Suzanne de Bruijn

112 Chapter 3.1 to rescue the stereocilia defects of Ripor2 knockout mice indicate a functional effect of the variant. Thirdly, neither other rare potentially causative variants in protein coding regions and splice sites of the shared haplotype region, nor structural variants affecting this region were revealed in exome or genome sequencing. RIPOR2 is localized at the taper region of themechanically sensitive stereocilia of murine hair cells 11,19,28 where it is organized in a ring-like fashion. 19 The latter is thought to be achieved by homo-oligomerization in a head-to-head and tail-to-tail manner, regulated by RHOC. 19 The oligomerization is essential for the structure of the taper region and for the morphology of the hair bundle as a whole, but the precise molecular mechanism is still elusive. The taper region is the specialized basal part of stereocilia that allows their deflection upon mechanical stimulation. 29 CLIC5, PTPRQ, MYO6, TPRN, RDX, GRXCR2, and RIPOR2 are described to concentrate and co-function in the taper region and to be crucial for its structure and/or for hair bundle development and maintenance in mice. 19,30-35 Direct interactions of these proteins are indicated, e.g., of CLIC5, RDX and TPRN, but not RIPOR2. 19,32 Also, interdependence for their concentration in the taper region was observed. 19,31,32 In RIPOR2-deficient hair cells, for example, TPRN is no longer concentrated at the stereociliary base. 19,30 Depletion of TPRN in Tprn knock-out mice leads to functional as well as (slowly) progressive morphological abnormalities of the stereocilia bundle. 30 Based on the above described molecular structure of the stereociliary taper, we hypothesize that p.(Gln566_Lys569)del RIPOR2 affects this taper region and thereby the durability of the hair bundle, potentially via an effect on TPRN. Additionally or alternatively, the RIPOR2 variant might affect the amount of the RIPOR2-interaction partner MYH9 in stereocilia, as well as the abundance of phosphorylated MYH9 and acetylated α-tubulin in the kinocilia, as these proteins are reduced in RIPOR2-deficient mice. 28 Interestingly, MYH9 defects in humans are also associated with progressive HL. 36 In light of developing therapeutic strategies, it is essential to determine whether the RIPOR2 variant has a loss-of-function, a dominant negative or toxic gain-of-function effect. A haploinsufficiency effect of the variant seems to be the least plausible, as a loss-of-function RIPOR2 variant in the heterozygous state was not indicated to be associated with HL. 11 Also, heterozygous Ripor2 knockout mice displayed no significant hearing loss at four weeks 19 and two months of age (Zhao, unpublished data). A dominant-negative effect of the p.(Gln566_Lys569del) variant cannot be excluded as an interaction between the mutant- and wildtype-RIPOR2 was detected in Co-IP assays.