Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila

Auditory neuropathy is a rare form of deafness characterized by an absent or abnormal auditory brainstem response with preservation of outer hair cell function. We have identified Diaphanous homolog 3 (DIAPH3) as the gene responsible for autosomal dominant nonsyndromic auditory neuropathy (AUNA1), which we previously mapped to chromosome 13q21-q24. Genotyping of additional family members narrowed the interval to an 11-Mb, 3.28-cM gene-poor region containing only four genes, including DIAPH3. DNA sequencing of DIAPH3 revealed a c.-172G > A, g. 48G > A mutation in a highly conserved region of the 5′ UTR. The c.-172G > A mutation occurs within a GC box sequence element and was not found in 379 controls. Using genome-wide expression arrays and quantitative RT-PCR, we demonstrate a 2- to 3-fold overexpression of DIAPH3 mRNA in lymphoblastoid cell lines from affected individuals. Likewise, a significant increase (≈1.5-fold) in DIAPH3 protein was found by quantitative immunoblotting of lysates from lymphoblastoid cell lines derived from affected individuals in comparison with controls. In addition, the c.-172G > A mutation is sufficient to drive overexpression of a luciferase reporter. Finally, the expression of a constitutively active form of diaphanous protein in the auditory organ of Drosophila melanogaster recapitulates the phenotype of impaired response to sound. To date, only two genes, the otoferlin gene OTOF and the pejvakin gene PJVK, are known to underlie nonsyndromic auditory neuropathy. Genetic testing for DIAPH3 may be useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy.