|Molecular Vision 2007;
Received 5 April 2007 | Accepted 28 September 2007 | Published 17 October 2007
N965S is a common ABCA4 variant in Stargardt-related retinopathies in the Danish population
Thomas Rosenberg,1 Flemming
Klie,1 Peter Garred,2 Marianne Schwartz3
1Gordon Norrie Center for Genetic Eye Diseases, National Eye Clinic for the Visually Impaired, Kennedy Center, Hellerup, Denmark; 2Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Denmark; 3Department of Clinical Genetics, Rigshospitalet, Copenhagen University Hospital, Denmark
Correspondence to: Thomas Rosenberg, Gordon Norrie Center for Genetic Eye Diseases, National Eye Clinic for the Visually Impaired 1 Rymarksvej, DK-2900 Hellerup, Denmark; Phone: +45 3945 2422; FAX: +45 3945 2420; email: email@example.com
Purpose: The study was conducted to resolve the spectrum of ABCA4 mutations in a cohort of unrelated Danish residents with early-onset macular dystrophy.
Methods: A microarray technique was used to analyze known ABCA4 mutations in genomic DNA from a selected group of 161 unrelated individuals referred to the national low vision clinic. The clinical observation time varied from a single examination to follow-up over 35 years.
Results: Fifty-nine allegedly disease-associated ABCA4 variants were found in 197 alleles (61.2%) from 124 (77.0%) patients. Two or three mutations were present in 73 (45.3%) patients, and only one mutation was found in 51 (31.7%) patients. The mutation spectrum included 45 missense mutations, five nonsense mutations, two frame shift deletions, and seven splice site mutations. The relative frequency among the mutations varied considerably. Twenty-eight mutations occurred only once among 197 alleles, while the five most abundant mutations were encountered in 50% of the mutation-carrying alleles. The rate of mutation detection, assessed as the fraction of individuals carrying at least one ABCA4 mutation, varied from 27% to 90% among seven phenotypic groups, and a single mutation, p.N965S (c.2894A>G) in the first nucleotide-binding domain accounted for 16.2% of 197 disease-associated alleles. The mutation causes moderate to serious phenotypes and eventually blindness.
Conclusions: Our study is the first mutation analysis of Stargardt-related retinopathies in a large cohort of patients from a Scandinavian population. The mutation detection rate, performed with an array-based technique, was comparable to that of other microarray-based ABCA4 studies as well as studies using more laborious techniques involving screening methods followed by sequencing. Four out of five of the most prevalent ABCA4 mutations are reported to be frequent in other Western European populations as well. However, the prevalence of the most common Danish mutation, N965S, significantly deviates from the one found in other studies. This underscores that the ABCA4 mutation spectrum within relatively stable populations might be skewed due to founder effects. The clinical spectrum of patients, who are either homozygous or compound heterozygous for the N965S mutation, indicates that this mutation has an early and profound effect on retinal function.
Autosomal recessive Stargardt disease (OMIM 248200) and fundus flavimaculatus are allelic phenotypes within the spectrum of ABCA4 retinopathies and constitute the most frequent early-onset macular dystrophies leading to irreversible loss of central vision. Since the cloning of the ABCA4 gene (originally named ABCR) , the phenotypic spectrum has expanded to include autosomal recessive cone-rod dystrophies and a small fraction of retinitis pigmentosa (RP19) [2-5], which implies additional involvement of the peripheral vision and ultimately may lead to total blindness. Furthermore, certain ABCA4 variants have also been associated with age-related macular degeneration (AMD) [6,7].
ABCA4 encodes a retinal specific ATP-binding transporter protein located at the rim of the photoreceptor discs in rod and cone outer segments [8,9]. It is composed of two homologous N and C terminal halves. Each contains three transmembrane domains and a cytosolic nucleotide binding domain (NBD) with a central, conserved, and highly hydrophobic domain, a lumenal loop near the amino terminus is situated between the first and second transmembrane domains . The ABCA4 protein facilitates the transport of the intradiscal photobleached rhodopsin product all-trans-retinal across the disc membrane. This action supports the regeneration of 11-cis-retinal in the retinoid cycle. The impediment of this transport is hypothesized to result in the accumulation of toxic retinoic substances in the discs, leading to the formation of lipofuscin followed by phagocytosis by the retinal pigment epithelium (RPE), and the eventual apoptosis of RPE and photoreceptor cells .
The ABCA4 gene spans 50 exons. Conventional screening methods leave about 40% disease-causing variants undetected , which together with a wide spectrum of possibly pathogenic variants and many common and rare polymorphisms, complicates the interpretation of the clinical relevance of sequencing results. With the introduction of a micro-array mutation detection technique, including known ABCA4 variants, a cost-effective mutation screening has been developed; however, it does not detect novel mutations . The Human Gene Mutation Database enumerates a total of 477 genetic variations. The majority (343) of these mutations are single nucleotide substitutions (missense or nonsense) while more than 100 mutations are evenly distributed between splice mutations and small deletions. Some of these mutations show a wide distribution among many populations. Others are enriched in certain areas due to suggested founder effects [12-17]. The majority of the reported variations are only present in a limited number of cases and in numerous compound heterozygous combinations, which has impeded the recognition of firm genotype-phenotype relations. Further difficulties are associated with a relatively high rate of undetected mutations and the abundant presence of polymorphisms with a possible influence on phenotype, which hypothetically in part may be due to the action in cis of some of the unknown mutations. Stargardt-related retinopathies show an extreme phenotypic diversity with regard to morphology, age of appearance and the life-long impact on visual functions. Nevertheless, a model for genotype-phenotype correlations based on a simple grading of sequence variants into three categories according to disease severity and suggested residual ABCA4 protein activity has proved to be a useful tool in the prognostic counseling of certain patients with ABCA4-related retinopathies [18-22]. Some experimental data supports this classification. Sun et al. measured in vitro protein yield, ATP binding, and all-trans retinal stimulated ATPase activity of 33 common recombinant missense ABCA4 mutations , and found differential effects among the variants. Some mutations (two in-frame deletions and G851D and G1886E introducing charged amino acids into predicted transmembrane domains) resulted in severely reduced amounts of protein. Abolished ATP binding among variants expressed with normal or nearly normal yield occurred with some mutations within either of the two NBDs (T971N, L1971R, G1977S, and E2096K). Others, such as the common c.2588G>C variant, revealed both reduced yield and reduced ATP binding. Examination of mutations according to topography uncovered distinctive differences in the retinal-stimulated ATP hydrolysis. Low activity was observed for S206R and L541P in the predicted lumenal domain, W1408R and T1526M in the cytosolic loop connecting the two homologous halves of ABCA4, and N965S, T971N, A1038V, G1961E, L1971R, G1977S, and E2096K in the two NBDs. Functional analysis of selected missense mutations were performed in vivo by Wiszniewski and collaborators who used the tadpole models to demonstrate retention of the gene product in the photoreceptor inner segment, which resulted in the total absence of physiological protein function . The mutation spectrum in Scandinavian patients with Stargardt-related retinopathies has not been reported before. However, large emigration waves between 100 and 150 years ago would predict a certain overlap of genotypes between Scandinavians and Americans of European descent.
Study participants were recruited from individuals who were referred to the Danish National Eye Clinic (NEC) for diagnosis, rehabilitation, and counseling. The recruitment period covered a period of 20 years during which examination techniques varied. Some of the patients were followed for 20-35 years, while others were seen only once. All patients underwent a standard ophthalmological examination supplemented by fundus photography, color vision assessment, and analysis for dark adaptation. A full-field electroretinogram (ERG) was performed on most of the participants. The medical files and retinal photographs of the participants were scrutinized in order to obtain further manageable measures to characterize the clinical picture. Data on age of onset were recorded. The clinical classification used in this study (Table 1) was mainly based on Fishman's staging  but extended to cover five groups and two subgroups enclosing the whole spectrum of Stargardt- and ABCA4-related "juvenile" retinopathies including cone-rod dystrophies and unclassified cases of macular lesions (bull's eye and other central RPE dystrophies; Table 1). The patients were grouped according to the clinical picture at the latest examination. Blood samples were collected for DNA banking for the explicit purpose of mutation screening when this possibility arose. After the finding of the ABCA4 gene patients previously diagnosed at NEC were asked to participate in a planned screening project. DNA from 161 apparently unrelated patients were selected for study. All patients gave their signed consent to participate in the study, which adhered to the tenets of the Helsinki Declaration. DNA was extracted by conventional salting-out and stored in freezers at -80 °C. A pilot study included DNA from 25 patients with disease groups 1 and 2 (Table 1). The samples were screened by single strand conformation polymorphism analysis (SSCP) followed by direct sequencing of exons of interest. From November 2002 through January 2003 200 samples from families with Stargardt-related retinopathies and controls with atypical RP underwent mutation screening on a commercial basis with a recently developed micro-array technique (Asper Biotech AS, Tartu, Estonia) , which at that time included 386 ABCA4 alleles. The results were not controlled with other techniques. In 2004, however, 52 samples were re-analyzed in a second microarray assay, yielding results, that were identical to those from the first assay. Screening for N965S and c.1356+5delG was performed using the "primer-by-design" approach from ABI and the Taq-man 7000 Sequence Detection System, and by following the conditions recommended by the manufacturer. Linkage disequilibrium was estimated as D' using the SNPAlyze program version 4.2 (Dynacom, Yokohama, Japan).
Our studies detected 59 allegedly disease-causing ABCA4 variants in 124 of the 161 individuals (77.0%) and in 197/322 alleles (61.2%). Table 2 shows the number of detected mutations per individual and the assumed number of disease-associated alleles. Two "complex" alleles L541P/A1038V and c.5714+5G>A/E1885K were identified by offspring analyses. Table 3 lists all ascertained potential pathogenic variants and the number of disease-associated alleles. Most alleles carried from a few to several polymorphisms in association with many of the disease-associated variants. Some of the most frequent polymorphisms are shown in Table 4. In Table 5 the age of onset was related to the phenotypes, and the number and relative frequency of mutation detection was shown for subjects and alleles within each phenotype in Table 6. The mutation N965S was found in 28 patients (4 homozygous) and was the most common mutation among 161 unrelated patients. ABCA4 variations in the second allele among the 28 patients were related to phenotype and age of onset in Table 7. N965S was in strong linkage disequilibrium (LD) with polymorphism c.1356+5delG. The c.1356+5delG polymorphism was found on 20% of normal chromosomes (n=96) in controls, compared to 57% in patients with at least one N965S chromosome. Paired LD coefficient (D') for these two loci was found to be -0.8854, p=4.04E-7. The clinical appearances and disease courses of four patients homozygous for N965S are summarized as follows and fundus pictures are presented in Figure 1.
Case D065 (Phenotype 5a, Table 1, Figure 1A,B) was a male born in 1982 and the single child of unrelated parents. Reduced visual acuity (VA) was observed when he was 13 years old. Ophthalmic examination a year later showed a Snellen visual acuity of 0.08 on his right eye and 0.05 on his left eye. He also had a central ill-demarcated grainy pigment atrophy with a bull's eye pattern and peripheral involvement consisting of branching pigment epithelium atrophy and small rounded atrophic spots with a deeply situated punctiform central hyperpigmentation. No yellow flecks were present. The dark adaptation showed a delayed time course with a slightly elevated final threshold, Goldmann-perimetry with object size IV/4e and II/4e demonstrated normal outer limits. Ganzfeld-ERG revealed equally subnormal rod and cone responses. His last follow-up examination was performed when he was 18 years old. At that time, his fundus showed diffuse atrophic macular changes with accentuation in the periphery and large atrophic splotches as well as punctuate hyperpigmentations. The vessel diameter, however, was only slightly attenuated.
Case D137 (Phenotype 3b, Table 1, Figure 1C) was a male born in 1943 and who was the only affected child of unrelated parents. Visual problems were noticed when he was around 17-18 years old. Our first examination took place in 1968, when he was 25 years old. He reported that objects suddenly "disappeared" while during office work. His VA was 0.5 on both eyes, and his visual field-measurements showed central scotomas measuring 10 ° with preservation of the central 1-2 °. Color vision tests revealed a strong acquired-type defect. Dark-adaptometry showed a normal biphasic curve, and the dark-adapted ERG had low-to-normal b-wave amplitudes. The macular regions were described as atrophic with large aggregates of pigment. His retinal vessels were narrowed, and there were abundant yellow-gray spots occupying the peripheral fundus. Over the next 15 years, his visual function deteriorated and by 1983 his VA had dropped to 0.05. The dark adaptometry had a reduced slope and a 2 log-unit elevation of the final threshold and the central scotomas had enlarged with a break through to the periphery. In 1991, the ERG showed nearly extinct rod responses and severely reduced cone-b-wave amplitudes with prolonged implicit time. The last examination took place in 2002 when the patient was 59 years old. His VA was now 0.01 and Goldmann-perimetry with a large-size object showed a preserved low peripheral remnant on his right eye and a 50 ° scotoma on his left eye. The funduscopic examination revealed a profound, central, ill demarcated atrophy of the pigment epithelium and choriocapillary layer with large, irregular pigment aggregates. Beyond the vascular arcades, there were abundant irregular pigment epithelium atrophy, small atrophic spots, and round as well as perivascular hyperpigmentations. Universal vascular constriction completed the picture of a pan-choroidoretinal dystrophy.
Case D174 (Phenotype 4, Table 1, Figure 1D) was a male born in 1954. He was one of three children from a marriage between first-degree cousins. His siblings had normal vision. His visual difficulties started at the age of 13 years. When he was 16 years old, he underwent eye-examination elsewhere, which revealed VA of 0.1 in his right eye and 0.125 in his left eye. The macular region showed conspicuous pigment disturbances. From 1970 he was followed at NEC. Despite increasing visual problems his central vision remained stable for a period of 10 years. His last eye examination took place when he was 32 years old. His VA had dropped to 0.06 in the right eye and 0.08 in the left eye. Color vision tests revealed he had a strong unspecified red-green defect. A substantial diffuse pigment epithelial atrophy occupied the macular regions with interspersed coarse ill-defined hyperpigmentations. The vascular tree showed no gross constriction despite a diffuse atrophy of the pigment epithelium occupying the mid-periphery with small irregular hyperpigmentations. No yellow spots were present. The dark-adaption curve was diphasic with a delayed breakpoint and a slowed rod-phase with a 1- log elevation of the final threshold. Normal Goldmann visual fields were measured, and a Ganzfeld ERG showed a cone-rod dystrophy-pattern with severely reduced cone-amplitudes and prolonged cone-implicit times. The rod-b-waves were moderately reduced.
Case D199 (Phenotype 2, Table 1, Figure 1E,F) was a male born in 1970. When he was 15 years old, it was noted he had a reduced reading ability. Our first examination took place the following year. His VA was 0.05 on the right eye and 0.3 on the left eye. Numerous flavimaculatus spots were present in the peripheral fundus outside the vascular arcades and nasal to the optic discs and fine pigment granularity was present in both foveas. Besides small central scotomas with a small Goldmann object the visual fields appeared normal. Color vision tests revealed an unspecific acquired-type defect, and dark-adaptometry showed a diphasic curve with a delayed and slowed rod-phase and a slightly elevated final threshold. The ERG rod-responses had normal amplitudes while the cone b-wave amplitude were low normal with upper borderline implicit times. At his last examination the patient was 22 years old, and his VA had dropped to 0.05 on both eyes with eccentric fixation. The central scotomas were considerably enlarged, and the ERG presented with low normal rod- and cone amplitudes. The funduscopic changes were still most accentuated centrally with extensive atrophy and hyperpigmentation. The flavimaculatus elements were in transition of becoming pigment atrophies and singular small rounded atrophies with punctuate hyperpigmentations had appeared.
The extreme phenotypic variability of Stargardt-related retinopathies with regard to age of onset, lesion type and clinical course remains a standing challenge. Common clinical appearances early in the disease course may later in life turn into different phenotypes, making prognostic predictions unreliable at a time during which prognostic counseling is most needed. Any clinical classification of Stargardt-related retinopathies is deemed imperfect due to atypical cases and overlap between the groups. The classification used in this study (Table 1) is no exception but has been useful as a practical means of describing the phenotypic variation in a clinical setting. Furthermore a broad concept of the disease group is an essential basis for establishing potential genotype-phenotype relations. The term flavimaculatus is often used to designate both centrifugal and centripetal forms, which among others are clearly separated by differences in age of diagnosis and might represent allelic disparity. Likewise the cone-rod dystrophy group defined by different clinical centers comprises both primary photoreceptor dystrophies and choroidoretinal dystrophies with early macular involvement. The main obstacle for a relevant classification is, however, the progressive nature of the disorders, which ideally demands several decennia of follow-up to determine the final visual prognosis [24,25]. On this background, interpretation of the effect of specific mutations has considerable and immediate clinical corollaries. Screening for ABCA4 variants based on arrayed primer extension technology has proved successful in a number of studies [15,17,23,26], yielding detection rates, that are compatible with those of more laborious techniques e.g. SSCP, DGGE, or dHPLC followed by sequencing. The present investigation is no exception showing rather high detection rates between 70-90% in patients within the Fishman groups I-III  who are carrying at least one disease associated allele (Table 6). At the same time the results indicate that Stargardt-related retinopathies may be identified with a quite high precision in a specialized setting. It is also noteworthy that 67% of the patients with cone-rod dystrophies carried at least one ABCA4 mutation. This is in accordance with other studies concluding that ABCA4 mutations are major disease-causing factors in cone-rod dystrophies [27-29]. Even patients with the descriptive term, bull's eye retinopathy, who had normal ERG had presumed disease-associated variants in 60% of the cases while macular pigment atrophy without distinctive morphological characteristics had low nosological specificity showing ABCA4 mutations in only a third of the cases. Depending on the diagnostic group, 9-59% of the 161 patients (45.3% in average) had mutations in both alleles and a single disease associated allele was ascertained in 31.7% reflecting a deficit, which is common to all published studies and likely should be explained by unidentified ABCA4 mutations. In 23 among 132 patients (17%) with a high diagnostic precision (our groups 1-3) no disease-carrying alleles were identified. Taking into account mutations in cis and undetected mutations in linkage disequilibrium with known mutations, up to 40% of present ABCA4 mutations have yet to be discovered. The full list of identified disease-associated variants (Table 3) enumerates 59 simple and complex mutations, the majority of which are missense mutations that repeatedly have been demonstrated in other materials from Europe and the United States. In a number of cases the pathogenicity has been substantiated by experimental evidence [10,23]. Three of the most common ABCA4 variants in Europe, G863A/G863del (c.2588G>C), L541P/A1038V, and G1961E were also among the most prevalent mutations in this material (Table 3). Maugeri et al.  analyzed the prevalence and significance of c.2588G>C and showed that this variant has two effects, p.G863A and p.delG863, both accounting for 50%. The c.2588G>C is a mild European founder mutation occurring with the highest relative frequencies in the German (31.3%) and Dutch (31.9%) populations. However, in a later study on German patients a much lower relative share (10.2%) was encountered . For comparison the 30 alleles encountered in our material represents 14.4% of the identified 197 mutation-carrying alleles. This may reflect the close relationship between the two populations studied. Interestingly, two patients with a flavimaculatus phenotype were homozygous for c.2588G>C, which is contradictory to the allegedly mild effect of the mutation. Homozygosity for c.2588G>C is rare. Maugeri et al. concluded that it is a mild mutation, causing disease only in a compound heterozygous combination with a severe allele. It has also been suggested that c.2588G>C, also found in normal controls, may be a benign variant in linkage disequilibrium with another, yet to be identified and more serious disease-associated variant . However, these authors sequenced the entire ABCA4 in two patients compound heterozygous for c.2588G>C and another ABCA4 mutation, and were not able to detect other mutations. This is supported by the fact that c.2588G>C, which mainly has been implicated in the Stargardt phenotype in our study also occurred in compound heterozygous associations with missense mutations R219T and N965S in patients exhibiting the more severe flavimaculatus phenotypes. Moreover, accompanying polymorphisms indicated at least two different haplotype backgrounds. All c.2588G>C were found in association with R943Q but in the majority of cases also with the polymorphic variants N1868I, L1894L, and P1401P. The complex allele L541P/A1038 V and G1961E were ascertained among 6.7% and 5.8% of the disease-associated alleles. This is considerably lower than the figures from the neighboring German population (12.7% and 20.5%, respectively) . The variant c.5461-10C>T (IVS38-10C>T) is a splice acceptor variant and apparently has no effect on splicing , but the frequency in Stargardt patients as compared to unaffected controls is highly statistically significant . The c.5461-10C>T is strongly believed to be in linkage disequilibrium with a, still, unknown mutation. The variant c.5461-10C>T has been listed among the disease-associated variants (Table 3) and was found among 6.6%. The most significant finding in our material is the high frequency of the N965S variant detected in 32/197 (16.2%) of the alleles with identified mutations. N965S is virtually absent in several large Continental and British materials [14,15,17,19,30,31], and it is only found sporadically in studies in the American population [11,20,24]. N965S was identified in 28 patients among the 161 unrelated probands and in an additional 15 family members. It was also found to be in strong linkage disequilibrium with the polymorphism c.1356+5delG, which in addition to the geographic accumulation indicates that N965S is a founder mutation of Danish origin. The high prevalence of N965S offered an excellent opportunity to make genotype-phenotype comparisons as summarized in Table 7. Most N965S-carrying individuals had a relative early disease-onset with an average of 15.5 years. The age of onset for four patients homozygous for N965S was in their teens. Due to conspicuous macular atrophy with coarse pigment aggregates, these patients developed progressive visual deterioration resulting in blindness (VA 0.05 or lower) after ten to 20 years. Concomitantly a widespread peripheral dystrophy - with or without a passing stage of flavimaculatus elements - occurred, leading to a diffuse choroidoretinal dystrophy (Figure 1). Functional tests showed acquired color vision defects, slowed dark adaptation, and reduced ERG amplitudes. With reservation for undetected disease-causing ABCA4 variants our finding underscores that N965S is a mutation with a medium to severe impact on phenotype due to impairment of ATP hydrolysis by the mutant ABCA4 protein .
This study was supported by grants from the Danish Eye Research Foundation (Oejenfonden) and The Eye Protection Society (Vaern om synet). We thank Soren Norby, M.D. for kindly revising the manuscript.
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