Suzanne de Bruijn

102 Chapter 3.1 DNA sequencing Next generation sequencing was performed for identification of DNA variants. Details of employed sequencing techniques are provided in Supplementary Methods . Variant interpretation For exome sequencing and Molecular Inversion Probe (MIP) datasets, annotated variants were filtered based on a population AF of ≤0.5% in the gnomAD database V.2.1, and our in-house exome database (~15,000 alleles). Variants in coding and splice site regions (-14/+14 nucleotides) were analyzed. Interpretation of missense variants was performed using the in silico tools CADD-PHRED (≥15), 12 SIFT (≤0.05), 13 PolyPhen-2 (≥0.450) 14 and MutationTaster (deleterious) 15 to predict potential functional effects. Variants were considered if a pathogenic effect was predicted by at least two different tools. Potential effects on splicing of missense and synonymous variants were evaluated using the algorithms embedded in the AlamutVisual software (V.2.10, Interactive Biosoftware). A change of ≥5% in splice site scores predicted by at least two algorithms was considered significant. For candidate variants, segregation analysis was performed by Sanger sequencing. PCR conditions are available upon request. Clinical evaluation Medical history was taken from all participants with special attention paid to acquired and noise-induced HL. Both affected and unaffected participants underwent general Ear Nose and Throat examinations, or this medical information was taken from previous examinations. Age of onset of HL was reported by subjects themselves. Only reports of a specific age of onset were used in calculations. The audiometric data in this study are described according to GENDEAF guidelines. 16 Pure tone- and speech- audiometry and click-evoked auditory brainstem response (ABR) was performed in a sound-attenuated booth, according to current standards (International Organisation for Standardization; ISO 8253-1:2010, ISO 389-1, ISO 389-5 and ISO 389-6). 17 Individuals were considered affected when pure tone thresholds for at least three individual frequencies were below the frequency-specific 95 th percentile of age- and sex-specific thresholds (ISO7029:2017) for the best hearing ear. HL was considered asymmetric if pure tone audiometry showed a difference of more than 10 dB between both ears at two individual frequencies. 16 Longitudinal (individual) progression of HL was calculated if there was a follow-up duration of at least 10 years, after onset of HL. The progression rate is defined as the mean increase pure tone average at 0.5-4 kHz (PTA 0.5-4kHz ) in dB/year between first and last audiometry. For symmetric HL, the average of both ears was used to calculate progression; for asymmetric HL, the best-hearing ear at first audiometry was used. In case of profound HL at 0.5-4 kHz at the latest audiometry, the most recent audiometry