Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Oct 5;91(4):636-45.
doi: 10.1016/j.ajhg.2012.08.018. Epub 2012 Sep 13.

A mutation in CABP2, expressed in cochlear hair cells, causes autosomal-recessive hearing impairment

Isabelle Schrauwen  1 , Sarah Helfmann, Akira Inagaki, Friederike Predoehl, Mohammad Amin Tabatabaiefar, Maria Magdalena Picher, Manou Sommen, Celia Zazo Seco, Jaap Oostrik, Hannie Kremer, Annelies Dheedene, Charlotte Claes, Erik Fransen, Morteza Hashemzadeh Chaleshtori, Paul Coucke, Amy Lee, Tobias Moser, Guy Van Camp
Affiliations

A mutation in CABP2, expressed in cochlear hair cells, causes autosomal-recessive hearing impairment

Isabelle Schrauwen et al. Am J Hum Genet. .

Abstract

CaBPs are a family of Ca(2+)-binding proteins related to calmodulin and are localized in the brain and sensory organs, including the retina and cochlea. Although their physiological roles are not yet fully elucidated, CaBPs modulate Ca(2+) signaling through effectors such as voltage-gated Ca(v) Ca(2+) channels. In this study, we identified a splice-site mutation (c.637+1G>T) in Ca(2+)-binding protein 2 (CABP2) in three consanguineous Iranian families affected by moderate-to-severe hearing loss. This mutation, most likely a founder mutation, probably leads to skipping of exon 6 and premature truncation of the protein (p.Phe164Serfs(∗)4). Compared with wild-type CaBP2, the truncated CaBP2 showed altered Ca(2+) binding in isothermal titration calorimetry and less potent regulation of Ca(v)1.3 Ca(2+) channels. We show that genetic defects in CABP2 cause moderate-to-severe sensorineural hearing impairment. The mutation might cause a hypofunctional CaBP2 defective in Ca(2+) sensing and effector regulation in the inner ear.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Pedigrees (A–C) Pedigrees and segregation of the c.637+1G>T (p.Phe164Serfs∗4) mutation in Iranian families Sh11 (A), Sh10 (B), and He (C). (D) Sequencing results of the c.637+1G>T mutation. The affected individual sequenced with next-generation sequencing is indicated with an arrow. Nomenclature was based on NM_016366.2 and NP_057450.2.
Figure 2
Figure 2
CABP2 c.637+1G>T Leads to Exon Skipping (A) A schematic illustration of the CABP2-pSPL3 exon trap. CABP2 exon 6 and flanking introns (dark gray)— with a WT or MT (c.637+1G>T) 5′ donor site—were cloned into the pSPL3 vector (light gray). The pSPL3 vector contains a portion of the HIV-1 tat gene, an intron containing the multiple cloning site, and functional 5′ splice donor (5′ sD) and 3′ splice acceptor (3′ sA) sites. (B) COS-7 cells were transfected with WT or MT CABP2 or an empty pSPL3 vector (“E”). After RNA isolation, splicing products were analyzed by RT-PCR and visualized by agarose-gel electrophoresis. In case of an empty vector (“E”), only vector-vector splicing (2) will occur, leading to a 177 bp secondary product. If exon 6 contains functional 5′ sD and 3′ sA sites (WT), both vector-exon splicing (1) and vector-vector splicing (2) will occur and will lead to 177 bp and 325 bp secondary PCR products, respectively. Mutant plasmids containing the c.637+1G>T (MT) 5′ donor splice-site mutation only showed vector-vector splicing (2), confirming that this mutation results in exon skipping of exon 6. Secondary PCR products were confirmed by sequencing. The following abbreviation is used: Bl, blank.
Figure 3
Figure 3
Audiogram Mean air conduction (AC) thresholds of the left ear for each family. Hearing loss is symmetric.
Figure 4
Figure 4
Isothermal Titration Calorimetry of WT and Truncated CaBP2 (A) Trace of the calorimetric titration of 5 μl aliquots (after one initial injection of 2 μl) of 5 mM CaCl2 into 1.3 ml of 110 μM recombinant WT apo-CaBP2. A robust endothermic binding reaction was observed. (B) Trace of the calorimetric titration of 5 μl aliquots (after one initial injection of 2 μl) of 5 mM CaCl2 into 1.3 ml of 114 μM recombinant truncated apo-CaBP2 (MT CaBP2 [ΔEF hands 3 and 4]). In contrast to the WT apo-CaBP2, the truncated protein leads to a weaker and exothermic reaction. (C) Trace of the blank titration curve for both experiments A and B with the same parameters but without protein in the sample cell.
Figure 5
Figure 5
c.637+1G>T Mutation Impairs CaBP2 Regulation of Cav1.3 Ca2+ Channels (A) The MT CaBP2 shows greater protein levels in transfected HEK 293T cells. Lysates of cells cotransfected with Cav1.3 subunits and WT or MT CaBP2 were subject to immunoblotting with CaBP2 antibodies (top panel). WT and MT CaBP2 were cotransfected at different molar ratios as indicated. The lower panel was probed with β-actin antibodies for confirming equal loading between groups. (B) The MT CaBP2 shows weaker regulation of CDI. HEK 293T cells were cotransfected as in (A). Ca2+ currents were evoked by 0.5 s pulses from −90 mV to −10 mV. Inactivation was measured as r500, which was the current amplitude at the end of the pulse normalized to the peak current amplitude. So that different protein levels (also in C) could be accounted for, comparisons to WT were made with cells transfected with cDNA encoding MT CaBP2 at levels ten times lower than those of WT CaBP2. Parentheses indicate numbers of cells. ∗p < 0.05. Error bars represent the SEM. (C and D) The MT CaBP2 shows a weaker ability to suppress Cav1.3 current density than does the WT CaBP2. Ca2+ currents were evoked by 50 ms pulses to variable voltages from −90 mV. Current amplitude was normalized to cell capacitance and plotted against test voltage. Error bars represent the SEM.
See this image and copyright information in PMC

References

    1. Morton C.C., Nance W.E. Newborn hearing screening—a silent revolution. N. Engl. J. Med. 2006;354:2151–2164. - PubMed
    1. Meyer N.C., Alasti F., Nishimura C.J., Imanirad P., Kahrizi K., Riazalhosseini Y., Malekpour M., Kochakian N., Jamali P., Van Camp G. Identification of three novel TECTA mutations in Iranian families with autosomal recessive nonsyndromic hearing impairment at the DFNB21 locus. Am. J. Med. Genet. A. 2007;143A:1623–1629. - PubMed
    1. Alasti F., Sanati M.H., Behrouzifard A.H., Sadeghi A., de Brouwer A.P., Kremer H., Smith R.J., Van Camp G. A novel TECTA mutation confirms the recognizable phenotype among autosomal recessive hearing impairment families. Int. J. Pediatr. Otorhinolaryngol. 2008;72:249–255. - PubMed
    1. Francey L.J., Conlin L.K., Kadesch H.E., Clark D., Berrodin D., Sun Y., Glessner J., Hakonarson H., Jalas C., Landau C. Genome-wide SNP genotyping identifies the Stereocilin (STRC) gene as a major contributor to pediatric bilateral sensorineural hearing impairment. Am. J. Med. Genet. A. 2012;158A:298–308. - PMC - PubMed
    1. Tabatabaiefar M.A., Alasti F., Shariati L., Farrokhi E., Fransen E., Nooridaloii M.R., Chaleshtori M.H., Van Camp G. DFNB93, a novel locus for autosomal recessive moderate-to-severe hearing impairment. Clin. Genet. 2011;79:594–598. - PubMed

Publication types

MeSH terms