GDF6, a novel locus for a spectrum of ocular developmental anomalies

Abstract

Colobomata represent visually impairing ocular closure defects that are associated with a diverse range of developmental anomalies. Characterization of a chromosome 8q21.2-q22.1 segmental deletion in a patient with chorioretinal coloboma revealed elements of nonallelic homologous recombination and nonhomologous end joining. This genomic architecture extends the range of chromosomal rearrangements associated with human disease and indicates that a broader spectrum of human chromosomal rearrangements may use coupled homologous and nonhomologous mechanisms. We also demonstrate that the segmental deletion encompasses GDF6, encoding a member of the bone-morphogenetic protein family, and that inhibition of gdf6a in a model organism accurately recapitulates the proband's phenotype. The spectrum of disorders generated by morpholino inhibition and the more severe defects (microphthalmia and anophthalmia) observed at higher doses illustrate the key role of GDF6 in ocular development. These results underscore the value of integrated clinical and molecular investigation of patients with chromosomal anomalies.

Figure 1.

A–F, Phenotypes of proband with chromosome 8q segmental deletion who has colobomatous developmental anomalies affecting the anterior and posterior ocular segments (A). Note the junction between the normal and abnormal retina. B, Retinochoroidal colobomata, with extensive optic nerve involvement. C, Iris coloboma (right eye). D, Normal optic nerve and retina (for comparison). E and F, Syndactyly affecting the 2nd and 3rd toes. G–I, Phenotypes of the proband’s father, demonstrating milder ocular colobomata and syndactyly. G, Dysplastic optic nerve with anomalous vascular pattern and small inferior retinochoroidal coloboma. H, Similar but milder changes (left eye). I, Soft-tissue syndactyly. M-FISH and summary of microsatellite marker genotyping results confirmed chromosome 8q segmental deletion and approximate breakpoint positions. J, 4′,6-Diamidino-2-phenylindole staining demonstrating length difference between the undeleted and the segmentally deleted chromosome. K, Merged fluorescence signals from five M-FISH probes illustrating the segmental deletion. L, “False color” counterstaining, to illustrate with greater clarity the effect of the segmental deletion on length of chromosome 8q. M, Montage illustrating normal hybridization pattern for each probe, decreased hybridization for diethylamino-coumarin (DEAC) and SpO, the approximate extent of the segmental deletion (dotted blue line), and the microsatellite markers used to refine the breakpoint position. Undeleted markers are shown in bold, deleted markers are underlined, and uninformative markers are shown in plain type.

Figure 2.

A, Schematic representation of the proband’s segmental deletion, with coordinates of the centromeric and telomeric breakpoints shown above the representation. GDF6 is located at positions 97223736–97244196. B, Sequence of junctional fragment comprising TCCTGG from the centromeric end, fused to a 4-bp insertion (AGCT), and AGGTTT from the telomeric breakpoint >10 million bp away. The repetitive sequences adjacent to the breakpoints comprise 5.7 kb of repeats centromerically and 3.6 kb of repeats telomerically. (Some repeats extend beyond the depicted region.) C, Amplification of a 4.5-kb deletion-junction fragment in the proband that is not present in the unaffected parents (1.7-kb control fragment amplified in all subjects). F = father; M = mother; P = proband. D, Murine Tcm locus on chromosome 4, a 1.17-Mb region encompassing five genes (A = unknown; B = helicase-related; C = Cralbp-related; D = Asph; E = Gdf6) and their orthologues on human chromosome 8. Because of a synteny break, four genes are ∼35 Mb centromeric to the segmental deletion, with only GDF6 common to both.

Figure 3.

A and B, In situ analysis of gdf6a expression in developing wild-type zebrafish embryos at 18 and 24 hpf. C, RT-PCR used to measure spliced gdf6a transcripts from uninjected (WT), gdf6a^MO1-injected (MO1), and gdf6a^MO2-injected (MO2) embryos, illustrating reduced levels of spliced RNA product in morphant embryos. No change is noted in expression of ef1a in either MO1- or MO2-injected embryos. Retinal and lens differentiation was evaluated by examining expression of marker genes in the developing retina at 24 hpf. Expression of aldh1a2 (D–F) in the dorsal-temporal retina is eliminated in embryos injected with either gdf6a morpholino. Similarly, expression of hmx3b (G–I) in the dorsal retina is strongly reduced in gdf6a morpholino–injected embryos. Expression of hmx3b in the developing lens is largely unaffected in gdf6a morphants (G–I). Analysis of the nasal marker foxg1 (J–L) was expanded posteriorly to more temporal retinal regions in gdf6a morphants. Red brackets highlight the extent of retinal gene expression. Embryos are mounted in dorsal view, with the anterior to the left.

Figure 4.

Larval zebrafish retinal defects, caused by injection of splice-blocking gdf6a morpholinos. Wild-type images at 48 hpf (A) and 72 hpf (C), compared with morphant counterparts (B and D). Decrease in lenticular and ocular size is evident at later stages (C and D). Ventral colobomata and dorsal groove are highlighted in panel B (arrows). E–H, Images of 5-dpf zebrafish larvae, illustrating phenotypic variability observed with higher doses (10 ng) of gdf6a morpholino (injected at the one-cell stage). E, Uninjected control. F, Milder phenotype, with unilateral coloboma and lenticular extrusion. G, Unilateral coloboma and microphthalmia. H, Severe retinal degeneration, resembling human anophthalmia. Transverse histological sections from wild-type (I and K) and gdf6a morphant (J and L) 3-d-old embryos, with use of phalloidin-Alexa488 and Richardson’s stains. Phalloidin-stained images are composites of 12 equally spaced 1-μm sections throughout the eye (I and J). Note reduction in ocular and lenticular size, loss of normal retinal lamination, and vacuolation of the lens that protrudes anteriorly (L). Scale bar = 100 μm.

Figure 5.

Quantitative ocular phenotypic data from wild-type and gdf6a morphant (5 ng gdf6a^MO1 plus 3 ng p53^MO) embryos at 2–5 dpf.