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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.

Cyclic AMP (cAMP) production, which is important for mechanotransduction within the inner ear, is catalyzed by adenylate cyclases (AC). However, knowledge of the role of ACs in hearing is limited. Previously, a novel autosomal recessive non-syndromic hearing impairment locus DFNB44 was mapped to chromosome 7p14.1-q11.22 in a consanguineous family from Pakistan. Through whole-exome sequencing of DNA samples from hearing-impaired family members, a nonsense mutation c.3112C>T (p.Arg1038*) within adenylate cyclase 1 (ADCY1) was identified. This stop-gained mutation segregated with hearing impairment within the family and was not identified in ethnically matched controls or within variant databases. This mutation is predicted to cause the loss of 82 amino acids from the carboxyl tail, including highly conserved residues within the catalytic domain, plus a calmodulin-stimulation defect, both of which are expected to decrease enzymatic efficiency. Individuals who are homozygous for this mutation had symmetric, mild-to-moderate mixed hearing impairment. Zebrafish adcy1b morphants had no FM1-43 dye uptake and lacked startle response, indicating hair cell dysfunction and gross hearing impairment. In the mouse, Adcy1 expression was observed throughout inner ear development and maturation. ADCY1 was localized to the cytoplasm of supporting cells and hair cells of the cochlea and vestibule and also to cochlear hair cell nuclei and stereocilia. Ex vivo studies in COS-7 cells suggest that the carboxyl tail of ADCY1 is essential for localization to actin-based microvilli. These results demonstrate that ADCY1 has an evolutionarily conserved role in hearing and that cAMP signaling is important to hair cell function within the inner ear., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton., Competing Interests: The authors have declared that no competing interests exist.