Suzanne de Bruijn

113 A RIPOR2 deletion is a frequent cause of adult-onset hearing loss However, a strong dominant negative effect would be expected to result in early-onset HL, comparable to that associated with the homozygous loss-of-function variant. 11 Therefore, we hypothesize that the variant has a toxic gain-of-function effect. RIPOR2 is expressed in a wide-range of tissues and cell types. 19 It is a known inhibitor of the small G-proteinRHOA inneutrophils andT lymphocytes, where it regulatesmigration of these cells. 37 Additionally, RIPOR2 is upregulated during muscle cell differentiation and induces the formation of filopodia. 38 We did not observe an effect of the four amino acid-deletion on filopodia formation (de Bruijn, unpublished data) which is in line with the fact that the deleted residues are not part of the RHOA-interaction domain. 38 This might, at least in part, explain that the RIPOR2 variant leads to HL only. The variant could affect a cochlear-specific protein interaction that determines RIPOR2 localization in the hair bundle. Furthermore, in tissues other than the inner ear loss of RIPOR2 function might be compensated by RIPOR1 and RIPOR3 which are described to have redundant functions. 39,40 Indeed, RNA levels of both RIPOR1 and RIPOR3 are low in hair cells (gEAR). 41 The audiometric phenotype and age of onset of HL associated with c.1696_1707del RIPOR2 displayed variation. Such intrafamilial phenotypic variation has also been reported for defects in several of the genes that can be associated with adult-onset adNSHL, e.g. EYA4 , MYO6 and POU4F3 , and remains unexplained. 42-44 Non-penetrance is an extreme of phenotypic variability. In our study, four subjects with the c.1696_1707del RIPOR2 variant had normal hearing: V:2, IV:26, III:28, (W97-056), III:14 (W04-262), and III:4 (W15-0495). They are aged 23, 40, 51, 49, and 50 years, respectively, at the latest audiometric evaluation. The average reported age of onset in the studied families is 30 years (SD 14.9) and 70 years the highest reported onset age. Therefore, the unaffected subjects might develop HL in the future. However, incomplete penetrance of the variant cannot be excluded. With a k-means cluster analysis, four distinct audiometric clusters couldbe distinguished. It is possible that subjects, due to increasing age, may go from one cluster to another cluster, which is not captured by the k-means clustering algorithm, since no longitudinal data are used. As no clear patterns of age of onset or audiometric configurations were observed within families or family branches with the RIPOR2 variant, the phenotypic variability might well result from an interplay between environmental and genetic modifying factors. We have addressed differences in transcript levels of both wildtype andmutant RIPOR2 alleles as potential modifiers of age of onset but no clear correlations were observed. As the analysis was performed on RNA extracted from peripheral blood, we cannot exclude that RIPOR2 mRNA levels determined by cochlear-specific cis or trans regulatory elements modify the onset of HL. Other candidate genetic modifiers are