A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

Abstract

Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.

Author Summary

It is known that hearing impairment running in families can be caused by mutations in more than eighty different genes. However, there are still families where the responsible gene is unknown. By studying a large Danish family with dominant inherited hearing impairment, we found that the disorder cosegregates with genetic markers on chromosome 6, suggesting that the responsible mutation lies within this chromosomal region. By sequencing this genetic locus, we discovered a mutation in the CD164 gene that is passed on to all the affected individuals. In the mouse ear, we demonstrated that the CD164 protein is expressed in hair cells and other sites known to be important for correct hearing. The identified mutation is predicted to result in shortening of the protein, leading to loss of an evolutionary conserved sequence important for cellular trafficking of CD164. Using cell lines, we show that the truncated protein is trapped on the cell surface while the normal protein is internalized. This finding is important because it implicates for the first time a role for CD164 in the complex physiological processes of hearing and suggests that failed endocytosis may be a possible disease mechanism for some types of hearing impairment.