Mutations in TBC1D24, a gene associated with epilepsy, also cause nonsyndromic deafness DFNB86

Abstract

Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.

Figure 1

Refinement of the DFNB86 Linkage Interval The thick vertical bar represents human chromosome 16p. The linkage interval for the deafness segregating in each of the four families is indicated by a thin vertical bar. The gray shaded region highlights the DFNB86 linkage interval shared by the four families.

Figure 2

Four Families in which Deafness Cosegregates with Missense Mutations in TBC1D24 (A) Profound deafness in three families cosegregates with the c.208G>T (p.Asp70Tyr) variant in TBC1D24. Representative chromatograms from wild-type and mutant sequences are shown. Altered nucleotides and affected residues are highlighted in gray. Genotypes of the participating family members are shown below each symbol in single-letter amino acid nomenclature. Individuals diagnosed with either seizures or epilepsy are highlighted by a gray rectangle. In family PKDF799, 10 of 11 deaf individuals have no history of seizures. Deaf female IV-13 had febrile seizures at the age of 8 years. In family DEM4587, there is no history of seizures in any of the eight clinically investigated family members, indicated by asterisks. Clinical re-evaluation of families DEM4221 and DEM4476 was not possible. (B) Deaf individuals from family DEM4476 are homozygous for the c.878G>C (p.Arg293Pro) variant in TBC1D24, whereas normal-hearing parents and siblings of affected family members are heterozygous carriers of this mutation.

Figure 3

Structure of TBC1D24 and Amino Acid Conservation of the Affected TBC1D24 Residues (A) ClustalW2 alignment of TBC1D24 orthologs shows that the two DFNB86-associated mutations affect conserved residues (red font). Amino acid residue identity and similarity percentages were calculated for full-length TBC1D24. (B) Graphic representation of TBC1D24 structure (left) and its encoded protein (right). The red bar above the TLDc domain represents the epitope for the commercial antibody (Abcam, ab101933) that we used in Figure 4. DFNB86-associated mutations are shown above the diagram, and epilepsy-associated mutations are shown below. Tall and short gray boxes represent coding exons and UTRs of the transcribed gene, respectively. Horizontal lines joining the gray boxes denote introns. The longest isoform of TBC1D24 was used as the reference sequence for mutation nomenclature (RefSeq NM_001199107.1 and NP_001186036.1). c. 1544C>T (p.Ala515Val) is renamed here according to the longest isoform but was originally reported as c.1526C>T (p.Ala509Val) on the basis of a shorter isoform of TBC1D24 (Falace et al.²⁴).

Figure 4

TBC1D24 Is Found in Spiral Ganglion Neurons of the Mouse Inner Ear (A) TBC1D24 antibody binds to spiral ganglion cells in the P30 cochlea. (B) The corresponding signal is absent in a control experiment with no added primary antibody. (C) A higher-magnification image reveals TBC1D24 antibody binding to the cell body and axonal projections of spiral ganglion. Scale bars in (A) and (B) represent 40 μm, whereas that in (C) represents 10 μm.