Investigating the requirement of Clrn2 for mammalian hearing

Cochlear inner and outer hair cells are the functional units of mammalian hearing. At their apex, specialised microvilli known as stereocilia are arranged in three rows of ascending height, and the mechano-electrical transduction (MET) channels, crucial for converting sound-induced fluid waves into electrical signals, reside at the top of the two shorter rows. Each MET channel is tethered to the adjacent stereocilia in a taller row via a tip link, which mechanically gates the channel upon deflection of the stereocilia bundle. The genetic mechanisms involved in the development and maintenance of the hair bundle are poorly understood.
The Harwell Ageing Screen utilised an unbiased forward genetics approach in mice to identify Clrn2 as a novel deafness gene. Clrn2 encodes the clarin-2 protein, a small glycoprotein constituting 232 residues, exhibiting four transmembrane domains. At this time there were no publications relating to the function of clarin-2. However, its paralogue Clrn1 is the causative gene in Usher Syndrome type 3A, a rare genetic condition characterised by combined deafness and blindness.
Following complementation testing, which confirmed Clrn2’s requirement for hearing, comprehensive auditory phenotyping of the Clrn2clarniet mouse (characterised by a W4STOP mutation encoding a severely truncated clarin-2 peptide) revealed a profound, early-onset, progressive auditory deficit. Clrn2 was therefore confirmed as critical for hearing in mouse. More recently, two separate studies have reported CLRN2 to be associated with adult hearing function and the cause of hearing loss in a consanguineous Iranian family.
Utilising the clarinet mutant, ultrastructural analysis and electrophysiological studies demonstrate that while the hair bundle begins to develop normally in the early postnatal stages, the two shorter ‘transducing’ outer hair cell stereocilia rows regress before the onset of hearing. To further investigate the role of clarin-2 in the postnatal cochlea, an additional two novel mutants were generated using CRISPR/Cas9 technology: Clrn2del629, whereby Clrn2’s critical second exon is constitutively deleted; and Clrn2fl, in which exon 2 is floxed by loxP sites, allowing its conditional deletion. Morphological and functional investigations of the Clrn2fl;Myo15cre mouse revealed that expression of hair cell-specific Clrn2 in the early postnatal stages only of development is sufficient to maintain the normal architecture of the hair bundle, but results in a severe auditory phenotype that is slower to progress than that of the Clrn2clarinet or Clrn2del629 mutant.
The results presented in this thesis have demonstrated Clrn2 as essential for mammalian hearing, being crucial for the maintenance of auditory hair bundle integrity and function. Furthermore, these investigations have suggested a critical role for clarin-2 in maintenance of the MET channel current, and implicate clarin-2 as an interacting partner of the MET channel complex. These findings further elaborate upon the genetic landscape of mammalian hearing, and remove Clrn2 from the ‘ignorome’ of genes with unknown biological function.

Keywords: clarin2
clrn2
deafness
hearing loss
mammalian hearing
sensorineural hearing loss
genetics
Advisor: Bowl, M
Whitby, M
Brown, S