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. 2015 Oct 1;10(10):e0133082.
doi: 10.1371/journal.pone.0133082. eCollection 2015.

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

Kunjan Patel  1 , Arnaud P Giese  2 , J M Grossheim  3 , Rashmi S Hegde  4 , Maria Delio  5 , Joy Samanich  6 , Saima Riazuddin  2 , Gregory I Frolenkov  3 , Jinlu Cai  1 , Zubair M Ahmed  2 , Bernice E Morrow  1
Affiliations

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

Kunjan Patel et al. PLoS One. .

Erratum in

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pedigree and audiogram of JS6 proband.
(A) Pedigree of family JS6 (arrow to proband). The filled symbols represent affected individuals. (B) Audiogram from the female proband, JS6.001 indicating hearing loss ranging from severe to profound. The symbols ‘o’ and ‘x’ denote air conduction pure-tone thresholds, and the ‘A’ symbol denotes bone conduction thresholds. Downward arrow denotes no response on the audiogram.
Fig 2
Fig 2. Mutation in penultimate amino acid of CIB2.
(A) Chromatogram from Sanger sequencing showing that both affected children have a homozygous c.556C>T (p.Arg186Trp) mutation and the parents are heterozygous carriers. The chromatogram of the proband is shown and the sibling has an identical chromatogram (not shown). (B) The mutation affects the arginine residue at amino acid position 186 of CIB2, which is the penultimate amino acid of the protein. The genomic position of the nucleotide variant is on chromosome 15, position 78,397,660 on human February 2009, GRCh37/hg19 assembly. (C) The arginine residue at amino acid position 186 is conserved across a wide variety of species. Identical residues are highlighted in gray color.
Fig 3
Fig 3. Restriction enzyme digestion to validate the CIB2 mutation.
Lanes 2 and 3 on the agarose gel represent the restriction digest of a PCR product that was performed on both affected children. The presence of the c.556C>T mutation abolishes the restriction site and results in a single product of 206bp (Lanes 2 and 3, depict the proband and sibling, respectively). Lanes 4 and 5 contain both parental samples and as a result the there is a PCR product of 206bp representing the mutant allele as well as two additional digested fragments at 124bp and 82bp, which represent the normal allele. Lanes 6 and 7 are restriction enzyme digests from two normal, unrelated individuals, with no PCR product corresponding to the mutant allele of 206bp and only two digested PCR products corresponding to the normal allele. Lane 1 contains the DNA size standard ladder.
Fig 4
Fig 4. The p.Arg186Trp mutation does not affect the targeting of CIB2 to the stereocilia tips of vestibular system hair cells.
Gene gun transfection of P3 vestibular system with a CIB2WT-GFP expression vector shows targeting of CIB2 to the cell body, the cuticular plate (Pseudocolor, *) and also along the length of stereocilia of hair cells (top set of panels). As previously shown, CIB2 also accumulates to the stereocilia tips (Pseudocolor, arrows). The p.Arg186Trp mutation does not affect the localization of CIB2 in the cuticular plate or to the tip of stereocilia (pseudocolor, *, arrows) as shown in the bottom set of panels. Scale bars, 5μm.
Fig 5
Fig 5. 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.
Fig 6
Fig 6. The C-terminal helix of CIB2 mutation may be destabilized because of steric hindrance.
Molecular models using the Protein Data Bank (PDB) 1XO5 crystal structure of Ca2+-CIB1 as a template. A) The backbone ribbon of the C-terminal helix of CIB1 is highlighted in red, and the four Ca2+ ions are represented by white spheres. B) The side-chain of the Arg186 residue is represented in white and blue (dash line), and the Trp residue is overlapped in green at position 186.
Fig 7
Fig 7. The p.Arg186Trp mutation affects the calcium binding affinity of CIB2.
Calcium responses in COS-7 cells transfected with DsRed-tagged CIB2 constructs were recorded after ATP stimulation. The p.Arg186Trp mutation abolished the ability of CIB2 to decrease ATP-induced calcium release from the cell, whereas the p.Phe91Ser mutation did not. Data are normalized to the average response of mock-transfected cells and are shown as mean ± s.e.m. ***P < 0.001; **P < 0.01; *P < 0.05.
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References

    1. Morton CC, Nance WE. Newborn hearing screening—a silent revolution. The New England journal of medicine. 2006;354(20):2151–64. . - PubMed
    1. Marazita ML, Ploughman LM, Rawlings B, Remington E, Arnos KS, Nance WE. Genetic epidemiological studies of early-onset deafness in the U.S. school-age population. American journal of medical genetics. 1993;46(5):486–91. 10.1002/ajmg.1320460504 . - DOI - PubMed
    1. Friedman TB, Griffith AJ. Human nonsyndromic sensorineural deafness. Annual review of genomics and human genetics. 2003;4:341–402. 10.1146/annurev.genom.4.070802.110347 . - DOI - PubMed
    1. Duman D, Tekin M. Autosomal recessive nonsyndromic deafness genes: a review. Frontiers in bioscience. 2012;17:2213–36. - PMC - PubMed
    1. Cohen BE, Durstenfeld A, Roehm PC. Viral causes of hearing loss: a review for hearing health professionals. Trends in hearing. 2014;18 10.1177/2331216514541361 - DOI - PMC - PubMed

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