Application A the gene array for ABCA4associated retinal dystrophies

Several laboratories independently described ABCA4 (ABCR) in 1997 as the causal gene for Stargardt disease (STGD1, MIM 248200) (6-8). STGD1 is usually a juvenile-onset macular dystrophy associated with rapid central visual impairment, progressive bilateral atrophy of the foveal retinal





Figure 7.1.

Principle of APEX. (A) Oligonucleotides are arrayed on a glass slide via their 5' end;

(B) complementary fragment of PCR-amplified sample DNA is annealed to oligos;

(C) sequence-specific single nucleotide extension of the 3' ends of primers with dye-labeled nucleotide analogs (ddNTPs) by DNA polymerase; (D) sample DNA fragments and not incorporated ddNTPs are washed off followed by signal detection. The dye-labeled nucleotide, T (shown in bold), bound to the oligonucleotide on the slide is the nucleotide being typed.

3322 C>T Relative

(Arg1108Cys) intensities A C G T

3322 C>T Relative

(Arg1108Cys) intensities A C G T

Figure 7.2.

Three possible different genotypes of the same ABCA4 variant on the ABCR400 chip detected by Genoramaâ„¢ Basecaller genotyping software. The software compares fluorescence intensities (shown as bars in the second cell from left) of four different labels in each spot pair and translates them into the presence or absence of nucleotide(s) in the given position on the array. Every position is queried from both strands, the nucleotide(s) in sense and antisense strand appear as duplicate spots in the upper and lower row of the software window, respectively.

pigment epithelium, and the frequent appearance of yellowish flecks around the macula and/or in the central and near-peripheral areas of the retina. Subsequently, ABCA4 mutations were identified and co-segregated with retinal dystrophies of substantially different phenotypes, such as autosomal recessive cone-rod dystrophy (arCRD) (9, 10) and atypical autosomal recessive retinitis pigmentosa (arRP, RP19) (9, 11, 12).

Disease-associated ABCA4 alleles have shown an extraordinary heterogeneity (6, 13-17). Currently over 450 disease-associated ABCA4 variants have been identified (R. Allikmets and J. Zernant, unpublished data), allowing comparison of this gene to one of the best-known members of the ABC superfamily, CFTR, encoding the cystic fibrosis transmembrane conductance regulator (18). What makes ABCA4 a more difficult diagnostic target than CFTR is that the most frequent disease-associated ABCA4 alleles, for example G1961E, G863A/delG863, and A1038V, have each been described in only around 10% of STGD patients in a distinct population, whereas the delF508 allele of CFTR accounts for close to 70% of all cystic fibrosis alleles (19).

Allelic heterogeneity has substantially complicated genetic analyses of ABCA4-associated retinal disease. Even in the case of STGD1, where the role of the ABCA4 gene is indisputable, the mutation detection rate has ranged from around 25% (15, 20) to around 55-60% (13, 14, 17, 21). In each of these studies, conventional mutation detection techniques such as single strand conformational polymorphism (SSCP), heteroduplex analysis, and denaturing gradient gel electrophoresis (DGGE) were applied. Direct sequencing, which is still considered the 'gold standard' of all mutation detection techniques, enabled a somewhat higher percentage of disease-associated alleles to be identified, from 66% to 80% (22, 23).

To overcome these challenges and to generate a high-throughput, cost-effective screening tool, we developed the ABCA4 genotyping microarray (24). By systematic analysis of all published, reported, and communicated data, we compiled the most comprehensive database of ABCA4 variants, where only those sequence changes currently considered disease-associated exceed 400. By design, we included on this chip all variants from the coding region of ABCA4 and adjacent intronic sequences. The overall efficiency of the array was enhanced by designing primers with mismatched or modified bases for several variants where ABCA4 sequence presented additional challenges, that is hairpin loops, repeats, etc. Currently, from more than 400 variants only three (<1%) remain undetected by the last version of the chip; 93% of all variants are detected from both strands, whereas around 7% are detected reliably from one strand.

The array was validated on an extensive cohort of 136 confirmed STGD samples, which we had previously screened by SSCP and/or heteroduplex analyses (13). The initial SSCP screening had detected 55% of all disease-associated alleles. The microarray screening detected numerous additional alleles, bringing the total to more than 70% of all disease-associated alleles (24). Further evaluation of the ABCR400 array by screening several previously not analyzed STGD patient cohorts of diverse ethnicity (European American, Italian, Dutch, Hungarian and Slovenian) is summarized as Table 7.1. The screening efficiency of the ABCR400 microarray was remarkably similar in all six cohorts, yielding from around 53% to 60% of all possible disease-associated ABCA4 alleles (Table 7.1).

Table 7.1. Screening efficiency of the ABCR400 array on several cohorts

STGD patient






alleles (%)


North America 1


158 (52.7%)

2-34% 1-39% 0-27%



34 (54.8%)

2-33% 1-45% 0-22%



40 (55.6%)

2-47% 1-18% 0-35%

North America 2


24 (60%)

2-45% 1-30% 0-25%

The Netherlands


20 (55.6%)

2-39% 1-33% 0-28%



15 (53.6%)

2-29% 1-50% 0-21%

a Shows the percentage of the screened patients with both disease-alleles found (2), with one allele found (1) or no alleles found (0).

a Shows the percentage of the screened patients with both disease-alleles found (2), with one allele found (1) or no alleles found (0).

The allele distribution was also similar across all cohorts (Table 7.1, last column). We detected both disease-associated alleles in between 29% and 45% patients (average 36.6%). The fraction of patients with no apparent STGD alleles detected ranged from 21% to 35% (average 26.4%), most likely indicating inclusion of phenocopies, which cannot be avoided completely due to the selection methods. More robust results were obtained on smaller, carefully characterized, cohorts derived from a single clinical source (i.e., Italian), or cohorts with less allelic heterogeneity (Hungarian).

In summary, the ABCR400 array alone determined 55-65% of all possible disease-associated ABCA4 alleles and, in combination with SSCP analysis, 70-78% of disease-associated alleleles in random cohorts of Stargardt disease patients. These results suggest that: (i) the ABCAY400 array is an efficient screening tool for known variants; and (ii) its efficiency for screening patient populations with STGD is comparable to direct sequencing. The ABCA4 array supplies two major applications: (i) (pre-)screening of all patients with suspected ABCA4-associated retinal pathology; including diagnostic screening of patients with Stargardt disease and cone-rod dystrophy; and (ii) high throughput, cost-efficient, and single-standard screening of large cohorts in case-control association studies, for example, for the AMD complex trait.

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