Refinement of Molecular Diagnostic Protocol of Auditory Neuropathy Spectrum Disorder: Disclosure of Significant Level of Etiologic Homogeneity in Koreans and Its Clinical Implications

Abstract

Auditory neuropathy spectrum disorder (ANSD) is a sensorineural hearing disorder caused by dysfunction of auditory neural conduction. ANSD has a heterogeneous etiology, including genetic factors; the response to cochlear implantation significantly varies depending on the etiology. The results of timely cochlear implantation for OTOF-related ANSD (DFNB9) have been reported to be good. Therefore, identifying the causative gene of ANSD, especially OTOF, is an important issue to rehabilitate these patients.Six sporadic ANSD subjects without anatomical abnormality of the cochlear nerve, including the 4 subjects that were previously reported to be without detectable OTOF mutation, were included. We performed targeted resequencing (TRS) of known deafness genes and multiphasic bioinformatics analyses of the data that ensured detection of capture failure and structural variations. Exclusion of SNP was also double checked. The TRS data previously obtained from 2 subjects were reanalyzed. Through this study, we detected 2 mutant alleles of OTOF from 5 (83.3%) of 6 ANSD subjects. All of the 5 subjects carried at least 1 mutant allele carrying p.R1939Q. This variant was categorized as a simple SNP (rs201326023) in the database and it resided in the exon with frequent capture failures, which previously led to exclusion of this variant from eligible candidacy mistakenly. In addition, we detected a structural variation within OTOF from a previously undiagnosed ANSD subject, which was the second structural variation reported in DFNB9 subjects to date.We identify a strong etiologic homogeneity of prelingual ANSD in case of the anatomically normal cochlear nerve in Koreans and now report DFNB9 as the single overwhelming cause. Multiphasic analysis of TRS data ensuring detection of capture failure and structural variations would be expected to reveal DFNB9 from a substantial portion of previously undiagnosed ANSD subjects in Koreans. Based on our results, we propose a novel strategy that incorporates imaging studies, prevalent mutation screening and multiphasic analysis of TRS data in a stepwise manner to correctly detect DFNB9 in Koreans.

FIGURE 1

Hierarchical and multiphasic diagnostic pipeline. This iagnostic strategy involves imaging, screening of a prevalent mutation, and multiphasic analysis of TRS data for etiologic diagnosis of auditory neuropathy spectrum disorder. AR = autosomal recessive, PCR = polymerase chain reaction, SNP = single-nucleotide polymorphism, TRS = targeted resequencing.

FIGURE 2

Clinical features suggesting auditory neuropathy spectrum disorder with the intact cochlear nerve. Audiologic results from 6 probands (A: SH81–185, B: SH132–273, C: SB10–23, D: SB22–51, E: SB42–79, F: SB204–398) show normal otoacoustic emission (OAE) response but no response to 90 or 100 dB of click sound in the auditory brainstem response tests. Internal auditory canal magnetic resonance images from 5 probands (A: SH81–185, B: SH132–273, C: SB10–23, D: SB22–51, F: SB204–398) show the intact cochlear nerve. Temporal bone computed tomography of SB42–79 (E) revealed no narrow bony cochlear nerve canal.

FIGURE 3

Sanger sequencing traces of variants and conservation of mutant residues (A) Sanger sequencing traces of SH81–185: c.5816G > A (p.R1939Q)/c.2521G > A (p.E856K), SH132–273: c.5816G > A (p.R1939Q) homozygote, SB10–23: c.5816G > A (p.R1939Q) homozygote, SB22–51: c.5816G > A (p.R1939Q)/skipped exon 12, SB204–398: c.5816G > A (p.R1939Q)/c.5566C > T (p.R1856W). (B) Conservation of mutant residues among orthologs from several species. p. R1939, p.E856, and p.R1856 are conserved among human, mouse, rat, cattle, monkey, chicken, and zebrafish. ∗P: proband, F: father, M: mother.

FIGURE 4

The second phase of TRS-134 data analyses (SB10–23, SB22–51, and SB204–398). (A) Targeted resequencing (TRS) data for SB10–23 and SB204–398 by the IGV viewer. A red box shows TRS capture failure compared with the control. The black box indicates each exon (referred to as “E”). (B) The region where a large genomic deletion is suspected in OTOF for SB22–51. Supportive discordant readings are distributed between Exon 11–13. (C) The result of breakpoint polymerase chain reaction (PCR) using our primer set shows genomic deletion of exon 12 in SB22–51 (proband), which is derived from SB22–53 (mother)'s one, while SB22–52 (father) has a normal allele with no deletion as below. The rightmost one is water control. Long range PCR and sequence analysis of PCR product figure out the exact breakpoint (chr2:26,710,657–chr2:26,706,557) with skipped exon 12. TRS-134 = targeted resequencing of the known 134 deafness genes.