Suzanne de Bruijn

109 A RIPOR2 deletion is a frequent cause of adult-onset hearing loss Figure 4. Four audiometric patterns of RIPOR2 -associated hearing loss. Air conduction thresholds of all subjects were analyzed with a k-means clustering protocol. The thick black lines depict the average of each cluster, the transparent grey areas represent the ±2 standard deviations. Cluster 1: mild hearing loss (average (PTA 0.5-4 kHz )) 23 dB hearing level (HL) with an inverse U-shape audiogram. Cluster 2: moderate hearing loss (average 48 dB HL), with relatively worse hearing in the lower frequencies. Cluster 3: moderate (average 39 dB HL) high-frequency hearing loss with a gently down sloping audiogram configuration (average of 28 dB HL difference between the mean of 0.5-1 and 4-8 kHz). Cluster 4: moderate (average 60 dB HL), mid-frequency hearing loss with a U-shape audiogram, individual audiometry ( Figure S5 ) shows relatively faster deterioration of higher frequencies later in life, for example W97-056 IV:20. dB HL, decibel hearing level; kHz, kiloHertz. The Ripor2 variant prevents correct localization of the protein in mouse cochlear hair cells Previous studies have shown that RIPOR2 is specifically localized to the base of the stereocilia in mouse cochlear hair cells. 19 RIPOR2 is highly conserved between mouse and human (87% amino acid identity). To study whether the localization of mouse RIPOR2 with a deletion of the orthologous four amino acid residues (p.584_587del) is altered, plasmids encoding wildtype- or mutant-RIPOR2 were injectoporated into cochlear outer hair cells of wildtype mice (P2). Interestingly, mutant-RIPOR2 was detected in the stereocilia but in none of the 12 evaluated cells it was retained at the stereocilia base where the wildtype protein was found to be localized in all 11 evaluated