Suzanne de Bruijn

111 A RIPOR2 deletion is a frequent cause of adult-onset hearing loss Mutant RIPOR2 cannot rescue morphological defects in outer hair cells from Ripor2 knockout mice In Ripor2 knockout mice, morphological defects were previously observed in hair cells, which included hair bundle polarity and cohesion and length of stereocilia. 19 After injectoporation of the Ripor2 mutant construct into outer hair cells of these mice, these defects could not be rescued in any of the 13 cells expressing mutant-RIPOR2. The typical V-shaped hair bundle was not formed, in contrast to the rescue effect observed in five out of six cells expressing wildtype-RIPOR2 ( Figure 5B ). This, together with the aberrant localization of mutant-RIPOR2, confirms an effect of the 4-amino acid deletion on RIPOR2 function in outer hair cells. DISCUSSION This study identified an in-frame 12 nucleotide deletion in RIPOR2 as a prevalent and highly penetrant genetic factor for adult-onset HL in the Netherlands and beyond. HL associated with the deletion is highly variable in age of onset and audiometric characteristics. Our study exemplifies that an increasing contribution of environmental factors and of low-penetrance genetic factors to the hearing ability during life, complicates the identification of highly penetrant genetic factors in adult-onset HL. This is best illustrated by family W97-056 in which the linkage interval was falsely delimited by a phenocopy. The RIPOR2 variant was significantly enriched in an in-house dataset, previously coined “SE-NL” (Southeast Netherlands) with exomes of 22,952 unrelated individuals with unknown hearing abilities. 27 Eighteen individuals were heterozygotes for the variant (AF 0.0392%), as compared to 8 of 56,352 individuals (AF 0.0071%) and 5 of 32,287 individuals (AF 0.0077%) of non-Finnish European descent in the gnomAD exome database v2.1.1 and gnomAD genome database v3, respectively. As the variant was indicated to be inherited from a common ancestor, this individual might well be of Dutch origin or of neighboring regions. Several lines of evidence indicate the association of the c.1696_1707del RIPOR2 variant with HL. Firstly, the deletion affects four highly conserved amino acids of RIPOR2, which is known to have a crucial role in murine and zebrafish hair cell development, function, and maintenance. 11,19,28 Ripor2 knockout mice are already found to be deaf at four weeks of age due to impaired mechanotransduction. 19 Also, knockdown of ripor2 in zebrafish induced loss of hair cells, and consequently profound hearing loss. 11 Secondly, aberrant localization of the mutant RIPOR2 in early postnatal mouse hair cells, ex vivo, and failure