ABCB6 mutations cause ocular coloboma

Abstract

Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.

Figure 1

Mutations in ABCB6 and Clinical Photographs of Patients with Coloboma (A) Pedigree and segregation of the Leu811Val mutation in the Chinese family affected by coloboma. (B–E) Photographs of patients with coloboma are compared to those of the unaffected individual. The proband (II:5) of the Chinese family has iris coloboma in the lower papillary margin (C) and inferior chorioretinal coloboma (E). Photographs of the normal iris (B) and fundus (D) of the unaffected individual (II:9) in the same family are also shown. (F–H) Two mutations in ABCB6 and their sequencing tracing, including Leu811Val (F) in the Chinese family affected by coloboma and Ala57Thr (G) in the three Indian patients who have microphthalmia and coloboma. A partial sequence of ABCB6 was compared with other species' orthologs (H). Arrows indicate the location of the two mutations identified in patients with ocular coloboma.

Figure 2

Subcellular Localization of ABCB6 in RPE Cells Immunofluorescent images of the RPE cell that transiently expresses Myc-tagged ABCB6 and markers for subcellular compartments are shown. Neither WT nor mutant ABCB6, both in green, colocalized with a mitochondrial maker, in red (A). Both WT and mutant ABCB6, in green, colocalized with an ER marker, in red (B), and with a Golgi maker, in red (C).

Figure 3

Expression of ABCB6 in Cell Lines and Tissues and Whole-Mount RNA In Situ Hybridization in Zebrafish (A) RT-PCR analyses of ABCB6 expression in D407 and HEK 293 cells and the human spleen, lungs, kidneys, heart, and blood gave a 435 bp product. β-Actin was used as an internal control for cDNA quantification and gave a 285 bp product. (B) Dorsal views of embryos from 24–72 hpf are shown. WT (unstained) embryos were used as controls. The transcript for abcb6 was expressed in developing CNS tissue, including the eyes, at 24 hpf and later stages. The expression of abcb6 is detected circumferentially around the lens at 36 and 48 hpf. In addition, abcb6 is expressed in developing pectoral fins.

Figure 4

Zebrafish Eye Development Disturbed by Knockdown of abcb6 Expression and Rescue of abcb6 Morphants (A) RT-PCR verified that abcb6^MO2 altered normal abcb6 splicing by deleting exon 2. This deletion results in an abnormal 268 bp product as opposed to a normal 818 bp product. (B) Immunoblotting with the Myc antibody verified that equal amounts of abcb6 produced from the WT and mutant ABCB6 mRNA were coinjected with MOs in zebrafish. β-Actin was used as an internal control. (C–L) Compared with zebrafish embryos treated with standard control MOs (C and D), zebrafish embryos treated with abcb6^MO2 show coloboma and retarded development in 96 hpf (E and F). These phenotypes can be rescued by the coinjection of WT ABCB6 mRNA (G and H), but the phenotypes could not be rescued by coinjection of the human mRNA containing either the Leu811Val (I and J) or the Ala57Thr (K and L) mutation. (M) Graph depicting the proportions of embryos with coloboma associated with the injection of either abcb6^MO1 or abcb6^MO2 and the proportions rescued by coinjection with WT and mutant ABCB6 mRNA.

Figure 5

Iris Coloboma Produced by Morpholino Knockdown of abcb6 in Zebrafish Compared with the Patients with Coloboma in the Chinese Family (A) A normal iris is shown in an unaffected individual in the same family as the control. (B) A pupil shaped like a keyhole is seen in an affected patient in the Chinese family afflicted with coloboma. (C) The eye of a fish that was treated with a control morpholino. (D) Morphants treated with abcb6^MO1 result in a keyhole-shaped pupil similar to that seen in the patients with coloboma.