June 2004
Volume 45, Issue 6
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Biochemistry and Molecular Biology  |   June 2004
Mutation Spectrum and Founder Chromosomes for the ABCA4 Gene in South African Patients with Stargardt Disease
Author Affiliations
  • Alison V. September
    From the Division of Human Genetics, University of Cape Town, Observatory, Cape Town, South Africa.
  • Anna A. Vorster
    From the Division of Human Genetics, University of Cape Town, Observatory, Cape Town, South Africa.
  • Rajkumar S. Ramesar
    From the Division of Human Genetics, University of Cape Town, Observatory, Cape Town, South Africa.
  • L. Jacquie Greenberg
    From the Division of Human Genetics, University of Cape Town, Observatory, Cape Town, South Africa.
Investigative Ophthalmology & Visual Science June 2004, Vol.45, 1705-1711. doi:10.1167/iovs.03-1167
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      Alison V. September, Anna A. Vorster, Rajkumar S. Ramesar, L. Jacquie Greenberg; Mutation Spectrum and Founder Chromosomes for the ABCA4 Gene in South African Patients with Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2004;45(6):1705-1711. doi: 10.1167/iovs.03-1167.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To assess the mutation spectrum of ABCA4 underlying Stargardt disease (STGD) in South Africa (SA) and to determine whether there is a single or a few founder chromosomes in SA STGD families.

methods. Sixty-four probands exhibiting the STGD phenotype were screened for mutations in the 50 exons of ABCA4 by single-strand conformational polymorphism–heteroduplex analysis sequencing and restriction fragment length polymorphism analysis. Microsatellite marker haplotyping was used to determine the ancestry in 10 families.

results. Fifty-seven ABCA4 disease-associated alleles were identified that comprised 16 different sequence variants, of which two were novel, in 40 individuals of the cohort of 64 subjects. The most common variants identified included the C1490Y, L2027F, R602W, V256splice, R152X, and 2588G→C mutations. The C1490Y variant was the most common disease-associated variant identified (19/64 subjects) and was absent in 392 control chromosomes. At least 10 ABCA4 disease-associated haplotypes were identified. Two of these haplotypes, which carried the C1490Y mutation, were identified in three unrelated families.

conclusions. Results suggest that ABCA4 is the major gene underlying STGD in the cohort investigated. Five of the six common sequence variants identified were at a higher frequency in the SA cohort than reported in published data on individuals of similar ancestry. The mutation and haplotype data suggests that there are several ancestral haplotypes underlying STGD in SA. There seems to be at least two different origins for the common C1490Y mutation, as well as two for the R602W mutation, thereby suggesting several founder effects for STGD in SA.

Stargardt disease (STGD; Online Mendelian Inheritance in Man [OMIM] no. 248200; http://www.ncbi.nlm.nih.gov/Omim/; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) is the most common hereditary macular dystrophy (MD) affecting children, with a prevalence estimated to be approximately 1:10,000. 1 It is characterized by central visual loss, atrophy of the retinal pigment epithelium, which resembles a “beaten-bronze appearance” and the distribution of ill-defined, orange-yellow flecks around the macula and/or the midperiphery of the retina. 2 STGD is predominantly inherited as an autosomal recessive trait. However, a few families with individuals who have MD and flecks seem to display an autosomal dominant inheritance pattern. 3 The first genetic locus for STGD, also the first recessive form of MD, was mapped to the short arm of chromosome 1 (1p21-p13). 4 Four years later, the disease-causing gene, ABCR (retina-specific ABC transporter), was identified and is alternately referred to as ABCA4 (ATP binding cassette [ABC] transporter). 5 Mutations in ABCA4 have been implicated in several clinically distinct retinal phenotypes, which include autosomal recessive STGD (arSTGD), 5 autosomal recessive retinitis pigmentosa (RP), 6 autosomal recessive cone–rod dystrophy (CRD), 7 and age-related macular degeneration (AMD). 8 Mutation analyses of ABCA4 have provided evidence suggesting that it is perhaps the most polymorphic retinal disease gene studied thus far. 9 10 To date, more than 400 disease-causing mutations, ranging from single-base substitutions to deletions of several exons, have been identified in ABCA4. The majority of reported changes are missense mutations. 11 12 13 Disease-causing mutations in ABCA4 account for 66% to 80% of STGD-associated chromosomes investigated. 13 14 A few mutations have been found to be at a high frequency in certain populations from Europe and the United States, suggesting a founder effect. 15 The purpose of this study was to assess the mutation spectrum of the ABCA4 gene underlying STGD in South Africa (SA). 
Methods
Ascertainment of Subjects
Before the commencement of this study, the research protocol was reviewed and approved by the University Research and Ethics Committee of the University of Cape Town (URC REC reference 168/99). Recruitment of subjects and relatives involved obtaining informed consent from each individual from whom blood samples were obtained, in compliance with the Declaration of Helsinki, October 2000. A total of 64 probands affected with arSTGD were selected for investigation. A diagnosis of STGD was made by ophthalmologists based on bilateral central visual loss (age of onset [AO] was in general before 32 years) and a family history with a recessive mode of inheritance. The AO of visual impairment was used in this study as a measure of the clinical severity of the retinal condition in affected individuals. The affected cohort of 64 subjects represented families from the SA white (n = 59), indigenous black African (n = 3), mixed-ancestry (n = 1), and Asian-Indian (n = 1) populations. In addition, 193 unrelated individuals with no known history of retinopathy were recruited as the control cohort. This group comprised 116 individuals from the white, 40 from indigenous black African, 40 from mixed-ancestry, and 47 from Asian-Indian populations of SA. Individuals with STGD were recruited through either Retina South Africa (a lay support group) or through regional and national referrals from ophthalmologists and schools for the blind. SA is a large country that is predominantly rural, and this poses many challenges in patient recruitment. For this reason, an integrated network of healthcare professionals, both in the public and private sectors throughout SA were used in a coordinated effort in patient recruitment and clinical evaluation. 
Mutation Detection
Genomic DNA was extracted from peripheral blood lymphocytes with a kit (Genomix; Talent, Trieste, Italy) and amplified using primer pairs that allowed amplification of the complete coding region of the gene (50 exons) including the exon–intron boundaries. Primer pairs were designed to amplify exons 22, 23, 24, 27, 33, and 49 (22 forward [F] 5′-CTCTTCCTCACCCTCCACAGC-3′, 22 reverse [R] 5′-GCTAGGGCTGCAGTGAGA-3′; 23F 5′-TTTTTGCAACTATATAGCCAGG-3′, 23R 5′-AGCCTGTGTGAGTAGCCATG-3′; 24F 5′-CTGTCATGGAAGGGAGTGC-3′, 24R 5′-CGAATACTGGGAGATGGCTGC-3′; 27F 5′-GAGATCCAGACCTTATAGGC-3′, 27R 5′-ACTGAGCTCAGCTAAACACCG-3′; 33F 5′-GCTACTAGTAGGCGTGAAGTTC-3′, 33R 5′-CTCATTCATGGTAGAATTGC-3′; and 49F 5′-GTGTAGGGTGCTGTTTTCCTG-3′, 49R 5′-GCTCTGAGCCAAGGAACTG-3′). Polymerase chain reaction (PCR) amplification of these exons was performed in 20-μL volumes containing 200 ng genomic template DNA; 20 pmol of each primer; 1.5 MgCl2; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 200 μM dATP, dCTP, dGTP, and dTTP; 4% (wt/vol) dimethylsulfoxide (DMSO); and 0.5 U Taq DNA polymerase (Invitrogen Life Technologies, Paisley, UK). An overlay of liquid paraffin was added to each sample to prevent evaporation during PCR amplification. Amplifications were performed by denaturing for 1 cycle at 94°C for 3 minutes, followed by 30 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, followed by a final extension of 1 cycle at 72°C for 5 minutes on an thermal cycler (Omnigene; Thermo Hybaid, Ashford, UK). Primer pairs were synthesized to amplify the remaining exons. 16 PCR amplification of these exons was performed in 25-μL volumes containing 200 ng genomic template DNA; 10 pmol of each primer; 1.5 or 2 mM MgCl2; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 200 μM dATP, dCTP, dGTP, and dTTP; and 0.5 U Taq DNA polymerase (Invitrogen Life Technologies). Amplification was performed by denaturing for 1 cycle at 94°C for 3 minutes, followed by 25 cycles at 94°C for 30 seconds, 60°C (50°C or 55°C for some primers) for 30 seconds, and 72°C for 40 seconds, followed by a final extension of 1 cycle at 72°C for 5 minutes on a thermal cycler (Omnigene; Thermo Hybaid). 
Single-strand conformation polymorphism analysis together with heteroduplex analysis (SSCP-HD) and PCR amplification were used to screen the coding regions (which included the exons and exon–intron junctions) of the ABCA4 gene. The mutation detection method used was an adaptation of the original method described previously. 17 This method involved the use of a flat-bed discontinuous buffer system (Multiphor II Electrophoresis unit; Amersham Pharmacia Biotech, Amersham, UK). Three microliters of a 1:1 ratio of PCR product to denaturing loading buffer was loaded onto a 12% nondenaturing polyacrylamide gel. The gels were electrophoresed at 350 V for 90 to 120 minutes (depending on the size of the PCR fragment; model PS 3000; Hoefer Scientific Instruments, San Francisco, CA) at either 9°C or 12°C, which was kept constant by a cooled waterbath circulator (model LT13; Labcon, Pinetown, SA). The gels had a maximum capacity of 40 samples. DNA migratory products were visualized by silver staining the gels after electrophoresis, using a standard protocol. 18  
DNA Sequencing
The samples that exhibited altered mobility patterns after SSCP-HD analyses were selected for sequence analysis. The PCR products were purified with a gel extraction protocol (QIAquick; Qiagen, Crawley, UK). Cleaned fragments were sequenced in both directions using a dye terminator chemistry cycle sequencing kit (BigDye Terminator; Applied Biosystems, Foster City, CA). Sequence products were purified through fine columns (Sephadex G-50l; Princeton Separations, Adelphia, NJ) and resolved by automated gel electrophoresis (Prism 377 DNA sequencer; Applied Biosystems). 
Restriction Fragment Length Polymorphism Analysis
Restriction fragment length polymorphism (RFLP) analyses were performed by using the RsaI restriction endonuclease enzyme to screen for the C1490Y mutation. The PCR product of exon 30 (8 μL) from each of the 64 affected individuals forming the study cohort was digested with 5 U of the RsaI enzyme according to the manufacturer (Promega, Southampton, UK). In addition, PCR products of exon 30 from 196 control individuals (representative of the white, indigenous black African, and mixed-ancestry populations) were digested with the RsaI enzyme. Segregation studies using RFLP analysis were conducted in a few families. Digested PCR products were separated on 6% polyacrylamide gels at 220 V for 25 minutes, using the flat-bed discontinuous buffer system (Multiphor II Electrophoresis Unit; Amersham Pharmacia Biotech). Digested products were visualized by silver staining the gels after electrophoresis. 
Haplotype Analysis
Haplotypes were constructed to identify specific STGD-associated chromosomes segregating in 10 of the 64 families in whom ABCA4 mutations were identified. Three microsatellite markers distributed over an approximate physical distance of 2.3 Mb: D1S188, D1S406, D1S236 flanking the ABCA4 gene locus were genotyped for each subject, together with the disease-associated sequence variant or variants. Standard PCR conditions were used for the amplification of the microsatellite markers used in haplotype construction. 
Results
The SSCP-HD mutation and DNA sequence analysis of the ABCA4 gene led to the identification of 58 potential disease-associated alleles. These comprised 16 distinct sequence variants in 40 of the 64 subjects with arSTGD investigated (Table 1) . Except for one deletion, 6352ΔA, the remaining variants were all single-base substitutions. Of these substitutions, 11 were missense mutations, 2 were truncation mutations, and 2 involved a splice donor site. 
This study identified both ABCA4 disease-associated alleles underlying arSTGD in 18 (28%) of the cohort of 64 subjects. Only one putative disease-causing allele was identified in 22 (34%) subjects of the cohort of 64 arSTGD subjects, whereas no sequence variants were detected in the remaining 24. 
Fourteen of the 16 variants identified were previously published and predicted to be disease-associated. The remaining two disease-associated variants, R1443H and 6352ΔA (nucleotide position), have not been previously reported. The R1443H variant was identified in a sporadic individual of Asian-Indian ancestry and was not observed in 47 unaffected, unrelated, ethnically matched control subjects. The 6352ΔA sequence variant, which introduces a frameshift mutation that results in a premature termination codon, was identified in an individual of Afrikaner descent. The C1490Y sequence variant was the most frequently observed mutation in this study (19/64; 30%) but was absent in ethnically matched, unrelated, unaffected control individuals representative of the SA white (n = 116), indigenous black (n = 40), and mixed-ancestry (n = 40) populations. 
Several nondisease causing sequence variants were identified in the mutation screen of the ABCA4 gene and are listed in Table 2 . Most (6/14) of these sequence variants (40%) occurred within intronic regions of the gene. Some of the sequence variants identified were classified as non–disease-causing because they did not segregate with the disease phenotype or they have been reported to be non–disease associated. 
In a large proportion of the individuals with the C1490Y variant, the AO was under the age of 20 years. The mean age of onset for individuals with the C1490Y mutation was 11.1 years with a standard deviation of 5.2. Of note, individual 209.1 and 113.3 were compound heterozygotes for the C1490Y and L2027F variations; however, their AOs differ considerably (18 and 10 years, respectively). The novel sequence variant R1443H was associated with a late AO of 31 years (individual 372.1). In contrast, the other novel mutation (6352ΔA) identified in this study was associated with an early AO of 10 years (individual 374.1). 
The clinical phenotype in one individual (105.1) changed from arSTGD (AO of 10 years) to atypical arRP (age of diagnosis, 24 years) during the course of this study (Table 3) . This individual initially presented with the typical ophthalmic picture consistent with a diagnosis of STGD. At a second examination at age 24 years, bone spicules were observed in the periphery, consistent with the diagnosis of RP. Individual 105.1 was heterozygous for two different mutations, the V256splice variant and the R152X variant. A rapid clinical deterioration of STGD was also noted in two individuals (129.3 and 219.1). Individual 129.3 carried the V256splice variant as the previous individual (105.1); however, the second mutation has not yet been determined. Individual 219.1 in whom a single disease-associated allele carrying the C1490Y variant was identified, was diagnosed with STGD at an early age (AO of 5 years); bilateral extensive RPE atrophy was noted 4 years later (Table 3)
Allelic segregation studies in 10 families with arSTGD, using three microsatellite markers and the ABCA4 sequence variant revealed at least 10 disease-associated haplotypes (Table 4) . A haplotype was found in at least one family for each of the five most common sequence variants identified in this study (Table 1) . A single STGD-associated haplotype was identified for the (1) L2027F sequence variant, in two unrelated families; (2) V256splice variant, in two unrelated families; (3) R152X variant, three unrelated families; (4) 2588G→C variant, in one family; (5) F1440S variant, in one family; and (6) IVS45+7G→A variant, in one family. Two disease-associated haplotypes were identified for the frequent C1490Y variant in three unrelated STGD families, suggesting several founder effects for STGD in SA (Fig. 1) . Likewise, two STGD-associated haplotypes for the R602W variant were identified in two unrelated families. 
Discussion
DNA samples from 2247 individuals representing 694 families with a clinical history of various forms of hereditary retinopathies have been archived in the Division of Human Genetics, University of Cape Town. Nearly 30% of these families display a form of macular dystrophy. 19 Of interest is that 50% of these kindreds have an autosomal recessive form of STGD and the large majority are of Afrikaner ancestry. Genealogical studies have also suggested that the observed sizable proportion of STGD in the Afrikaner people may be due, in part, to a founder effect. 19 20 The Afrikaner population (Afrikaans speaking whites) comprises mainly Dutch, German, and French ancestry. In fact, several diseases in the SA Afrikaner population have been reported to be due to a founder mutation. 21 This hypothesis has more recently been confirmed in several conditions for example long QT syndrome, hypercholesterolemia, variegate porphyria, and Fanconi anemia. 22 23 24 25 “Die Groot trek” which translated to English: “the great move” is the name given to describe the SA historical migration (1835-1843) of the Afrikaner farmers (Boers) from the Cape colony (Cape Province region) to the interior where they settled in the North West Province, Limpopo Province, Mpumalanga, and Kwazulu Natal Province. More than 90% (59/64) of the individuals with STGD investigated in this study were of Afrikaner descent. The perceived increased frequency of STGD among the Afrikaner population in our archive is perhaps a result of a historical ascertainment bias, due to sample referrals largely from special schools for the visually impaired. These institutions have previously been restricted to specific subjects and populations. Furthermore, the observed clustering of STGD among the SA Afrikaner population in the Gauteng province, together with the segregation laws of the previous SA government, are possibly both confounding factors contributing to the apparent selective bias observed in this group. 
This investigation is the first to report on the identification of ABCA4 mutations in a SA STGD cohort. A total of 58 disease-associated alleles were identified that comprised 16 different sequence variants in 40 (63%) of the cohort of 64 subjects investigated. Most of these potential disease-causing mutations involved single-base substitutions resulting in purine–purine and pyrimidine–pyrimidine transitions. 
Two disease-associated alleles were identified in each of 18 of the 64 affected arSTGD probands. A single disease-associated allele was identified in a further 22 probands. It is evident that a significant number of mutations were not identified. However, this mutation detection rate is comparable with that of other ABCA4 studies in which similar mutation screening methods were used. 10 11 26 27 28 Methods such as direct sequencing of all arSTGD probands in other studies have, however, identified approximately 66% to 80% of ABCA4-associated STGD chromosomes. 13 14 The SSCP-HD mutation screening method used in this study has a reported sensitivity of 97%. 17 It is possible that allelic mutations have been missed because (1) of the sensitivity of the method used, or (2) the unidentified mutations may reside in parts of the gene—for example, the promotor or regulatory regions that have not yet been screened. Furthermore, the remaining unidentified STGD-associated alleles may reside in other genes yet to be identified. However, studies have suggested that ABCA4 is possibly the major gene underlying arSTGD. 29 30  
The spectrum of mutations in the SA STGD cohort was observed to be similar to that noted in populations from Europe. This was an expected finding because of the common ancestry shared between the SA STGD cohort and many populations in Europe. However, the frequencies of specific mutations were observed to be distinctly different. The C1490Y sequence variant was the most common disease-associated variant identified in this study (19/64; 30%), followed by the L2027F (8/64; 13%), the R602W variant (6/64; 9%), the V256splice variant (5/64; 8%), and the 2588G→C and R152X sequence variants occurred at equal frequencies (4/64; 6%). The large majority of the individuals investigated in this study were of Western European origin, mainly from Dutch, French, German, and British stock. Five (C1490Y, L2027F, R602W, V256splice and R152X) of the six common sequence variants identified were at a higher frequency in the SA STGD cohort than in populations from Europe and the United States. Of interest, the C1490Y, R602W, V256splice, and R152X variants were found to be some of the rarer ABCA4 mutations observed in populations of Europe. Because of a founder effect, the 2588G→C mutation was observed at a particularly high frequency (37.5%) in individuals with arSTGD in populations from the Netherlands and Germany, and 7.3% in Northern and Central European patients with STGD. 15 31 However, the 2588G→C mutation was observed to be present at a low frequency of 6% (4/68) in the SA cohort. Finally, variants R1443H and 6352ΔA identified in this SA study, had not been reported previously. 
This study presents further supporting evidence that mutations such as R152X and V256splice variants within ABCA4 can cause recessive panretinal degeneration, which is typified by changes in the clinical presentation from STGD to a more severe arRP phenotype over time. 32 Individual 105.1 was heterozygous for two different mutations: the V256splice variant and the R152X sequence variant. The R152X change has been associated with a mild clinical phenotype of fundus flavimaculatus, which has a late AO and a slow progressive clinical phenotype. 13 33 In this study, the R152X sequence variant is associated with an earlier AO (<28 years) than the published data of 70 and 52 years. In contrast, the V256splice variant has previously been associated with RP, which is regarded as a severe clinical phenotype, having an early AO and a more rapid degenerative progression. 15 This report suggests that the V256splice variant has a severe effect on protein function. Without in vitro functional analyses of the effects of these two mutations (R152X and V256splice) on the ABCA4 protein, it is difficult to predict accurately whether both variants contribute equally to the severe clinical phenotype or whether the severity of the phenotype is determined by the effects of the V256splice mutation on the protein function. However, the findings of the present investigation suggest that the V256splice variant and the R152X variant both affected the ABCA4 protein function severely and that this could explain the severe clinical phenotype. It is therefore reasonable to propose that arRP is not only caused by homozygous null mutations in ABCA4 but also by a combination of different null mutations. It has previously been hypothesized that two null mutations cause atypical RP and combinations of a null and a moderately severe mutation cause CRD. 15  
The novel R1443H involves a substitution of a conserved arginine residue with histidine, another basic amino acid, at position 1443 within extracellular domain 2 (ECD 2) of the ABCA4 protein. 34 It is therefore predicted that R1443H does not have a dramatic functional effect within the intradiscal space of the photoreceptor cells where ECD-2 is localized. Basic amino acids are usually found on the exterior surfaces of proteins because of their strong polar properties. It has been suggested that mutations toward the 3′ end of the gene may not have a significant effect on protein function and hence can be more often associated with milder phenotypes (referring specifically to AO). 34 The novel R1443H and R2030X sequence variants, located toward the 3′ end of the gene may therefore explain the milder phenotype (AO = 31 years) observed in individual 372.1, who was heterozygous for these mutations. The 6352ΔA mutation, which is also very close to the carboxyl terminal of the protein, however, was associated with a more severe phenotype (AO = 10 years) in individual 374.1. The severe phenotype noted in this patient was therefore attributed to the subject’s heterozygosity for the 6352ΔA and L2027F mutations, with the latter being the major contributor to the disease phenotype. Further investigation into the AO reported to be associated with L2027F (data not presented), however, highlights a correlation between the L2027F variant with a milder clinical phenotype associated with a late AO (second and third decade of life). It is therefore reasonable to hypothesize that regarding individual 374.1, who was heterozygous for the two mutations, L2027F and the novel mutation 6352ΔA, (1) both mutations contribute equally to the severity of the phenotype, (2) modifying sequences within ABCA4 or other genes modulate the effect of the compound heterozygous mutations 6352ΔA/L2027F on the ABCA4 protein, and hence the phenotype, or (3) environmental factors such as diet, smoking, and UV radiation may all contribute to the scenario in (2). This therefore suggests that the two novel mutations 6352ΔA and R1443H may have moderate and mild effects on ABCA4 protein function, respectively. It is evident that in vitro functional analysis is required to elucidate the consequences of the singular and combined effects of mutations on the ABCA4 protein structure, processing, and function. 
From the published data, the C1490Y mutation was noted to be present at a particularly low frequency (1%) in the European population. 9 This is in contrast, to the notably high frequency (30%) observed in the SA Afrikaner population who are of Dutch, German, and French ancestry. It was therefore reasonable to expect this sequence variant in the background population of SA. However, the C1490Y variant was absent in the 196 ethnically unrelated, unaffected control individuals investigated. This absence in the control group and the high association with the affected cohort therefore suggests that the high frequency of the C1490Y sequence variant in the SA STGD cohort is due to a founder effect. Currently, efforts are concentrated on acquiring more control samples to extend the frequency study in the SA background population. 
The 16 different disease-associated sequence variants identified together with the high association of C1490Y with STGD in SA and its absence in 392 control chromosomes suggests the presence of several ancestral haplotypes underlying STGD in SA and, possibly, several founder effects for the C1490Y in the SA cohort of subjects. Haplotype construction revealed 10 disease-associated haplotypes in 10 families. Of note, two disease-associated haplotypes were identified for the frequent C1490Y variant in three unrelated families with STGD, suggesting several founder effects for STGD in SA. Likewise, two STGD-associated haplotypes for the R602W variant were identified in two unrelated families. Recruitment of more subjects from SA families in whom the C1490Y variant has been identified will facilitate the interrogation of a founder effect for this mutation in the SA STGD cohort. 
In conclusion, in this study we sought to assess the mutation spectrum of the ABCA4 gene underlying STGD in SA and to determine whether there is a single or a few (if any) founder chromosomes in SA families with STGD. The study has illustrated that ABCA4 is the major gene underlying STGD in SA (>60% mutations identified). More important, there seems to be at least two different origins for the same “disease-predisposing” allele carrying the common C1490Y mutation in this study population. 
It is anticipated that improved identification and classification of ABCA4 sequence variants and subsequent clinical management of families will facilitate informed reproductive choices that could, in turn, decrease the burden of STGD in SA. 
Table 1.
 
List of 16 Different Potential Disease-Associated Sequence Variants Identified in 64 SA Subjects with arSTGD
Table 1.
 
List of 16 Different Potential Disease-Associated Sequence Variants Identified in 64 SA Subjects with arSTGD
Nucleotide Change Amino Acid Change Families (N = 64) Exon Reference
C454T R152X 4 5 3 33
G455A R152Q 1 5 35
C634T R212C 1 6 16 27
G768T (Splice donor) V256splice 5 6 15
C1885T R602W 6 13 9
2588G→C G863A 4 17 8
T3047C V989A 1 20 11
T4319C F1440S 1 29 9
G4328A* R1443H 1 29 This study
G4469A C1490Y 19 30 15 9
G5077A V16931 1 36 36
C6079T L2027F 8 44 8
C6088A R2030X 1 44 9 37
C6112T R2038W 2 44 5
IVS45+7G→A Splice donor 1 45 26
6352ΔA* Frameshift 1 46 This study
Table 2.
 
The 14 Non–Disease-Causing Sequence Variants Identified in the Study
Table 2.
 
The 14 Non–Disease-Causing Sequence Variants Identified in the Study
Nucleotide Change Amino Acid Change Families (n = 64)
G1009C E310Q 2
G989A G330D 1
IVS18-38ΔG 3
G2828A R943Q 6
C4184T P1395L 1
IVS27-71T→A 1
IVS28-38G→A 1
IVS38-10T→C 1
IVS39-17T→A 1
G5682C L1894L 1
TG5925CA L1984L 2
IVS43-16G→A 3
C6329T 12083I 1
G6355A E2119K 2
Table 3.
 
Ages of Onset Associated with the Potential Disease-Associated Mutations in 40 Individuals with arSTGD
Table 3.
 
Ages of Onset Associated with the Potential Disease-Associated Mutations in 40 Individuals with arSTGD
Identity No. AO (y) Phenotype Mutation 1 Mutation 2
224.1 9 STGD C1490Y R602W
170.2 10 STGD C1490Y R602W
241.1 9 STGD C1490Y 2588→C
448.1 20 STGD C1490Y 2588G→C
113.3 10 STGD C1490Y L2027F
209.1 18 STGD C1490Y L2027F
165.4 10 STGD C1490Y V256splice
166.3 27 STGD C1490Y R152X
151.4 5 STGD C1490Y ND
219.1 5 (rapid clinical progression was observed by 9 years) STGD C1490Y ND
223.1 9 STGD C1490Y ND
307.1 9 STGD C1490Y ND
319.3 9 STGD C1490Y ND
385.1 10 STGD C1490Y ND
226.1 10 STGD C1490Y ND
142.2 10 STGD C1490Y ND
273.1 11 STGD C1490Y ND
382.1 12 STGD C1490Y ND
449.1 14 STGD C1490Y ND
344.2 ND STGD C1490Y ND
374.1 10 STGD L2027F 6352ΔA, †
305.1 18 STGD L2027F R2038W
377.1 25 STGD L2027F R2038W
276.1 27 STGD L2027F R212C
204.4 8 STGD L2027F ND
135.4 13 STGD L2027F ND
446.1 9 STGD R602W ND
109.3 11 STGD R602W ND
110.7 13 STGD R602W ND
438.3 12 STGD R602W ND
123.1 9 STGD V256splice R152X
105.1* 10 STGD AND atypical RP V256splice R152X
24
129.3* 10 (rapid clinical progression was observed) STGD V256splice ND
163.22 10 STGD V256splice ND
173.1 8 STGD 2588G→C ND
9.4 27 STGD 2588G→C R152X
330.2 29 STGD R152Q V989A
372.1 31 STGD R1443H, † R2030X
141.3 11 STGD F1440S IVS45+7G→A (splice site mutation)
206.3 ND STGD V1693I ND
Table 4.
 
The 10 ABCA4 Disease-Associated Haplotypes Identified in the 10 STGD Families Investigated
Table 4.
 
The 10 ABCA4 Disease-Associated Haplotypes Identified in the 10 STGD Families Investigated
Family D1S188 Marker ABCA4 Mutation
D1S406 D1S236
166 14 6 12 C1490Y
170 15 4 9 C1490Y
151 15 4 9 C1490Y
204 9 5 14 L2027F
135 9 5 14 L2027F
105 8 4 14 V256splice
129 8 4 14 V256splice
170 10 5 12 R602W
110 16 6 3 R602W
9 7 5 14 2588G→C
9 16 5 4 R152X
105 16 5 4 R152X
166 16 5 4 R152X
141 8 3 6 F1440S
141 9 5 14 IVS45+7G→A
Figure 1.
 
Haplotype segregation analysis at the ABCA4 locus. Filled symbols represent individuals with diagnosed STGD. The markers reflect the physical position relative to the ABCA4 gene. Two haplotypes were identified for the C1490Y variant in three families: (a) 166, (b) 170, and (c) 151.
Figure 1.
 
Haplotype segregation analysis at the ABCA4 locus. Filled symbols represent individuals with diagnosed STGD. The markers reflect the physical position relative to the ABCA4 gene. Two haplotypes were identified for the C1490Y variant in three families: (a) 166, (b) 170, and (c) 151.
 
The authors thank the genetic nurse Sister Lecia Bartmann for visiting STGD family members and collecting blood specimens for DNA studies and to Ari Ziskind for his assistance in the clinical assessment of the study. 
Blacharski PA. Fundus flavimaculatus. Newsome DA eds. Retinal Dystrophies and Degenerations. 1988;135–159. Raven Press New York.
Anderson KL, Baird L, Lewis RA, et al. A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p. Am J Hum Genet. 1995;57:1351–1363. [PubMed]
Zhang K, Kniazeva M, Han M, et al. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat Genet. 2001;27:89–93. [PubMed]
Kaplan J, Gerber S, Larget-Piet D, et al. A gene for Stargardt’s disease (fundus flavimaculatus) maps to the short arm of chromosome 1. Nat Genet. 1993;5:308–311. [CrossRef] [PubMed]
Allikmets R, Singh N, Sun H, et al. A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet. 1997a;15:236–246. [CrossRef]
Martinez-Mir A, Paloma E, Allikmets R, et al. Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR. Nat Genet. 1998;18:11–12. [CrossRef] [PubMed]
Cremers FP, van de Pol DJ, van Driel M, et al. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt’s disease gene ABCR. Hum Mol Genet. 1998;7:355–362. [CrossRef] [PubMed]
Allikmets R, Shroyer NF, Singh N, et al. Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science. 1997;277:1805–1807. [CrossRef] [PubMed]
Lewis RA, Shroyer NF, Singh N, et al. Genotype/Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am J Hum Genet. 1999;64:422–434. [CrossRef] [PubMed]
Rivera A, White K, Stohr H, et al. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am J Hum Genet. 2000;67:800–813. [CrossRef] [PubMed]
Briggs CE, Rucinski D, Rosenfeld PJ, Hirose T, Berson EL, Dryja TP. Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration. Invest Ophthalmol Vis Sci. 2001;42:2229–2236. [PubMed]
Gerth C, Andrassi-Darida M, Bock M, Preising MN, Weber BH, Lorenz B. Phenotypes of 16 Stargardt macular dystrophy/fundus flavimaculatus patients with known ABCA4 mutations and evaluation of genotype-phenotype correlation. Graefes Arch Clin Exp Ophthalmol. 2002;240:628–638. [CrossRef] [PubMed]
Yatsenko AN, Shroyer NF, Lewis RA, Lupski JR. Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4). Hum Genet. 2001;108:346–355. [CrossRef] [PubMed]
Shroyer NF, Lewis RA, Yatsenko AN, Wensel TG, Lupski JR. Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that manifest both Stargardt disease and age-related macular degeneration. Hum Mol Genet. 2001;10:2671–2678. [CrossRef] [PubMed]
Maugeri A, van Driel MA, van de Pol DJ, et al. The 2588G→C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am J Hum Genet. 1999;64:1024–1035. [CrossRef] [PubMed]
Gerber S, Rozet JM, van de Pol TJ, et al. Complete exon-intron structure of the retina-specific ATP binding transporter gene (ABCR) allows the identification of novel mutations underlying Stargardt disease. Genomics. 1998;48:139–142. [CrossRef] [PubMed]
Liechti-Gallati S, Schneider V, Neeser D, Kraemer R. Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease. Eur J Hum Genet. 1999;7:590–598. [CrossRef] [PubMed]
Spritz RA, Holmes SA, Ramesar R, Greenberg J, Curtis D, Beighton P. Mutations of the KIT (mast/stem cell growth factor receptor) proto-oncogene account for a continuous range of phenotypes in human piebaldism. Am J Hum Genet. 1992;51:1058–1065. [PubMed]
Greenberg J, Rebello G, Ramesar R. Ophthalmic genetics: a review of the molecular genetics of familial retinal dystrophies in Southern Africa. Special Med. 1999;21:109–112.
Ramesar R, September A, Rebello G, Greenberg J, Goliath R. Migratory history of populations and its use in determining research direction for retinal degenerative disorders. Anderson RE La Vail MM Hollyfield JGet al eds. New Insights into retinal degenerative diseases. 2001;335–338. Kluwer Academic Plenum New York.
Beighton P. Sclerosteosis. J Med Genet. 1988;25:200–203. [CrossRef] [PubMed]
de Jager T, Corbett CH, Badenhorst JC, Brink PA, Corfield VA. Evidence of a long QT founder gene with varying phenotypic expression in South African families. J Med Genet. 1996;33:567–573. [CrossRef] [PubMed]
Defesche JC, Van Diermen DE, Hayden MR, Kastelein JP. Origin and migration of an Afrikaner founder mutation FHAfrikaner-2 (V408M) causing familial hypercholesterolemia. Gene Geogr. 1996;10:1–10. [PubMed]
Warnich L, Kotze MJ, Groenewald IM, et al. Identification of three mutations and associated haplotypes in the protoporphyrinogen oxidase gene in South African families with variegate porphyria. Hum Mol Genet. 1996;5:981–984. [CrossRef] [PubMed]
Tipping AJ, Pearson T, Morgan NV, et al. Molecular and genealogical evidence for a founder effect in Fanconi anemia families of the Afrikaner population of South Africa. Proc Natl Acad Sci USA. 2001;98:5734–5739. [CrossRef] [PubMed]
Papaioannou M, Ocaka L, Bessant D, et al. An analysis of ABCR mutations in British patients with recessive retinal dystrophies. Invest Ophthalmol Vis Sci. 2000;41:16–19. [PubMed]
Rozet JM, Gerber S, Souied E, et al. Spectrum of ABCR gene mutations in autosomal recessive macular dystrophies. Eur J Hum Genet. 1998;6:291–295. [CrossRef] [PubMed]
Simonelli F, Testa F, de Crecchio G, et al. New ABCR mutations and clinical phenotype in Italian patients with Stargardt disease. Invest Ophthalmol Vis Sci. 2000;41:892–897. [PubMed]
Glazer LC, Dryja TP. Understanding the etiology of Stargardt’s disease. Ophthalmol Clin North Am. 2002;15:93–100. [CrossRef] [PubMed]
Koenekoop RK. The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review. Ophthalmic Genet. 2003;25:75–80.
Maugeri A, Flothmann K, Hemmrich N. The ABCA4 2588G→C Stargardt mutation: single origin and increasing frequency from South-West to North-East Europe. Eur J Hum Genet. 2002;10:197–203. [CrossRef] [PubMed]
Fukui T, Yamamoto S, Nakano K, et al. ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2002;43:2819–2824. [PubMed]
Souied EH, Ducroq D, Rozet JM, et al. A novel ABCR nonsense mutation responsible for late-onset fundus flavimaculatus. Invest Ophthalmol Vis Sci. 1999;40:2740–2744. [PubMed]
Peelman F, Labeur C, Vanloo B, et al. Characterization of the ABCA transporter subfamily: identification of prokaryotic and eukaryotic members, phylogeny and topology. J Mol Biol. 2003;325:259–274. [CrossRef] [PubMed]
Fumagalli A, Ferrari M, Soriani N, et al. Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients. Hum Genet. 2001;109:326–338. [CrossRef] [PubMed]
Webster AR, Heon E, Lotery AJ, et al. An analysis of allelic variation in the ABCA4 gene. Invest Ophthalmol Vis Sci. 2001;42:1179–1189. [PubMed]
Fishman GA, Stone EM, Grover S, Derlacki DJ, Haines HL, Hockey RR. Variation of clinical expression in patients with Stargardt dystrophy and sequence variations in the ABCR gene. Arch Ophthalmol. 1999l;117:504–510. [CrossRef]
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