The p.Arg186Trp mutation does not affect the targeting of CIB2 to the stereocilia tips of vestibular system hair cells.

<p>Gene gun transfection of P3 vestibular system with a CIB2^WT-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.</p

The p.Arg186Trp mutation affects the calcium binding affinity of CIB2.

<p>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.</p

Restriction enzyme digestion to validate the <i>CIB2</i> mutation.

<p>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.</p

The C-terminal helix of CIB2 mutation may be destabilized because of steric hindrance.

<p>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.</p

Mutation in penultimate amino acid of <i>CIB2</i>.

<p>(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.</p

Pedigree and audiogram of JS6 proband.

<p>(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.</p