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DIAPH3)/ Diaphanous Causes Hearing Defects in Humans with Auditory Neuropathy and in Drosophila

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DIAPH3)/ Diaphanous Causes Hearing Defects in Humans with Auditory Neuropathy and in Drosophila Increased activity of Diaphanous homolog 3 (DIAPH3)/ diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila Cynthia J. Schoena,b, Sarah B. Emeryc, Marc C. Thornec, Hima R. Ammanad,Elzbieta_ Sliwerska b, Jameson Arnettc, Michael Hortsche, Frances Hannand,f, Margit Burmeisterb,g,h, and Marci M. Lesperancec,1 aNeuroscience Graduate Program, bMolecular & Behavioral Neuroscience Institute, cDivision of Pediatric Otolaryngology, Department of Otolaryngology- Head and Neck Surgery, and Departments of eCell and Developmental Biology, gHuman Genetics, and hPsychiatry, University of Michigan Health System, Ann Arbor, MI 48109; and Departments of dCell Biology and Anatomy and fOtolaryngology, New York Medical College, Valhalla, NY 10595 Edited* by Mary-Claire King, University of Washington, Seattle, WA, and approved June 15, 2010 (received for review March 11, 2010) Auditory neuropathy is a rare form of deafness characterized by an We previously mapped the nonsyndromic auditory neuropathy, absent or abnormal auditory brainstem response with preservation autosomal dominant 1 (AUNA1) locus to chromosome 13q21-q24 of outer hair cell function. We have identified Diaphanous homolog in an American family of European descent (5). Affected individ- 3 (DIAPH3) as the gene responsible for autosomal dominant non- uals in this family develop hearing loss in the second decade of life syndromic auditory neuropathy (AUNA1), which we previously which rapidly progresses to profound deafness. Outer hair cell mapped to chromosome 13q21-q24. Genotyping of additional fam- function as measured by OAEs is preserved until approximately the ily members narrowed the interval to an 11-Mb, 3.28-cM gene-poor fifth decade of life. Electrically evoked responses of the auditory region containing only four genes, including DIAPH3. DNA sequenc- nerve return to normal after cochlear implantation, suggesting that ing of DIAPH3 revealed a c.-172G > A, g. 48G > A mutation in a highly the defect resides in the inner hair cells, the afferent synapses, or conserved region of the 5′ UTR. The c.-172G > A mutation occurs the terminal dendrites of the auditory nerve (6). Except for the within a GC box sequence element and was not found in 379 con- possibility of slightly earlier onset in homozygotes, no phenotypic trols. Using genome-wide expression arrays and quantitative RT- differences between homozygotes and heterozygotes were ob- GENETICS PCR, we demonstrate a 2- to 3-fold overexpression of DIAPH3 mRNA served in this family. in lymphoblastoid cell lines from affected individuals. Likewise, a sig- nificant increase (≈1.5-fold) in DIAPH3 protein was found by quan- Results titative immunoblotting of lysates from lymphoblastoid cell lines Genotyping of additional short tandem repeat markers as well as derived from affected individuals in comparison with controls. In single nucleotide polymorphisms in the linkage region refined the > fi addition, the c.-172G A mutation is suf cient to drive overexpres- telomeric boundary of AUNA1 to D13S1309, excluding the proto- sion of a luciferase reporter. Finally, the expression of a constitu- cadherin 9 gene (PCDH9) (7). Genotyping of a DNA sample from tively active form of diaphanous protein in the auditory organ of a distantly related affected family member revealed a recombi- Drosophila melanogaster recapitulates the phenotype of impaired nation event at marker D13S1492, which redefined the centromeric response to sound. To date, only two genes, the otoferlin gene end of the AUNA1 interval (Fig. S1). The refined AUNA1 interval OTOF and the pejvakin gene PJVK, are known to underlie nonsyn- spans an 11-Mb, 3.28-cM genomic region containing only four dromic auditory neuropathy. Genetic testing for DIAPH3 may be genes: protocadherin 17 (PCDH17), protocadherin 20 (PCDH20), useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy. Tudor domain-containing 3 (TDRD3), and DIAPH3. DNA sequencing of all exons and intron–exon junctions of DIAPH3 demonstrated a point mutation in the 5′ UTR, c.-172G > genetics | 5’UTR | formin | overexpression | DIAPH1 A, g.48G > A, occurring within a consensus GC box DNA recog- nition site (8). The 5′ UTR DNA sequence is highly conserved in uditory neuropathy (AN) is a rare form of deafness resulting the vertebrate orthologs of the human DIAPH3 gene (Fig. S2). The Afrom a disorder of inner hair cells, their synapses with the mutation is absent in 379 controls (758 control chromosomes), auditory nerve (VIII nerve), or the auditory nerve itself, with segregates with deafness, and, as predicted by the linkage analysis preservation of outer hair cell function (1). In contrast, most forms (5), is homozygous in two affected subjects. DNA sequencing of of deafness are associated with a loss of outer hair cell function, as PCDH17, PCDH20, and TDRD3 revealed no novel variants from typically measured by otoacoustic emissions (OAEs). Although the reference sequences. there are more than 60 genes for nonsyndromic deafness, the ge- Whole-genome expression arrays were used to identify genes that netic architecture of nonsyndromic auditory neuropathy is not well are differentially expressed in mRNA from lymphoblastoid cell lines fi Diaphanous ho- understood. We now report the identi cation of (LCLs) from affected individuals and controls. Signal intensity was molog 3 (DIAPH3) as a gene responsible for a dominant form of 2- to 3-fold higher in affected individuals than in controls for two nonsyndromic auditory neuropathy. probes representing the DIAPH3 gene (Hs. 576922 and DIAPH3; DIAPH3 is one of three human orthologs of Drosophila di- P = 0.000029 and P = 0.000713, respectively). To confirm and aphanous. A mutation in DIAPH1 underlies DFNA1, autosomal dominant nonsyndromic sensorineural hearing loss (2), whereas mutations in the X-linked DIAPH2 cause premature ovarian failure Author contributions: C.J.S., S.B.E., M.H., F.H., M.B., and M.M.L. designed research; C.J.S., (3). These genes encode diaphanous-related formin (DRF) pro- S.B.E., M.C.T., H.R.A., E.S., J.A., M.H., F.H., M.B., and M.M.L. performed research; M.H., F.H., teins, actin nucleation factors involved in maintenance of cell po- and M.B. contributed new reagents/analytic tools; C.J.S., S.B.E., M.C.T., E.S., M.H., F.H., M.B., larity and cell shape, intracellular transport, and vesicular trafficking and M.M.L. analyzed data; and C.J.S., S.B.E., M.H., F.H., M.B., and M.M.L. wrote the paper. (4). DRFs are maintained in an inactive state through an auto- The authors declare no conflict of interest. inhibitory interaction between the N-terminal GTPase-binding *This Direct Submission article had a prearranged editor. domain (GBD) and the C-terminal diaphanous autoinhibitory do- 1To whom correspondence should be addressed. E-mail: [email protected]. main (DAD) (4). Binding of Rho-GTP to the GBD activates DRFs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. through displacement of DAD, a process which is tightly regulated. 1073/pnas.1003027107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1003027107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 quantify the expression changes seen on microarrays, the threshold cycle (Ct) for DIAPH3, as compared with the Ct for a housekeeping gene, β-glucuronidase (GUSB), was determined for controls, het- erozygotes, and homozygotes by quantitative RT-PCR of mRNA from LCLs. The estimated difference of marginal means for ΔC (Ct GUSB – Ct DIAPH3)wassignificant for controls vs. hetero- zygotes (−0.4285 vs. 0.6496, P = 0.0028) and controls vs. homo- zygotes (−0.4285 vs. 1.1466, P < 0.001) (Fig. 1). Expression was increased 2.11-fold in heterozygotes compared with controls and 2.98-fold in homozygotes compared with controls, as reflected by an increase in ΔC values (Fig. 1). Results for homozygotes were not significantly different from those for heterozygotes (P = 0.2641). To determine the effect of increased mRNA expression on levels of protein expression, we used immunoblotting to compare relative amounts of DIAPH3 protein in LCL lysates from controls, heterozygotes, and homozygotes (Fig. 2A). GADPH was used as a loading control, and the DIAPH3/GAPDH ratio was calculated. Fig. 2. Expression of DIAPH3 protein is significantly increased in lysates from There was a significant difference in the estimated marginal means LCLs from heterozygotes and homozygotes vs. controls. (A) Immunoblot of for the DIAPH3/GAPDH ratio in controls vs. heterozygotes (4.83 LCL lysates from two wild types, two homozygotes, and two heterozygotes vs.7.14, P = 0.045) and controls vs. homozygotes (4.83 vs. 7.85, P = using DT154 antibody (43, 44). The DIAPH3 band is at 137 kDa (UniProt da- tabase); cross-reactivity is seen with DIAPH1 (140 kDa) and DIAPH2 (126 kDa). 0.0093) (Fig. 2B). Protein levels were increased 1.48-fold in het- GAPDH was used as a loading control, and DIAPH3 densitometry was nor- erozygotes and 1.62-fold in homozygotes relative to controls (Fig. malized to GAPDH densitometry. (B) Estimated marginal means and SE for the 2B). There was no significant difference in protein levels between DIAPH3/GAPDH ratio for wild types, homozygotes, and heterozygotes tested homozygotes and heterozygotes (P = 0.68). in four batches of cells. There was a significant difference between controls Given the increased expression of DIAPH3 observed in mRNA and heterozygotes (4.83 vs.7.14, P = 0.045, a 1.48-fold increase) and between from affected family members, we investigated whether increased controls and homozygotes (4.83 vs. 7.85, P = 0.0093, a 1.62-fold increase). expression could be explained by the presence of the 5′ UTR mu- There was no significant difference in protein levels between homozygotes tation. Although promoter and/or enhancer sequences for DIAPH3 and heterozygotes (P = 0.68).
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