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Genetic defects of GDF6 in the zebrafish out of sight mutant and in human eye developmental anomalies.

den Hollander AI, Biyanwila J, Kovach P, Bardakjian T, Traboulsi EI, Ragge NK, Schneider A, Malicki J - BMC Genet. (2010)

Bottom Line: A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size.The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis.In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Craniofacial and Molecular Genetics, Tufts University, 136 Harrison Ave, Boston MA 02111, USA.

ABSTRACT

Background: The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the outm233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans.

Results: In this study, we show that the severe reduction of eye size in the outm233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in outm233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in outm233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed.

Conclusions: This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype.

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Positional cloning of the zebrafish out of sight mutation. (A) The out locus maps to chromosome 16 between markers Z6293 and Z8819. Sequence analysis of the gdf6a gene from this interval identified a mutation affecting its start codon (c.1A > G, p. Met1Val) in mutant (C) but not wild-type (B) individuals. SP = signal peptide, ASD = active signaling domain. (D, E) The overexpression of wild-type (E) but not mutant (D) gdf6a causes ventral eye defects. Shown are lateral (left panels) and ventro-lateral (right panels) views of zebrafish larvae at 3 dpf. Anterior is to the left, asterisks indicate the lens. (F) The frequency of ventral eye defects, following the overexpression of wild-type or mutant gdf6a in four independent experiments, as indicated on the horizontal axis (1 through 4). Numbers above the bar graph indicate sample sizes.
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Figure 2: Positional cloning of the zebrafish out of sight mutation. (A) The out locus maps to chromosome 16 between markers Z6293 and Z8819. Sequence analysis of the gdf6a gene from this interval identified a mutation affecting its start codon (c.1A > G, p. Met1Val) in mutant (C) but not wild-type (B) individuals. SP = signal peptide, ASD = active signaling domain. (D, E) The overexpression of wild-type (E) but not mutant (D) gdf6a causes ventral eye defects. Shown are lateral (left panels) and ventro-lateral (right panels) views of zebrafish larvae at 3 dpf. Anterior is to the left, asterisks indicate the lens. (F) The frequency of ventral eye defects, following the overexpression of wild-type or mutant gdf6a in four independent experiments, as indicated on the horizontal axis (1 through 4). Numbers above the bar graph indicate sample sizes.

Mentions: Mapping analysis revealed that the out locus is located on chromosome 16 between markers Z6293 and Z8819 (Figure 2). This region contains around 41 annotated genes, including gdf6a (radar, LOC566470). Sequence analysis of gdf6a in the outm233 mutant identified a c.1A > G transition affecting the start codon (p.Met1Val). This is the first ATG in the gdf6a transcript, and is preceded by a termination codon 42 bp upstream, confirming that this is the translation start site. The first ATG following the affected start codon is located four base pairs downstream (c.5A), and if used would result in a shift in the open reading frame. However, that ATG might not be used as a translation start site since it is not embedded in a Kozak consensus sequence. Another translation start site that is predicted to be functional by NetStart [27] is located at position c.223A, and its use would produce a protein in the correct reading frame but lacking the first 74 amino acids, including the signal peptide.


Genetic defects of GDF6 in the zebrafish out of sight mutant and in human eye developmental anomalies.

den Hollander AI, Biyanwila J, Kovach P, Bardakjian T, Traboulsi EI, Ragge NK, Schneider A, Malicki J - BMC Genet. (2010)

Positional cloning of the zebrafish out of sight mutation. (A) The out locus maps to chromosome 16 between markers Z6293 and Z8819. Sequence analysis of the gdf6a gene from this interval identified a mutation affecting its start codon (c.1A > G, p. Met1Val) in mutant (C) but not wild-type (B) individuals. SP = signal peptide, ASD = active signaling domain. (D, E) The overexpression of wild-type (E) but not mutant (D) gdf6a causes ventral eye defects. Shown are lateral (left panels) and ventro-lateral (right panels) views of zebrafish larvae at 3 dpf. Anterior is to the left, asterisks indicate the lens. (F) The frequency of ventral eye defects, following the overexpression of wild-type or mutant gdf6a in four independent experiments, as indicated on the horizontal axis (1 through 4). Numbers above the bar graph indicate sample sizes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992036&req=5

Figure 2: Positional cloning of the zebrafish out of sight mutation. (A) The out locus maps to chromosome 16 between markers Z6293 and Z8819. Sequence analysis of the gdf6a gene from this interval identified a mutation affecting its start codon (c.1A > G, p. Met1Val) in mutant (C) but not wild-type (B) individuals. SP = signal peptide, ASD = active signaling domain. (D, E) The overexpression of wild-type (E) but not mutant (D) gdf6a causes ventral eye defects. Shown are lateral (left panels) and ventro-lateral (right panels) views of zebrafish larvae at 3 dpf. Anterior is to the left, asterisks indicate the lens. (F) The frequency of ventral eye defects, following the overexpression of wild-type or mutant gdf6a in four independent experiments, as indicated on the horizontal axis (1 through 4). Numbers above the bar graph indicate sample sizes.
Mentions: Mapping analysis revealed that the out locus is located on chromosome 16 between markers Z6293 and Z8819 (Figure 2). This region contains around 41 annotated genes, including gdf6a (radar, LOC566470). Sequence analysis of gdf6a in the outm233 mutant identified a c.1A > G transition affecting the start codon (p.Met1Val). This is the first ATG in the gdf6a transcript, and is preceded by a termination codon 42 bp upstream, confirming that this is the translation start site. The first ATG following the affected start codon is located four base pairs downstream (c.5A), and if used would result in a shift in the open reading frame. However, that ATG might not be used as a translation start site since it is not embedded in a Kozak consensus sequence. Another translation start site that is predicted to be functional by NetStart [27] is located at position c.223A, and its use would produce a protein in the correct reading frame but lacking the first 74 amino acids, including the signal peptide.

Bottom Line: A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size.The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis.In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Craniofacial and Molecular Genetics, Tufts University, 136 Harrison Ave, Boston MA 02111, USA.

ABSTRACT

Background: The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the outm233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans.

Results: In this study, we show that the severe reduction of eye size in the outm233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in outm233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in outm233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed.

Conclusions: This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype.

Show MeSH
Related in: MedlinePlus