID: PMC2553007  PMID: 17277736
3.  Defective Lipid Transport and Biosynthesis in Recessive and Dominant Stargardt Macular Degeneration 
Progress in lipid research  2010;49(4):476-492.
Stargardt disease is a common inherited macular degeneration characterized by a significant loss in central vision in the first or second decade of life, bilateral atrophic changes in the central retina associated with degeneration of photoreceptors and underlying retinal pigment epithelial cells, and the presence of yellow flecks extending from the macula. Autosomal recessive Stargardt disease, the most common macular dystrophy, is caused by mutations in the gene encoding ABCA4, a photoreceptor ATP binding cassette (ABC) transporter. Biochemical studies together with analysis of abca4 knockout mice and Stargardt patients have implicated ABCA4 as a lipid transporter that facilitates the removal of potentially toxic retinal compounds from photoreceptors following photoexcitation. An autosomal dominant form of Stargardt disease also known as Stargardt-like dystrophy is caused by mutations in a gene encoding ELOVL4, an enzyme that catalyzes the elongation of very long chain fatty acids in photoreceptors and other tissues. This review focuses on the molecular characterization of ABCA4 and ELOVL4 and their role in photoreceptor cell biology and the pathogenesis of Stargardt disease.
PMCID: PMC2946192  PMID: 20633576
Stargardt Disease; ABCA4; ABC Transporters; Retinoids; ELOVL4; Elongase; Very long chain fatty acids
4.  Exome Sequencing Analysis Identifies Compound Heterozygous Mutation in ABCA4 in a Chinese Family with Stargardt Disease 
PLoS ONE  2014;9(3):e91962.
Stargardt disease is the most common cause of juvenile macular dystrophy. Five subjects from a two-generation Chinese family with Stargardt disease are reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family initiated the disease during childhood, developing progressively impaired central vision and bilateral atrophic macular lesions in the retinal pigmental epithelium (RPE) that resembled a “beaten-bronze” appearance. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. Exome sequencing was used to analyze the exome of two patients II1, II2. A total of 50709 variations shared by the two patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in all family members by PCR and Sanger sequencing. Compound heterozygous variants p.Y808X and p.G607R of the ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4) gene, which encodes the ABCA4 protein, a member of the ATP-binding cassette (ABC) transport superfamily, were identified as causative mutations for Stargardt disease of this family. Our findings provide one novel ABCA4 mutation in Chinese patients with Stargardt disease.
PMCID: PMC3954841  PMID: 24632595
5.  Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa 
Molecular Vision  2008;14:262-267.
Stargardt disease (STGD), characterized by central visual impairment, is the most common juvenile macular dystrophy. All recessively inherited cases are thought to be due to mutations in the ABCA4 gene. Early-onset autosomal recessive retinitis pigmentosa (arRP) is a severe retinal degeneration that presents before the patient is ten years old. It has been associated with mutations in different genes, including CRB1. The aim of this study was to determine the genetic causes for two different retinal dystrophies, STGD and early-onset arRP, both segregating in one Spanish family.
Mutational analyses were performed using the ABCR400 and Leber congenital amaurosis (LCA) genotyping microarrays. Additional scanning for mutations was conducted by denaturing high performance liquid chromatography (dHPLC); results were confirmed by direct sequencing.
A patient, who exhibited a STGD phenotype, was found to be homozygous for the p.Asn1805Asp (c.5413A>G) mutation in ABCA4. However, his affected sister, who had the arRP phenotype, was found to be heterozygous for this allele; no other sequence change could be found in ABCA4. Analysis using the LCA chip revealed the p.Cys948Tyr mutation in CRB1 in heterozygous state. A second mutation (p.Trp822ter) was found in the CRB1 gene in the affected female by denaturing high performance liquid chromatography (dHPLC) and direct sequencing.
Two distinct retinal dystrophies with mutations affecting two different genes cosegregated in this family. The presence of two different phenotypes associated with mutations in two distinct genes in one single family must be considered especially when dealing with retinal dystrophies which bear high carrier frequencies in general population.
PMCID: PMC2258218  PMID: 18334942
6.  Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy 
Gene therapy  2008;15(19):1311-1320.
Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4−/− mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4−/− mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4−/− mice were injected subretinally with 1 µl (~5.0 × 105 TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4−/− mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4−/− mice accumulated 8–12 pmol per eye (s.d. = 2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3–5 times more A2E (27–39 pmol per eye, s.d. = 1.5; P = 0.001–0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.
PMCID: PMC3110063  PMID: 18463687
Stargardt disease; ABCA4; EIAV lentivirus; mouse model; A2E
7.  Novel ABCA4 compound heterozygous mutations cause severe progressive autosomal recessive cone-rod dystrophy presenting as Stargardt disease 
Molecular Vision  2009;15:638-645.
To identify the gene causing a severe form of progressive autosomal recessive cone-rod dystrophy presenting as Stargardt disease and to characterize clinical features in a large American family.
We characterized an American family who had an unusual retinal dystrophy with clinical features of Stargardt disease and severe progressive cone-rod dystrophy. Family members underwent complete ocular examinations with evaluation of visual acuity, visual fields, fundus examination, fluorescein angiography, and electroretinography. Genome-wide linkage analysis of the family was performed using 408 microsatellite markers spanning the entire human genome. Direct DNA sequence analysis was used for mutational analysis of the ABCA4 gene in all exons and exon-intron boundary regions and for testing cosegregation of the mutations with the disease in the family. DNA sequence analysis was used to determine the presence of the mutations in 200 unrelated controls.
The proband presented with a clinical phenotype that was initially compatible with Stargardt disease, only to progress to a severe cone-rod dystrophy over the course of a few years. The disease-causing gene in the family was linked to the ABCA4 locus on chromosomal 1p22. One novel mutation, c.655A>T, was identified in exon 6 and another novel splicing mutation, c.5312+3A>T, was identified in intron 37 of ABCA4. The mutations were not present in 200 controls. The two affected sisters in this pedigree were compound heterozygotes for the mutations. Unaffected family members either did not carry either or had only one of the two mutations.
We have identified two novel ABCA4 mutations, c.655A>T and c.5312+3A>T. When present as a compound heterozygous state, the mutations cause a phenotype of retinal dystrophy that initially manifests as Stargardt disease and slowly progresses to a severe cone-rod dystrophy. These results expand the wide range of clinical manifestations of ABCA4 mutations.
PMCID: PMC2665199  PMID: 19352439
8.  Exome Analysis of Two Limb-Girdle Muscular Dystrophy Families: Mutations Identified and Challenges Encountered 
PLoS ONE  2012;7(11):e48864.
The molecular diagnosis of muscle disorders is challenging: genetic heterogeneity (>100 causal genes for skeletal and cardiac muscle disease) precludes exhaustive clinical testing, prioritizing sequencing of specific genes is difficult due to the similarity of clinical presentation, and the number of variants returned through exome sequencing can make the identification of the disease-causing variant difficult. We have filtered variants found through exome sequencing by prioritizing variants in genes known to be involved in muscle disease while examining the quality and depth of coverage of those genes. We ascertained two families with autosomal dominant limb-girdle muscular dystrophy of unknown etiology. To identify the causal mutations in these families, we performed exome sequencing on five affected individuals using the Agilent SureSelect Human All Exon 50 Mb kit and the Illumina HiSeq 2000 (2×100 bp). We identified causative mutations in desmin (IVS3+3A>G) and filamin C (p.W2710X), and augmented the phenotype data for individuals with muscular dystrophy due to these mutations. We also discuss challenges encountered due to depth of coverage variability at specific sites and the annotation of a functionally proven splice site variant as an intronic variant.
PMCID: PMC3498247  PMID: 23155419
9.  Hetero-oligomeric interactions of an ELOVL4 mutant protein: implications in the molecular mechanism of Stargardt-3 macular dystrophy 
Molecular Vision  2010;16:2438-2445.
Stargardt disease 3 (STGD3) is a juvenile macular dystrophy caused by mutations in the elongase of very long-chain fatty acids-like 4 (ELOVL4) gene, which encodes an elongase involved in the production of extremely long-chain fatty acids. The STGD3-related mutations cause production of C-terminally truncated proteins (ELOVL4ΔC). STGD3 is transmitted in an autosomal dominant manner. To date, molecular mechanisms of this pathology have been proposed based solely on the interaction between wild-type ELOVL4 and ELOVL4ΔC. However, analyses of Elovl4ΔC knockin mice revealed reduced levels of not only ELOVL4 substrates, but also of fatty acids with a broad spectrum of chain lengths. Therefore, we investigated the molecular mechanisms responsible for ELOVL4ΔC affecting the entire very long-chain fatty acid (VLCFA) elongation pathway.
The ELOVL4ΔC protein was expressed in HEK 293T cells, and its effect on elongase activities toward several acyl-CoAs were examined. We also investigated the homo- and hetero-oligomerization of ELOVL4ΔC with other elongases (ELOVL1–7) or with other enzymes involved in VLCFA elongation using coimmunoprecipitation experiments.
We found that ELOVL4ΔC forms a homo-oligomer more strongly than wild-type ELOVL4. ELOVL4ΔC also interacts strongly with other elongases, although similar interactions for wild-type ELOVL4 were observed as only weak. In addition, ELOVL4ΔC is able to form an elongase complex by interacting with other components of the VLCFA elongation machinery, similar to wild-type ELOVL4.
We propose that not only the ELOVL4-ELOVL4ΔC homo-oligomeric interaction, but also several hetero-oligomeric interactions, may contribute to the pathology of STGD3.
PMCID: PMC2996883  PMID: 21139992
10.  Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice 
Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with ω-hydroxy very long chain fatty acids (≥C28) and accumulation of ceramides with non ω-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.
PMCID: PMC1796950  PMID: 17311087
ELOVL4; lipid; epidermis; keratinocyte differentiation; stratum corneum
11.  Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease 
Human Molecular Genetics  2013;22(25):5136-5145.
Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.
PMCID: PMC3842174  PMID: 23918662
12.  Mutations of the retinal specific ATP binding transporter gene (ABCR) in a single family segregating both autosomal recessive retinitis pigmentosa RP19 and Stargardt disease: evidence of clinical heterogeneity at this locus 
Journal of Medical Genetics  1999;36(6):447-451.
Stargardt disease (STGD) is an autosomal recessive macular dystrophy of childhood characterised by bilateral loss of central vision over a period of several months. STGD has been mapped to chromosome 1p22.1 and recently ascribed to mutations in the retinal specific ATP binding transporter gene (ABCR). The fundus flavimaculatus with macular dystrophy (FFM), an autosomal recessive condition responsible for gradual loss of visual acuity in adulthood (second to third decade) has also been mapped to the same locus. However, a gene for autosomal recessive retinitis pigmentosa with distinctive features of choriocapillaris atrophy at an advanced stage (RP19) has been mapped to the genetic interval encompassing the STGD gene on chromosome 1p (D1S435-D1S236), raising the question of whether, despite striking differences in clinical course and presentation, RP19 and STGD might be allelic disorders at the ABCR locus.
In a family segregating RP and STGD in two first cousins, we found that heterozygosity for a splicing mutation in the ABCR gene (1938-1 G→A) resulted in STGD while hemizygosity for this splice mutation resulted in RP, and when studying the RP patient's parents, we found a maternal non-contribution with apparent segregation of a null allele ascribed to a partial deletion of the ABCR gene.
The present study shows that, despite striking clinical differences, RP19 and STGD are allelic disorders at the ABCR locus.

Keywords: Stargardt disease; RP19; ABCR gene
PMCID: PMC1734380  PMID: 10874631
13.  Mutations in the peripherin/RDS gene are an important cause of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus 
The British Journal of Ophthalmology  2007;91(11):1504-1511.
To describe the phenotype and to analyse the peripherin/RDS gene in 10 unrelated families with multifocal pattern dystrophy simulating Stargardt disease (STGD1).
The probands of 10 families and 20 affected family members underwent an ophthalmic examination including dilated fundus examination, fundus autofluorescence imaging and optical coherence tomography (OCT). In all probands and in selected family members, fluorescein angiography, electrophysiological testing and visual field analysis were performed. Blood samples were obtained from affected and unaffected family members for analysis of the peripherin/RDS gene.
All 10 probands carried mutations in the peripherin/RDS gene. Nine different mutations were identified, including six mutations that were not described previously. All probands showed a pattern dystrophy with yellow–white flecks in the posterior pole that strongly resembled the flecks seen in STGD1, on ophthalmoscopy as well as on autofluorescence and OCT. Clinical findings in the family members carrying the same mutation as the proband were highly variable, ranging from no visible abnormalities to retinitis pigmentosa.
Mutations in the peripherin/RDS gene are the major cause of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus. This autosomal dominant disorder should be distinguished from autosomal recessive STGD1, in view of the different inheritance pattern and the overall better visual prognosis.
PMCID: PMC2095453  PMID: 17504850
14.  Molecular analysis of the ABCA4 gene for reliable detection of allelic variations in Spanish patients: identification of 21 novel variants 
Mutations in ABCA4 have been associated with autosomal recessive Stargardt disease (STGD), a few cases with autosomal recessive cone–rod dystrophy (arCRD) and autosomal recessive retinitis pigmentosa (arRP). The purpose of the study was threefold: to molecularly characterise families with no mutations or partially characterised families; to determine the specificity and sensitivity of the genotyping microarray; and to evaluate the efficiency of different methodologies.
23 STGD, five arCRD and three arRP Spanish patients who were previously analysed with the ABCR400 microarray were re-evaluated. Results were confirmed by direct sequencing. In patients with either none or only one mutant allele, ABCA4 was further analysed by denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA). Haplotype analysis was also performed.
In the first analysis performed with the microarray, 27 ABCA4 variants (27/62; 43.5%) were found. By dHPLC scanning, 12 novel mutations were additionally identified. In addition, two previously described mutations, one false negative (1/62; 1.6%) and one false positive (1.6%), were detected. MLPA analysis did not reveal additional substitutions. The new strategy yielded an increment of 21% compared with the approach used in the first round.
ABCA4 should be analysed by optimal combination of high-throughput screening techniques such as microarray, dHPLC and direct sequencing. To the best of our knowledge, this strategy yielded significant mutational spectrum identification in Spanish patients with ABCA4-associated phenotypes. Follow-up of patients, presenting an early onset of the disease and severe mutations, seems essential to perform accurate genotype–phenotype correlations and further characterisation of pathological ABCA4 alleles.
PMCID: PMC2668911  PMID: 19028736
15.  Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice 
Vision research  2007;47(5):714-722.
ELOVL4 was first identified as a disease-causing gene in Stargardt macular dystrophy (STGD3, MIM 600110.) To date, three ELOVL4 mutations have been identified, all of which result in truncated proteins which induce autosomal dominant juvenile macular degenerations. Based on sequence homology, ELOVL4 is thought to be another member within a family of proteins functioning in the elongation of long chain fatty acids. However, the normal function of ELOVL4 is unclear. We generated Elovl4 knockout mice to determine if Elovl4 loss affects retinal development or function. Here we show that Elovl4 knockout mice, while perinatal lethal, exhibit normal retinal development prior to death at day of birth. Further, postnatal retinal development in Elovl4 heterozygous mice appears normal. Therefore haploinsufficiency for wildtype ELOVL4 in autosomal dominant macular degeneration likely does not contribute to juvenile macular degeneration in STGD3 patients. We found, however, that Elovl4+/− mice exhibit enhanced ERG scotopic and photopic a and b waves relative to wildtype Elovl4+/+ mice suggesting that reduced Elovl4 levels may impact retinal electrophysiological responses.
PMCID: PMC2583245  PMID: 17254625
STGD3; Elovl4; knockout; mouse; ERG
16.  DHA does not protect ELOVL4 transgenic mice from retinal degeneration 
Molecular Vision  2009;15:1185-1193.
Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3.
Transgenic mice expressing mutant human ELOVL4 (TG2) were bred to mice expressing the fat-1 protein, which can convert n6 to n3 PUFA. Mice were maintained on an n3-deficient diet containing 10% safflower oil (SFO, enriched in n6 PUFA; n6/n3=273) so that four experimental groups were produced that differed only in levels of n3 PUFA and expression of the hELOVL4 transgene. These groups were identified by genotyping and named Fat1+/TG2+, Fat1–/TG2+, Fat1+/TG2–, and Fat1–/TG2–. All were continued on the SFO diet for 4 to 16 weeks such that those not expressing Fat1 would be deficient in n3 fatty acids. At both time points, animals were analyzed for retinal function by electroretinography (ERG), photoreceptor cell viability by outer nuclear layer (ONL) thickness measurements, fatty acid profiles in several tissues, and rhodopsin levels.
Mice expressing the fat-1 transgene had significantly higher levels of n3 PUFA, primarily DHA, in retina, liver, and plasma lipids at 4 and 16 weeks of age. Retinal DHA levels in fat-1 mice were twice those of controls. By 16 weeks of age, mice expressing the mutant hELOVL4 transgene had a significantly greater loss of photoreceptor cells, reduced ERG amplitudes, and lower rhodopsin levels than control mice. There was no effect of retinal fatty acids on the rate of degeneration of retinas expressing the ELOVL4 transgene.
We found no evidence that high levels of DHA in retinal membranes protected photoreceptor cells expressing mutant ELOVL4 from retinal degeneration. We conclude that DHA is not beneficial for the treatment of retinal degeneration in this animal model of human STGD3 macular dystrophy.
PMCID: PMC2697457  PMID: 19536303
17.  Stargardt macular dystrophy: common ABCA4 mutations in South Africa—establishment of a rapid genetic test and relating risk to patients 
Molecular Vision  2012;18:280-289.
Based on the previous indications of founder ATP-binding cassette sub-family A member 4 gene (ABCA4) mutations in a South African subpopulation, the purpose was to devise a mechanism for identifying common disease-causing mutations in subjects with ABCA4-associated retinopathies (AARs). Facilitating patient access to this data and determining the frequencies of the mutations in the South African population would enhance the current molecular diagnostic service offered.
The majority of subjects in this study were of Caucasian ancestry and affected with Stargardt macular dystrophy. The initial cohort consisted of DNA samples from 181 patients, and was screened using the ABCR400 chip. An assay was then designed to screen a secondary cohort of 72 patients for seven of the most commonly occurring ABCA4 mutations in this population. A total of 269 control individuals were also screened for the seven ABCA4 mutations.
Microarray screening results from a cohort of 181 patients affected with AARs revealed that seven ABCA4 mutations (p.Arg152*, c.768G>T, p.Arg602Trp, p.Gly863Ala, p.Cys1490Tyr, c.5461–10T>C, and p.Leu2027Phe) occurred at a relatively high frequency. The newly designed genetic assay identified two of the seven disease-associated mutations in 28/72 patients in a secondary patient cohort. In the control cohort, 12/269 individuals were found to be heterozygotes, resulting in an estimated background frequency of these mutations in this particular population of 4.46 per 100 individuals.
The relatively high detection rate of seven ABCA4 mutations in the primary patient cohort led to the design and subsequent utility of a multiplex assay. This assay can be used as a viable screening tool and to reduce costs and laboratory time. The estimated background frequency of the seven ABCA4 mutations, together with the improved diagnostic service, could be used by counselors to facilitate clinical and genetic management of South African families with AARs.
PMCID: PMC3275638  PMID: 22328824
18.  Cone Photoreceptor Abnormalities Correlate with Vision Loss in Patients with Stargardt Disease 
Adaptive optics scanning laser ophthalmoscopy demonstrated abnormal cone spacing in regions of abnormal fundus autofluorescence and reduced visual function in 12 patients with Stargardt disease.
To study the relationship between macular cone structure, fundus autofluorescence (AF), and visual function in patients with Stargardt disease (STGD).
High-resolution images of the macula were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography in 12 patients with STGD and 27 age-matched healthy subjects. Measures of retinal structure and AF were correlated with visual function, including best-corrected visual acuity, color vision, kinetic and static perimetry, fundus-guided microperimetry, and full-field electroretinography. Mutation analysis of the ABCA4 gene was completed in all patients.
Patients were 15 to 55 years old, and visual acuity ranged from 20/25–20/320. Central scotomas were present in all patients, although the fovea was spared in three patients. The earliest cone spacing abnormalities were observed in regions of homogeneous AF, normal visual function, and normal outer retinal structure. Outer retinal structure and AF were most normal near the optic disc. Longitudinal studies showed progressive increases in AF followed by reduced AF associated with losses of visual sensitivity, outer retinal layers, and cones. At least one disease-causing mutation in the ABCA4 gene was identified in 11 of 12 patients studied; 1 of 12 patients showed no disease-causing ABCA4 mutations.
AOSLO imaging demonstrated abnormal cone spacing in regions of abnormal fundus AF and reduced visual function. These findings provide support for a model of disease progression in which lipofuscin accumulation results in homogeneously increased AF with cone spacing abnormalities, followed by heterogeneously increased AF with cone loss, then reduced AF with cone and RPE cell death. ( number, NCT00254605.)
PMCID: PMC3109028  PMID: 21296825
19.  Subretinal Fibrosis in Stargardt's Disease with Fundus Flavimaculatus and ABCA4 Gene Mutation 
Case Reports in Ophthalmology  2012;3(3):410-417.
To report on 4 patients affected by Stargardt's disease (STGD) with fundus flavimaculatus (FFM) and ABCA4 gene mutation associated with subretinal fibrosis.
Four patients with a diagnosis of STGD were clinically examined. All 4 cases underwent a full ophthalmologic evaluation, including best-corrected visual acuity measured by the Snellen visual chart, biomicroscopic examination, fundus examination, fundus photography, electroretinogram, microperimetry, optical coherence tomography and fundus autofluorescence. All patients were subsequently screened for ABCA4 gene mutations, identified by microarray genotyping and confirmed by conventional DNA sequencing of the relevant exons.
In all 4 patients, ophthalmologic exam showed areas of subretinal fibrosis in different retinal sectors. In only 1 case, these lesions were correlated to an ocular trauma as confirmed by biomicroscopic examination of the anterior segment that showed a nuclear cataract dislocated to the superior site and vitreous opacities along the lens capsule. The other patients reported a lifestyle characterized by competitive sport activities. The performed instrumental diagnostic investigations confirmed the diagnosis of STGD with FFM in all patients. Moreover, in all 4 affected individuals, mutations in the ABCA4 gene were found.
Patients with the diagnosis of STGD associated with FFM can show atypical fundus findings. We report on 4 patients affected by STGD with ABCA4 gene mutation associated with subretinal fibrosis. Our findings suggest that this phenomenon can be accelerated by ocular trauma and also by ocular microtrauma caused by sport activities, highlighting that lifestyle can play a role in the onset of these lesions.
PMCID: PMC3551412  PMID: 23341817
Stargardt's disease; ABCA4 gene; Subretinal fibrosis; Ocular trauma; Lifestyle
20.  ABCA4 mutations in Portuguese Stargardt patients: identification of new mutations and their phenotypic analysis 
Molecular Vision  2009;15:584-591.
To resolve the spectrum of causative retina-specific ATP-binding cassette transporter gene (ABCA4) gene mutations in Portuguese Stargardt (STGD) patients and compare allele frequencies obtained in this cohort with those of previous population surveys.
Using a microarray technique (ABCR400 gene chip), we screened all previously reported ABCA4 gene mutations in the genomic DNA of 27 patients from 21 unrelated Stargardt families whose phenotypes had been clinically evaluated using psychophysics and electrophysiological measurements. Furthermore, we performed denaturing high performance liquid chromatography whenever one or both mutant alleles failed to be detected using the ABCR gene chip.
A total of 36 mutant alleles (out of the 54 tested) were identified in STGD patients, resulting in a detection rate of 67%. Two mutant alleles were present in 12 out of 21 STGD families (57%), whereas in four out of 21 (19%) of the families, only one mutant allele was found. We report the presence of 22 putative pathogenic alterations, including two sequence changes not found in other populations, c.2T>C (p.Met1Thr) and c.4036_4037delAC (p.Thr1346fs), and two novel disease-associated variants, c.400C>T (p.Gln134X) and c.4720G>T (p.Glu1574X). The great majority of the mutations were missense (72.7%). Seven frameshift variants (19.4%), three nonsense mutations (8.3%), and one splicing sequence change (2.7%) were also found in STGD chromosomes. The most prevalent pathologic variant was the missense mutation p.Leu11Pro. Present in 19% of the families, this mutation represents a quite high prevalence in comparison to other European populations. In addition, 23 polymorphisms were also identified, including four novel intronic sequence variants.
To our knowledge, this study represents the first report of ABCA4 mutations in Portuguese STGD patients and provides further evidence of different mutation frequency across populations. Phenotypic characterization of novel putative mutations was addressed.
PMCID: PMC2660377  PMID: 19365591
21.  Familial discordance in Stargardt disease 
Molecular Vision  2012;18:227-233.
To report genetic and phenotypic discordance across two generations of a family with autosomal recessive Stargardt disease (STGD1) and to compare pathogenicities of the G1961E and A1038V alleles of the ATP-binding cassette transporter, subfamily A, member 4 (ABCA4) gene.
Five members of a family with STGD1 (patients 1–4, affected; patient 5, carrier) were included. Clinical assessment was performed together with fundus autofluorescence and spectral domain-optical coherence tomography. Patients were stratified based on the results of electroretinogram testing. Genotyping of the ABCA4 gene was performed with the ABCR500 microarray.
STGD1 was diagnosed in the male proband and his female sibling (patients 1 and 2, respectively). Two children of patient 2 (patients 3 and 4) were also affected. Genotyping revealed the W663X stop mutation in all affected patients. Patients 3 and 4, who were compound heterozygous for the G1961E mutation, had earlier ages of onset than patients 1 and 2, who were compound heterozygous for the A1038V mutation. Patient 1 had an age of onset 28 years younger than patient 2, whose delayed onset can be explained by relative foveal sparing, while patient 4 had an age of onset 44 years younger than patient 2.
The G1961E mutation, which has been considered “mild,” yields a more severe phenotype in this family than the A1038V mutation, which has been considered “severe.” Marked intrasibship discordance in clinical course is described, suggesting an additional role for modifying factors in ABCA4 pleiotropism.
PMCID: PMC3272278  PMID: 22312191
22.  Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt’s dystrophy 
Biochimica et Biophysica Acta  2011;1822(7):1169-1179.
Autosomal recessive Stargardt macular dystrophy is caused by mutations in the photoreceptor disc rim protein ABCA4/ABCR. Key clinical features of Stargardt disease include relatively mild rod defects such as delayed dark adaptation, coupled with severe cone defects reflected in macular atrophy and central vision loss. In spite of this clinical divergence, there has been no biochemical study of the effects of ABCA4 deficiency on cones vs. rods. Here we utilize the cone-dominant Abca4−/−/Nrl−/− double knockout mouse to study this issue. We show that as early as post-natal day (P) 30, Abca4−/−/Nrl−/− retinas have significantly fewer rosettes than Abca4+/+/Nrl−/− retinas, a phenotype often associated with accelerated degeneration. Abca4-deficient mice in both the wild-type and cone-dominant background accumulate more of the toxic bisretinoid A2E than their ABCA4-competent counterparts, but Abca4−/−/Nrl−/− eyes generate significantly more A2E per mole of 11-cis-retinal (11-cisRAL) than Abca4−/− eyes. At P120, Abca4−/−/Nrl−/− produced 340±121 pmoles A2E/nmol 11-cisRAL while Abca4−/− produced 50.4±8.05 pmoles A2E/nmol 11-cisRAL. Nevertheless, the retinal pigment epithelium (RPE) of Abca4−/−/Nrl−/− eyes exhibits fewer lipofuscin granules than the RPE of Abca4−/− eyes; at P120: Abca4−/−/Nrl−/− exhibit 0.045±0.013 lipofuscin granules/μm2 of RPE vs. Abca4−/− 0.17±0.030 lipofuscin granules/μm2 of RPE. These data indicate that ABCA4-deficient cones simultaneously generate more A2E than rods and are less able to effectively clear it, and suggest that primary cone toxicity may contribute to Stargardt’s-associated macular vision loss in addition to cone death secondary to RPE atrophy.
PMCID: PMC3351560  PMID: 22033104
ABCA4; Cone; Retinal degeneration; Stargardt’s; A2E
23.  ABCA4-associated retinal degenerations spare structure and function of the human parapapillary retina 
To study the parapapillary retinal region in patients with ABCA4-associated retinal degenerations.
Patients with Stargardt disease or cone-rod dystrophy and disease-causing variants in the ABCA4 gene were included. Fixation location was determined under fundus visualization and central cone-mediated vision was measured. Intensity and texture abnormalities of autofluorescence (AF) images were quantified. Parapapillary retina of an eye donor with ungenotyped Stargardt disease was examined microscopically.
AF images ranged from normal, to spatially homogenous abnormal increase of intensity, to spatially heterogenous speckled pattern, to variably sized patches of low intensity. A parapapillary ring of normal-appearing AF was visible at all disease stages. Quantitative analysis of the intensity and texture properties of AF images showed the preserved region to be an annulus, at least 0.6 mm wide, surrounding the optic nerve head. A similar region of relatively preserved photoreceptor nuclei was apparent in the donor retina. In patients with foveal fixation, there was better cone sensitivity at a parapapillary locus in the nasal retina compared to the same eccentricity in the temporal retina. In patients with eccentric fixation, ~30% had a preferred retinal locus in the parapapillary retina.
Human retinal degenerations caused by ABCA4 mutations spare the structure of retina and RPE in a circular parapapillary region which commonly serves as the preferred fixation locus when central vision is lost. The retina between fovea and optic nerve head could serve as a convenient, accessible and informative region for structural and functional studies to determine natural history or outcome of therapy in ABCA4-associated disease.
PMCID: PMC2579900  PMID: 16303974
Image analysis; macular degeneration; optic nerve head; retinal degeneration; retinal pigment epithelium
24.  From a single whole exome read to notions of clinical screening: primary ciliary dyskinesia and RSPH9 p.Lys268del in the Arabian Peninsula 
Annals of human genetics  2012;76(3):211-220.
Primary ciliary dyskinesia (PCD) is a genetic disorder, usually autosomal recessive, causing early respiratory disease and later subfertility. Whole exome sequencing may enable efficient analysis for locus heterogeneous disorders such as PCD. We whole exome sequenced one consanguineous Saudi Arabian with clinically diagnosed PCD and normal laterality, to attempt ab initio molecular diagnosis.
We reviewed thirteen known PCD genes and potentially autozygous regions (extended homozygosity) for homozygous exon deletions, non-dbSNP codon, splice-site base variants or small indels. Homozygous non-dbSNP changes were also reviewed exome-wide.
One single molecular read representing RSPH9 p.Lys268del was observed, with no wildtype reads, and a notable deficiency of mapped reads at this location. Among all observations, RSPH9 was the strongest candidate for causality. Searching unmapped reads revealed seven more mutant reads. Direct assay for p.Lys268del (MboII digest) confirmed homozygosity in the affected individual, then confirmed homozygosity in three siblings with bronchiectasis. Our finding in southwest Saudi Arabia indicates that p.Lys268del, previously observed in two Bedouin families (Israel, UAE) is geographically widespread in the Arabian Peninsula. Analogous with cystic fibrosis CFTR p.Phe508del, screening for RSPH9 p.Lys268del (which lacks sentinel dextrocardia) in those at risk would help in early diagnosis, tailored clinical management, genetic counselling and primary prevention.
PMCID: PMC3575730  PMID: 22384920
high-throughput nucleotide sequencing; primary ciliary dyskinesia; screening
25.  The Role of the Photoreceptor ABC Transporter ABCA4 in Lipid Transport and Stargardt Macular Degeneration 
Biochimica et biophysica acta  2009;1791(7):573-583.
ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders.
PMCID: PMC2746242  PMID: 19230850

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