All patients were screened for genetic variation in the
ABCA4 gene with the ABCR400 microarray,
26 which includes all currently known (>450) disease-associated variants, followed by validation by direct sequencing. The
ABCA4 genotyping microarray enables simultaneous detection of all known
ABCA4 variants in one reaction. The array utilizes the arrayed primer extension (APEX) technology and is constructed by synthesis and application of sequence-specific oligonucleotides for every known
ABCA4 allele. For template preparation, all exons of the
ABCA4 gene were PCR amplified, as described previously.
27 In the amplification mixture, 20% of the dTTP was replaced with dUTP.
27 The amplification products were concentrated and purified with a PCR kit (MIPSpin PCR kit; Genotein, Inc., Seoul, Korea). Fragmentation of amplification products was achieved by adding thermolabile uracil
N-glycosylase (Epicentre Biotechnologies, Madison, WI) and the following heat treatment.
27 For the APEX-based genotyping, one third of every amplification product was used in the primer extension reaction on the ABCR400 microarray. Each APEX reaction consisted of a fragmented and denatured PCR product, 4 units of DNA polymerase (ThermoSequenase; GE Healthcare, Amersham, UK), 1× reaction buffer, and a 1.4-μM final concentration of each fluorescently labeled ddNTP: Texas red-ddATP, fluorescein-ddGTP (GE Healthcare), Cy3-ddCTP, and Cy5-ddUTP (NEN, Boston, MA). The reaction mixture was applied to the ABCR400 microarray slide for 15 minutes at 58°C, and the reaction was stopped by washing the slide at 95°C in distilled water
28 (Milli-Q; Millipore, Bedford, MA). The slides were imaged (Genorama QuattroImager; Asper Biotech, Ltd., Tartu, Estonia), and the
ABCA4 sequence variants were identified by the accompanying software
28 (Genorama Genotyping Software; Asper Biotech, Ltd.). Array-identified variants were confirmed by direct sequencing with a dye-termination cycle sequencing kit (
Taq Dyedeoxy Terminator Cycle Sequencing; Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Sequencing reactions were resolved on an automated sequencer (model 377; Applied Biosystems, Inc.). Segregation analysis was performed in all families, to determine the phase for all mutations.