A RIPOR2 in-frame deletion is a frequent and highly penetrant cause of adult-onset hearing loss

Abstract

Background: Hearing loss is one of the most prevalent disabilities worldwide, and has a significant impact on quality of life. The adult-onset type of the condition is highly heritable but the genetic causes are largely unknown, which is in contrast to childhood-onset hearing loss.

Methods: Family and cohort studies included exome sequencing and characterisation of the hearing phenotype. Ex vivo protein expression addressed the functional effect of a DNA variant.

Results: An in-frame deletion of 12 nucleotides in RIPOR2 was identified as a highly penetrant cause of adult-onset progressive hearing loss that segregated as an autosomal dominant trait in 12 families from the Netherlands. Hearing loss associated with the deletion in 63 subjects displayed variable audiometric characteristics and an average (SD) age of onset of 30.6 (14.9) years (range 0-70 years). A functional effect of the RIPOR2 variant was demonstrated by aberrant localisation of the mutant RIPOR2 in the stereocilia of cochlear hair cells and failure to rescue morphological defects in RIPOR2-deficient hair cells, in contrast to the wild-type protein. Strikingly, the RIPOR2 variant is present in 18 of 22 952 individuals not selected for hearing loss in the Southeast Netherlands.

Conclusion: Collectively, the presented data demonstrate that an inherited form of adult-onset hearing loss is relatively common, with potentially thousands of individuals at risk in the Netherlands and beyond, which makes it an attractive target for developing a (genetic) therapy.

Keywords: genetics; human genetics; molecular genetics.

Figure 1

Pedigree of family W97-056 and segregation of RIPOR2 variant c.1696_1707del for affected and unaffected family members. The age of onset of hearing loss or the age at the most recent audiometric evaluation are indicated in the pedigree symbols, respectively. Subjects who did not report an age of onset are indicated with a question mark. The index case is marked by an arrow. Exome sequencing was performed in subjects with an underlined genotype. Subjects determined to be affected by heteroanamnesis are indicated with a vertical black bar. The subject marked in grey is diagnosed with intellectual disability and excluded from further participation in this study. Subject identifiers correspond to those in de Brouwer et al.. M, c.1696_1707del; +, wild-type.

Figure 2

Family pedigrees and segregation of RIPOR2 variant c.1696_1707del for affected and unaffected family members, the age of onset of hearing loss or the age at the most recent audiometric evaluation are indicated in the pedigree symbols, respectively. Subjects who did not report an age of onset are indicated with a question mark. Index cases are marked by arrows. Exome sequencing was performed in subjects with an underlined genotype. Subjects determined to be affected by heteroanamnesis are indicated with a vertical black bar. Based on the information provided in the questionnaires, an autosomal dominant inheritance pattern of hearing loss is likely for each of the single cases. M, c.1696_1707del; +, wild-type; PS, primary school.

Figure 3

Selection of audiograms and the ARTA. (A–C) Air conduction thresholds of three selected individuals with the c.1696_1707del RIPOR2 variant are depicted. For the individuals in panels A and B, hearing loss was symmetric and the average of left and right ear thresholds are depicted. For the individual in panel C, hearing loss was asymmetric and the thresholds for both right and left ears are depicted. The p95 values are matched to the individuals’ sex and age at most recent audiometry, according to the iso 7029:2017 standard. (D) The age related typical audiogram (ARTA): cross-sectional linear regression analysis of last visit audiograms of affected subjects with the c.1696_1707del RIPOR2 variant. dB HL, decibel hearing level; kHz; kilohertz; L, left; R, right; y, age in years; .

Figure 4

Four audiometric patterns of RIPOR2-associated hearing loss. Air conduction thresholds of all subjects were analysed with a k-means clustering protocol. The thick black lines depict the average of each cluster, the transparent grey areas represent the ±2 SD. Cluster 1: mild hearing loss (average (PTA_{0.5-4 kHz}) 23 dB HL) with an inverse U-shape audiogram. Cluster 2: moderate hearing loss (average 48dB 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 (online supplementary figure 5) shows relatively faster deterioration of higher frequencies later in life, for example W97-056 IV:20. dB HL, decibel hearing level; hl, kHz, kilohertz; PTA, pure tone average.

Figure 5

Functionality of mutant RIPOR2 is altered in mouse cochlear outer hair cells. (A) Mutant RIPOR2 differed in localisation from wild-type RIPOR2 in mouse cochlear outer hair cells. Outer hair cells of wild-type mice were injectoporated at P2 to express murine N-terminally HA-tagged wild-type RIPOR2 (RIPOR2wt) or mutant RIPOR2 (RIPOR2mut). Expression was evaluated after 2 days by immunohistochemistry and three representative images of cells expressing the mutant RIPOR2 are provided. Eleven cells expressing the wild-type construct and 12 cells expressing the mutant construct were evaluated. (B) Mutant RIPOR2 did not rescue stereocilia defects in RIPOR2-deficient hair cells. Cochlear explants of RIPOR2-deficient mice were prepared at P2 and injectoporated with constructs RIPOR2wt or RIPOR2mut. After culturing for 2 days, five out of six cells expressing the wild-type RIPOR2 construct demonstrated rescued hair bundle morphology but none of the 13 cells expressing the mutant RIPOR2 construct. Cells expressing the constructs are boxed. HA-tagged protein was stained in green, stereocilia were stained using phalloidin (phal) conjugated with Alexa Fluor 568 (red). HA, haemagglutinin. Scale bar represents 5 μm.