A homozygous SLITRK6 nonsense mutation is associated with progressive auditory neuropathy in humans

Abstract

Objectives/hypothesis: SLITRK family proteins control neurite outgrowth and regulate synaptic development. In mice, Slitrk6 plays a role in the survival and innervation of sensory neurons in the inner ear, vestibular apparatus, and retina, and also influences axial eye length. We provide the first detailed description of the auditory phenotype in humans with recessive SLITRK6 deficiency.

Study design: Prospective observational case study.

Methods: Nine closely related Amish subjects from an endogamous Amish community of Pennsylvania underwent audiologic and vestibular testing. Single nucleotide polymorphism microarrays were used to map the chromosome locus, and Sanger sequencing or high-resolution melt analysis were used to confirm the allelic variant.

Results: All nine subjects were homozygous for a novel nonsense variant of SLITRK6 (c.1240C>T, p.Gln414Ter). Adult patients had high myopia. The 4 oldest SLITRK6 c.1240C>T homozygotes had absent ipsilateral middle ear muscle reflexes (MEMRs). Distortion product otoacoustic emissions (DPOAEs) were absent in all ears tested and the cochlear microphonic (CM) was increased in amplitude and duration in young patients and absent in the two oldest subjects. Auditory brainstem responses (ABRs) were dys-synchronised bilaterally with no reproducible waves I, III, or V at high intensities. Hearing loss and speech reception thresholds deteriorated symmetrically with age, which resulted in severe-to-profound hearing impairment by early adulthood. Vestibular evoked myogenic potentials were normal in three ears and absent in one.

Conclusion: Homozygous SLITRK6 c.1240C>T (p.Gln414Ter) nonsense mutations are associated with high myopia, cochlear dysfunction attributed to outer hair cell disease, and progressive auditory neuropathy.

Keywords: Amish; Auditory neuropathy; SLITRK6.

Figure 1. Gene discovery in autosomal recessive auditory neuropathy

We mapped and identified the gene using a single sibship which in isolated populations such as the Old Order Amish can be accomplished with as few as 2 affected individuals, then subsequently identified additional affected individuals from separate sibships. A) We performed a genome-wide homozygosity mapping study using Affymetrix GeneChip Mapping 10K SNP Arrays with a single Old Order Amish sibship with three affected. Two large, shared blocks of homozygosity were identified on chromosomes 13 and 14. However, the location score was highest for the chromosome 13q31 region and a subsequent affected child in the family was not homozygous for the chromosome 14 SNP markers. The common homozygous segment was flanked by SNPs rs722023 and rs958373, spanned 9.3 Mb and contained 33 known or predicted genes based on NCBI annotations. B) Sanger sequencing of the SLITRK6 gene revealed homozygosity for a nonsense variant in exon 2 in all affected individuals (c.1240C>T). The three panels depict sequencing of a normal homozygote, a carrier, and a mutation homozygote, respectively.

Fig. 2. Cochlear Microphonics and Auditory brainstem responses to condensation and rarefaction clicks

A) Right ear from subject 2. The CM is robust and continue over 3 milliseconds B) Waveforms from right and left ears from subject 4 are superposed for each stimulus polarity to show ear symmetry of the CM. C) Same as B) for subject 3.

Fig. 3. Auditory Brainstem Responses

A) ABR tracing from the right ear of Subject 2 that depicts the presence of wave I and II obscured by the large ringing CM. The waves are revealed by adding both condensation and rarefaction waveforms B) shows wave I in the right ear of Subject 6 when both condensation and rarefaction waveforms are added together. C) CM and neural synchrony were absent in subject 8.

Fig. 4. Behavioral Audiology

A) Averaged pure tone audiograms for 3 different age groups. B) Speech reception thresholds as a function of pure tone average (0.5, 1, 2, 4 kHz). C) Pure tone average as a function of age. D) Speech reception thresholds as a function of age. E) Pure tone average correlation between right and left ears. F) Correlation of speech reception thresholds between right and left ears.

Fig. 5. In Vitro Expression and Localization

A) SLITRK6 has an extracellular N-terminal extension that contains two SLIT-like leucine-rich repeat domains and an intracellular C-terminus with two conserved tyrosine residues that are subject to phosphorylation in a sequence homologous to the Ntrk neurotrophin receptors. The c.1240C>T (p.Gln414Ter) change introduces a premature termination codon predicted to truncate the protein after Leu⁴¹³ Conserved domains in human wild-type SLITRK6 (top) (Uniprot Q9H5Y7) and SLITRK6 Gln414Ter (bottom). LRRNT – leucine–rich repeat N-terminal domain, LRR – leucine-rich repeat, LRRCT – leucine-rich repeat C-terminal domain, TMD – transmembrane domain. C-terminal tyrosine residues 820 and 833 are phosphotyrosine residues in a conserved Ntrk-like domain.B) Western blot of lysates of UB/OC-2 cells overexpressing human SLITRK6 C-terminal V5 epitope-tagged constructs. SLITRK6 Gln414Ter is stably expressed as an approximately 50-kDa cytosolic protein. The increased abundance of SLITRK6 Gln414Ter relative to wild-type SLITRK6 despite equal total protein loading may result from lower efficiency in extracting wild-type SLITRK6 from the membrane relative to cytosolic SLITRK6 Gln414Ter. C & D) Heterologous overexpression of wild-type SLITRK6 in UB/OC-2 cells suggests that SLITRK6 traffics normally through the major mFembrane system to the plasma membrane. By contrast, SLITRK6 Gln414Ter appears to be relatively uniformly distributed throughout the cytosol without the membrane puncta characteristic of SLITRK family expression. Scale bar in E = 10 μm in C-E. Data in B are representative of 6 independent transfections. Data in C-E are representative of 4 independent transfections.