Hum Mol Genet
October 1, 2005;
ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies.
, also called ABCR
, is a retinal-specific member of the ATP-binding cassette (ABC) family that functions in photoreceptor outer segments as a flipase of all-trans retinal. Homozygous and compound heterozygous ABCA4
mutations are associated with various autosomal recessive retinal dystrophies, whereas heterozygous ABCA4
mutations have been associated with dominant susceptibility to age-related macular degeneration in both humans and mice. We analyzed a cohort of 29 arRP families for the mutations in ABCA4
with a commercial microarray, ABCR
-400 in addition to direct sequencing and segregation analysis, and identified both mutant alleles in two families (7%): compound heterozygosity for missense (R602W) and nonsense (R408X) alleles and homozygosity for a complex [L541P; A1038V] allele. The missense mutations were analyzed functionally in the photoreceptors of Xenopus laevis tadpoles, which revealed mislocalization of ABCA4
protein. These mutations cause retention of ABCA4
in the photoreceptor inner segment, likely by impairing correct folding, resulting in the total absence of physiologic protein function. Patients with different retinal dystrophies harboring two misfolding alleles exhibit early age-of-onset (AO) (5-12 years) of retinal disease. Our data suggest that a class of ABCA4
mutants may be an important determinant of the AO of disease.
Hum Mol Genet
Disease Ontology terms:
STARGARDT DISEASE 1; STGD1
[+] show captions
Figure 2. Transgenic expression of WT and mutated human ABCA4 in X. laevis tadpoles. (A and B) Cryosections of the retina from 2-week-old tadpoles expressing human ABCA4 were immunostained with anti-ABCA4 Rim 3F4 antibody and subsequently visualized with goat anti-mouse antibody conjugated to Alexa Fluor 488 (green). The sections were stained additionally with Texas Red—WGA (red) to label the photoreceptor outer segments (A). The nuclei in both sections were stained with DAPI (blue). Human ABCA4 is recognized by 3F4 monoclonal antibody and found exclusively in the ROS rims as shown by the yellow signal (marked with arrow). (C–L) Microphotographs of 2-week-old X. laevis tadpoles expressing R602W (C and D), [L541P; A1038V] (E and F), L541P (G and H), A1038V (I and J) and C1490Y (K and L). Rod photoreceptors expressing each of the mutant proteins, except A1038V, demonstrated localization of the transgenic protein to the rod inner segment and cell body. Misfolded proteins are homogenously distributed in the inner segments and cell bodies, although the presence of fine aggregates was noted for L541P. Mutation A1038V does not influence the localization of ABCA4 and the mutant protein was found in the ROS. OS, outer segments; IS, inner segment; N, nucleus.
Figure 1. Haplotype and segregation analysis of ABCA4 mutations in two arRP families. Pedigrees are drawn using standard conventions; filled symbols denote RP-affected individuals. Haplotypes for the ABCA4 genomic region are given below for each family member. ABCA4 mutations are abbreviated by ‘ABCA4′ with their predicted amino acid changes shown. Segregation is denoted with background gray shading.
Figure 3. ATP hydrolytic activity assay. WT and mutant constructs [L541P; A1038V], R602W and C1490Y ABCA4 were expressed in COS7 cells. The proteins were affinity-purified and assayed for basal ATP hydrolytic activity at identical protein concentrations. All investigated mutants resulted in decreased ATPase activity.
Figure 4. Folding mutations affect conserved residues from extracellular loops—ECD I and II. Shown is a proposed topological model of ABCA4 with its membrane spanning domains and the mutations: L541P, R602W, A1038V and C1490 analyzed. The comparison of ABCA4 sequences among species revealed that mutated residues: L541, R602 and C1490 are evolutionary conserved. Hs, human; Bt, bovine; Mm, murine; St, frog (Silurana tropicalis); Xl, frog (Xenopus laevis); Tr, fish (Takifugu rubripes) ABCA4.