Suzanne de Bruijn

108 Chapter 3.1 Longitudinal analysis of HL in individual subjects revealed a large variation inprogression of HL between subjects ( Table S3 ). We could not identify a specific pattern, such as a certain progression (in dB/y) in certain decades. There was a median progression of 1.2 dB/y (range 0.5-2.7 dB/y), for the frequencies 0.5-4 kHz. Cross-sectional linear regression was applied to calculate ARTA ( Figure 3D ). Progression ranged from 0.7 dB/y (0.25 kHz) to 1.3 dB/y (8 kHz). Progression of HL was significant for all frequencies ( F -test, p-value: <0.0001). Speech reception thresholds were generally lower than, or comparable to, PTA 0.5-2kHz ( Table S3 ). This indicates absence of retrocochlear pathology and is in line with normal results of click-evoked ABR in four subjects ( Table S3 ). CT and/or MRI of the bilateral temporal bones and cerebellopontine angle in six subjects revealed normal inner and middle ear anatomy ( Table S3 ). Vestibular testing, performed in 11 randomly selected subjects with the RIPOR2 variant, aged 29 to 71 years, led to the conclusion that c.1696_1707del RIPOR2 is not associated with vestibular dysfunction ( Table S4 ). Further details are provided in the Supplementary Results . Transcript levels of RIPOR2 do not correlate with age of onset in affected subjects We hypothesized that the variability in age of onset of the HL associated with the c.1696_1707del RIPOR2 variant might be explained by differences in expression levels of the wildtype allele. Alternatively, variants in cis regulatory elements of the affected allele more distantly located from RIPOR2 , could influence expression levels of the mutant allele and might thereby modulate the age of onset. To test these hypotheses, allele-specific transcript levels of RIPOR2 were determined in peripheral blood cells of 33 subjects using quantitative RT-PCR. Subjects were divided into three groups based on self-reported age of onset: <20 years (n=7), 20-39 years (n=15) and ≥ 40 years (n=6). No significant differences were observed between the different subject groups, neither for the wildtype or c.1696_1707del variant RIPOR2 alleles nor for total RIPOR2 transcript levels ( Figure S7 ). Also, no difference was observed between the ratios of RIPOR2 mutant to wildtype relative transcript levels. A small difference was observed in total RIPOR2 transcript levels between subjects with an early onset of HL and controls (p=0.0241). This could suggest a trend between low expression levels and an early onset of HL, however, considering the overall variability in transcript levels it is more likely that other factors play a role. A larger sample size would be required to confirm or negate the observed trend.