Suzanne de Bruijn

121 A RIPOR2 deletion is a frequent cause of adult-onset hearing loss to select clusters based on average hearing threshold, while retaining the overall shape of the audiogram. Next, the optimal number of clusters in the data was obtained by using the Elbow method; 10 for a number of k=1 to k=15 clusters. The distortion, i.e. the sum of squared distances from each point to its assigned center, was determined for each value of k. The smallest number of k clusters with the lowest distortion (i.e. the elbow) was then taken as the optimal number of clusters. Finally, the audiograms of all the patients within each cluster were visualized and an average cluster prototype was obtained by averaging all audiograms within a cluster. Vestibular testing Vestibular function was assessed by electronystagmography, caloric irrigation testing, rotary chair stimulation and video head impulse tests, as described previously. 11 Cervical and ocular vestibular-evoked myogenic potentials (cVEMP/oVEMP) were measured to assess saccular and utricular function, respectively. 12,13 When responses were seen at or below 100 dB Hearing Level during (air conducted) cVEMP testing, saccular function was considered to be present, otherwise absent. 12 For (bone conducted) oVEMP stimulation, this normal value is ≤140 dB Force Level. 13 Allele-specific expression analysis Peripheral blood (2.5 ml) was collected in PAXgene Blood RNA tubes (BD Biosciences). RNAwas isolatedusing thePAXgenebloodRNAkit (Qiagen) following themanufacturer’s protocol. Subsequently, cDNA was prepared using the iScript cDNA synthesis kit (Bio- Rad) with 500 ng RNA. Allele-specific primer sets were designed and validated; the design was based on a forward primer that specifically hybridizes to either the mutant or wildtype RIPOR2 alleles. Additionally, primers were designed for exons 3-4 of RIPOR2 , as well as for exons 2-3 of the reference gene GUSB (NM_000181). Primer sequences are provided in Table S7 . All qPCR reaction mixtures were prepared with the GoTaq qPCR Master Mix (Promega) according to the manufacturer’s protocol. Amplifications were performed with the Applied Biosystem Fast 7900 System (Applied Biosystems). For all RNA samples, cDNA was synthesized twice, and all qPCR reactions were performed in duplicate. Relative gene expression levels, as compared to the internal reference gene GUSB , were determined with the ΔCt method. 14 Statistical analyses were performed using a one-way ANOVA followed by Tukey’s multiple comparison test to test for significance between the groups.