Suzanne de Bruijn

103 A RIPOR2 deletion is a frequent cause of adult-onset hearing loss at which all thresholds at 0.5-4 kHz could be measured, was selected. Cross-sectional linear regression analysis was applied on pure tone thresholds to calculate an Age Related Typical Audiogram (ARTA), 18 using Prism 6.0 software (GraphPad). A k-means clustering analysis was performed as described in Supplementary Methods . Injectoporation of Ripor2 -constructs and immunostaining The generation of Ripor2 LacZ/LacZ mice has been described previously. 19 For Ripor2 DNA construct generation, Ripor2 cDNA (NM_029679.2, without exon 13) was amplified from a mouse-cochlear cDNA library and cloned into a pEGFP-N3-derived vector from which the EGFP coding sequence was deleted. Procedures for injectoporation and immunostaining have been described previously, 19 and are detailed in Supplementary Methods. Immunoprecipitations and western blots Cell culture, immunoprecipitations and western blots were carried out as described. 19,20 Experiments were carried out at least three times. Antibodies used are listed in Supplementary Methods . Methods and materials for VNTR marker analysis, vestibular testing and allele-specific expression analysis are provided in Supplementary Methods. RESULTS Exome sequencing revealed an in-frame deletion in RIPOR2 To identify the genetic defect underlying the HL in family W97-056 ( Figure 1 ), exome sequencing was performed in three affected family members (III:22, IV:20 and IV:25). After applying the variant filtering and prioritization described above, two variants were shared between the three affected individuals ( Table S1 ). A SPATS1 variant (c.419G>A; p.(Gly140Glu); NM_145026.3), did not completely segregate with HL within the family as 7 out of 23 affected subjects did not harbor the variant ( Figure S1 ). Also, SPATS1 expression was not detected in the mammalian cochlea 21,22 and SPATS1 function has only been related to spermatogenesis. 23 Therefore, this variant was deemed non- causative. The in-frame deletion was present in exon 14 of RIPOR2 (c.1696_1707del; p.(Gln566_Lys569del); NM_014722.3; Chr6:g.24,843,303_24,843,314del; rs760676508). It affects a highly conserved protein region of RIPOR2 which is present in all RIPOR2