Figure 3: Protein yield and ATP-binding capacity of 37 naturally occurring ABCR variants produced in transiently transfected 293 cells.Membranes were analysed by immunoblotting with affinity-purified anti-ABCR antibodies (top) and photoaffinity labelling with α-32P azido-ATP (bottom). We loaded 1 μg (immunoblotting) or 2.5 μg (azido-ATP labelling) of total membrane protein, as determined by Bradford assay, per track. The mutations that reside in NBD-1 and NBD-2 are indicated above the corresponding lanes. The relative levels of the different variant proteins and the extent of azido-ATP labelling were observed to be highly reproducible in multiple independent experiments. ABCR (large arrowhead); an endogenous 55-kD protein (small arrowhead) serves as an internal control for azido-ATP labelling. Molecular mass standards are shown on the left in kD.Full size imageThe combination of immunoblotting and azido-ATP labelling revealed defects in more than 75% of the variants tested. Among the variants with reduced yield and/or ATP binding are G863A and delG863, the two protein products of a guanosine2588→cytosine mutation that both generates a glycine-to-alanine substitution at codon 863 and activates a cryptic splice acceptor site in exon 17 that results in the removal of codon 863 from approximately 50% of the transcripts10. This is the most common allele among STGD patients in Northern Europe, representing roughly 20% of disease-associated alleles. It is also present at a frequency of approximately 3% in the general population in Northern Europe and approximately 1% in the United States population7,9,10. Genotype-phenotype correlations suggest that it is a mild allele and that it leads to STGD only when paired with a more severe allele10. Relative to wild type, the G863A variant is subtantially impaired and the delG863 variant is mildly impaired (Fig. 3).

Over 200 ABCA4 sequence variants have been reported so far in patients with STGD and other retinopathies1,5,6,7,8,9,10,11,12,13,14,15,16,17. We have examined 33 missense mutations, 3 small in-frame deletions and 1 frameshift near the carboxy terminus (Table 1 and Fig. 2), including those mutations most commonly encountered in STGD patients1,5,6,7,8,9,10,11 and several that were reported in AMD patients15. As an initial step in assessing protein folding and stability, we analysed each ABCR variant by immunoblotting and azido-ATP labelling (Fig. 3). Mutations that cause small deletions (delVVAIC1681 and delPAL1761) or introduce charged amino acids into predicted transmembrane domains (G851D and G1886E) produce greatly reduced amounts of protein. Among the ABCR variants that are expressed with normal or nearly normal yield, azido-ATP labelling revealed a subset that is defective in ATP binding. A variety of mutations that lie outside of the nucleotide-binding domains (NBDs) can impair azido-ATP labelling, including L541P, predicted to reside adjacent to a transmembrane domain, and W1408R, which resides between the homologous halves of ABCR (Fig. 2). These data suggest that ATP binding to the NBDs is allosterically coupled to conformational changes in or near the transmembrane regions. Moreover, some mutations within either of the two NBDs abolish or nearly abolish all azido-ATP labelling, as seen, for example, with variants T971N, L1971R, G1977S and E2096K, implying allosteric coupling between the two NBDs, as described for P-glycoprotein22,23.Table 1 Naturally occurring ABCR variants produced in transfected 293 cellsFull size tableFigure 2: Locations of 37 naturally occurring ABCR sequence variants and 4 synthetic mutations.The predicted transmembrane topography and domain structure of ABCR is based on the hydropathy profile and sequence alignment with other ABC transporters. The cytosolic face of the membrane is downward. NBD, nucleotide binding domain; HH, highly hydrophobic domain shared with other members of the ABC1/ABCR subfamily of ABC transporters. A, B and C indicate the sequence motifs characteristic of nucleotide binding folds. Asterisks denote the four synthetic mutations.Full size imageFigure 3: Protein yield and ATP-binding capacity of 37 naturally occurring ABCR variants produced in transiently transfected 293 cells.Membranes were analysed by immunoblotting with affinity-purified anti-ABCR antibodies (top) and photoaffinity labelling with α-32P azido-ATP (bottom). We loaded 1 μg (immunoblotting) or 2.5 μg (azido-ATP labelling) of total membrane protein, as determined by Bradford assay, per track. The mutations that reside in NBD-1 and NBD-2 are indicated above the corresponding lanes. The relative levels of the different variant proteins and the extent of azido-ATP labelling were observed to be highly reproducible in multiple independent experiments. ABCR (large arrowhead); an endogenous 55-kD protein (small arrowhead) serves as an internal control for azido-ATP labelling. Molecular mass standards are shown on the left in kD.Full size imageThe combination of immunoblotting and azido-ATP labelling revealed defects in more than 75% of the variants tested. Among the variants with reduced yield and/or ATP binding are G863A and delG863, the two protein products of a guanosine2588→cytosine mutation that both generates a glycine-to-alanine substitution at codon 863 and activates a cryptic splice acceptor site in exon 17 that results in the removal of codon 863 from approximately 50% of the transcripts10. This is the most common allele among STGD patients in Northern Europe, representing roughly 20% of disease-associated alleles. It is also present at a frequency of approximately 3% in the general population in Northern Europe and approximately 1% in the United States population7,9,10. Genotype-phenotype correlations suggest that it is a mild allele and that it leads to STGD only when paired with a more severe allele10. Relative to wild type, the G863A variant is subtantially impaired and the delG863 variant is mildly impaired (Fig. 3).

Patient 2 (P2), previously described in a large IRD cohort study [1], is also of Somali origin and was seen in the retina clinic at the University of Iowa at age 11

Patient 1 (P1) experienced reduced vision from age 5 and was referred to ophthalmology testing at Haukeland University Hospital at age 12.

Patient 4 with an early frameshift mutation in one allele and a downstream p.Val552Ile mutation in the second allele has a mild form of STGD1. Because the frameshift mutation is likely to result in a null allele, any residual functional activity of ABCA4 would arise from ABCA4 harboring the p.Val552Ile missense mutation. Our in vitro studies showing that the p.Val552Ile variant expresses at close to WT levels, exhibits normal N-Ret-PE binding properties, and has only a modest reduction in ATPase activity (Table 2) are consistent with the mild disease phenotype of patient 4. Another study has also reported that the p.Val552Ile mutation is associated with a STGD1 disease phenotype.50 However, in silico predictions on the pathological relevance of this mutation have been variable.20,13 On the basis of allele frequencies in controls versus patients, it has been argued that the p.Val552Ile is most likely benign.20 At a protein level, a hydrophobic amino acid residue valine is replaced with another hydrophobic residue isoleucine. Accordingly, this substitution would be predicted to have only a marginal impact on ABCA4 protein structure and function. However, valine at position 552 of ABCA4 is invariable among vertebrate species, including other mammals, chicken, Xenopus, and Japanese puffer fish (Takifugu rubripes), attesting to the likely importance of valine at this position. Collectively, these studies suggest that the p.Val552Ile is a mild mutation in which the pathogenicity may only be displayed in selected cases. More specifically, the p.Val522Ile would display a mild STGD1 phenotype when combined with a null allele as in the case of patient 4 or a missense mutation with little or no activity such as the p.Asn965Ser mutation,37,51 but would not display a disease phenotype in a patient homozygous for this mutation or patients in which this mutation is combined with a mutation that shows significant ABCA4 function because under these circumstances sufficient ABCA4 activity would be realized to prevent the accumulation of toxic retinoids.

Next, we measured the effect of disease-associated mutations on the ATPase activity of ABCA4. WT and ABCA4 variants were solubilized in CHAPS, purified by immunoaffinity chromatography, and subsequently reconstituted into PE-containing liposomes at similar protein concentrations. The ATPase activity of the mutants in the presence and absence of N-Ret-PE substrate is shown in Figure 6A, ​A,6B.6B. As previously reported,25,47 addition of 40 μM all-trans retinal to WT ABCA4 resulted in a 1.8- to 2.5-fold increase in ATPase activity (Fig. 6). The ATPase activity of the mutants was measured at the same protein concentration as WT ABCA4 to determine the effect of the mutation on the functional activity of ABCA4. Five mutants (p.Val552Ile, p.Ala1038Val, p.Ala1357Thr, p.Ala1794Pro, and p.Leu2027Phe) showed reduced basal ATPase activity relative to WT ABCA4 (∼40%–85%), but this activity was stimulated 1.6- to 3.0-fold by the addition of all-trans retinal. On the other hand, p.Gly72Arg, p.Met448Lys, p.Leu541Pro, p.Gly1091Glu, p.Gly1961Glu, and p.Arg2077Trp variants showed drastically reduced basal activity with little or no substrate stimulation.Open in a separate windowFigure 6ATPase activity of ABCA4 variants. The ATPase activity of immunopurified and reconstituted ABCA4 variants was measured in the presence or absence of all-trans retinal. (A) Quantification of the basal and retinal-stimulated ATPase activity of ABCA4 variants normalized to WT basal ATPase activity. ATPase assays were carried out using similar concentrations of purified ABCA4. Data expressed as an average ± SD for n ≥ 3 independent experiments. (B) Representative curves of specific ATPase activity as a function of all-trans retinal concentration for WT and ABCA4 variants. (C) Relative basal ATPase activity of WT and A1794P using equal amounts of transfected HEK293T cells. Data expressed as an average ± SD. Measurements were done in triplicate.To more directly evaluate the expression and function of the p.Ala1794Pro variant, we transfected HEK293T cells separately with WT ABCA4 and the p.Ala1794Pro mutant cDNAs at similar levels. After solubilization in CHAPS buffer, the samples were subjected to high-speed centrifugation and the supernatant was reconstituted into liposomes for analysis of its basal and substrate activated ATPase activity. As shown in Figure 6C, the p.Ala1794Pro had a significantly reduced activity due largely to the low expression of this variant. These studies indicate that only a small fraction of the p.Ala1794Pro mutant folds into a functionally active protein and correlates well with the phenotype of patient 5.

G818EER51 ± 1363 ± 5111 ± 825 ± 312 ± 32Moderate/Severe