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

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\u3eT; 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

Enhanced Maternal Origin of the 22q11.2 Deletion in Velocardiofacial and DiGeorge Syndromes

Velocardiofacial and DiGeorge syndromes, also known as 22q11.2 deletion syndrome (22q11DS), are congenital-anomaly disorders caused by a de novo hemizygous 22q11.2 deletion mediated by meiotic nonallelic homologous recombination events between low-copy repeats, also known as segmental duplications. Although previous studies exist, each was of small size, and it remains to be determined whether there are parent-of-origin biases for the de novo 22q11.2 deletion. To address this question, we genotyped a total of 389 DNA samples from 22q11DS-affected families. A total of 219 (56%) individuals with 22q11DS had maternal origin and 170 (44%) had paternal origin of the de novo deletion, which represents a statistically significant bias for maternal origin (p = 0.0151). Combined with many smaller, previous studies, 465 (57%) individuals had maternal origin and 345 (43%) had paternal origin, amounting to a ratio of 1.35 or a 35% increase in maternal compared to paternal origin (p = 0.000028). Among 1,892 probands with the de novo 22q11.2 deletion, the average maternal age at time of conception was 29.5, and this is similar to data for the general population in individual countries. Of interest, the female recombination rate in the 22q11.2 region was about 1.6–1.7 times greater than that for males, suggesting that for this region in the genome, enhanced meiotic recombination rates, as well as other as-of-yet undefined 22q11.2-specific features, could be responsible for the observed excess in maternal origin

Enhanced Maternal Origin of the 22q11.2 Deletion in Velocardiofacial and DiGeorge Syndromes

Velocardiofacial and DiGeorge syndromes, also known as 22q11.2 deletion syndrome (22q11DS), are congenital-anomaly disorders caused by a de novo hemizygous 22q11.2 deletion mediated by meiotic nonallelic homologous recombination events between low-copy repeats, also known as segmental duplications. Although previous studies exist, each was of small size, and it remains to be determined whether there are parent-of-origin biases for the de novo 22q11.2 deletion. To address this question, we genotyped a total of 389 DNA samples from 22q11DS-affected families. A total of 219 (56%) individuals with 22q11DS had maternal origin and 170 (44%) had paternal origin of the de novo deletion, which represents a statistically significant bias for maternal origin (p = 0.0151). Combined with many smaller, previous studies, 465 (57%) individuals had maternal origin and 345 (43%) had paternal origin, amounting to a ratio of 1.35 or a 35% increase in maternal compared to paternal origin (p = 0.000028). Among 1,892 probands with the de novo 22q11.2 deletion, the average maternal age at time of conception was 29.5, and this is similar to data for the general population in individual countries. Of interest, the female recombination rate in the 22q11.2 region was about 1.6–1.7 times greater than that for males, suggesting that for this region in the genome, enhanced meiotic recombination rates, as well as other as-of-yet undefined 22q11.2-specific features, could be responsible for the observed excess in maternal origin

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