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A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family.

Patel K, Giese AP, Grossheim JM, Hegde RS, Hegde RS, Delio M, Samanich J, Riazuddin S, Frolenkov GI, Cai J, Ahmed ZM, Morrow BE - PLoS ONE (2015)

Bottom Line: The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling.Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia.However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Albert Einstein College of Medicine, 1301 Morris Park Avenue, Bronx, New York, United States of America.

ABSTRACT
Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.

No MeSH data available.


Related in: MedlinePlus

The p.Arg186Trp mutation does not affect the Myosin 15a/Whirlin/CIB2 tripartite complex.COS-7 cells were co-transfected with GFP-Myosin 15a, Dsred-CIB2WT, Dsred-CIB2R186W and Whirlin constructs. A) Co-transfection of GFP-Myosin 15a, Dsred-CIB2WT and Whirlin shows that the Myosin 15a-Whirlin complex is able to transport CIB2 to the tip of the filopodia and form a tripartite complex. The linescan analysis shows co-localization of the three proteins. B, C) The p.Arg186Trp mutation does not affect transport of CIB2 as Dsred-CIB2R186W co-localizes with Whirlin and Myosin 15a at the tip of the filopodia. D) In vitro co-immunoprecipitation of CIB2R186W-GFP and Dsred-Whirlin constructs showing that CIB2R186W variant interacts with Whirlin. GFP construct is used as a negative control. Scale bars, 10μm.
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pone.0133082.g005: The p.Arg186Trp mutation does not affect the Myosin 15a/Whirlin/CIB2 tripartite complex.COS-7 cells were co-transfected with GFP-Myosin 15a, Dsred-CIB2WT, Dsred-CIB2R186W and Whirlin constructs. A) Co-transfection of GFP-Myosin 15a, Dsred-CIB2WT and Whirlin shows that the Myosin 15a-Whirlin complex is able to transport CIB2 to the tip of the filopodia and form a tripartite complex. The linescan analysis shows co-localization of the three proteins. B, C) The p.Arg186Trp mutation does not affect transport of CIB2 as Dsred-CIB2R186W co-localizes with Whirlin and Myosin 15a at the tip of the filopodia. D) In vitro co-immunoprecipitation of CIB2R186W-GFP and Dsred-Whirlin constructs showing that CIB2R186W variant interacts with Whirlin. GFP construct is used as a negative control. Scale bars, 10μm.

Mentions: We previous have shown that CIB2, Whirlin and Myosin XVa forms a tripartite complex located at the tip of filopodia of COS-7 cells [30]. To determine if the deafness causing mutation affects the ability to localize to the tips of filopodia, we over-expressed DsRed tagged CIB2WT (Fig 5A) and CIB2R186W constructs of human CIB2 (Fig 5B and 5C) along with non-tagged human Whirlin and GFP-Myosin 15a in COS-7 cells. Confocal imaging of transfected COS-7 cells revealed that CIB2 and CIB2R186W variants are located at the tip of filopodia (Fig 5), indicating the persistence of the interaction between CIB2 and Whirlin despite the presence of the mutation. The interaction between CIB2R186W variant and Whirlin was further confirmed by in vitro co-immunoprecipitation assay (Fig 5D).


A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family.

Patel K, Giese AP, Grossheim JM, Hegde RS, Hegde RS, Delio M, Samanich J, Riazuddin S, Frolenkov GI, Cai J, Ahmed ZM, Morrow BE - PLoS ONE (2015)

The p.Arg186Trp mutation does not affect the Myosin 15a/Whirlin/CIB2 tripartite complex.COS-7 cells were co-transfected with GFP-Myosin 15a, Dsred-CIB2WT, Dsred-CIB2R186W and Whirlin constructs. A) Co-transfection of GFP-Myosin 15a, Dsred-CIB2WT and Whirlin shows that the Myosin 15a-Whirlin complex is able to transport CIB2 to the tip of the filopodia and form a tripartite complex. The linescan analysis shows co-localization of the three proteins. B, C) The p.Arg186Trp mutation does not affect transport of CIB2 as Dsred-CIB2R186W co-localizes with Whirlin and Myosin 15a at the tip of the filopodia. D) In vitro co-immunoprecipitation of CIB2R186W-GFP and Dsred-Whirlin constructs showing that CIB2R186W variant interacts with Whirlin. GFP construct is used as a negative control. Scale bars, 10μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591343&req=5

pone.0133082.g005: The p.Arg186Trp mutation does not affect the Myosin 15a/Whirlin/CIB2 tripartite complex.COS-7 cells were co-transfected with GFP-Myosin 15a, Dsred-CIB2WT, Dsred-CIB2R186W and Whirlin constructs. A) Co-transfection of GFP-Myosin 15a, Dsred-CIB2WT and Whirlin shows that the Myosin 15a-Whirlin complex is able to transport CIB2 to the tip of the filopodia and form a tripartite complex. The linescan analysis shows co-localization of the three proteins. B, C) The p.Arg186Trp mutation does not affect transport of CIB2 as Dsred-CIB2R186W co-localizes with Whirlin and Myosin 15a at the tip of the filopodia. D) In vitro co-immunoprecipitation of CIB2R186W-GFP and Dsred-Whirlin constructs showing that CIB2R186W variant interacts with Whirlin. GFP construct is used as a negative control. Scale bars, 10μm.
Mentions: We previous have shown that CIB2, Whirlin and Myosin XVa forms a tripartite complex located at the tip of filopodia of COS-7 cells [30]. To determine if the deafness causing mutation affects the ability to localize to the tips of filopodia, we over-expressed DsRed tagged CIB2WT (Fig 5A) and CIB2R186W constructs of human CIB2 (Fig 5B and 5C) along with non-tagged human Whirlin and GFP-Myosin 15a in COS-7 cells. Confocal imaging of transfected COS-7 cells revealed that CIB2 and CIB2R186W variants are located at the tip of filopodia (Fig 5), indicating the persistence of the interaction between CIB2 and Whirlin despite the presence of the mutation. The interaction between CIB2R186W variant and Whirlin was further confirmed by in vitro co-immunoprecipitation assay (Fig 5D).

Bottom Line: The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling.Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia.However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Albert Einstein College of Medicine, 1301 Morris Park Avenue, Bronx, New York, United States of America.

ABSTRACT
Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.

No MeSH data available.


Related in: MedlinePlus