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 (approximately 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.

A novel paradigm to evaluate conditioned pain modulation in fibromyalgia.

INTRODUCTION: Application of noxious stimulation to one body area reduces pain sensitivity in a remote body area through activation of an endogenous pain-inhibitory network, a behavioral phenomenon referred to as conditioned pain modulation (CPM). The efficiency of CPM is predictive of a variety of health outcomes, while impaired CPM has been associated with various chronic pain conditions. Current methods used to assess CPM vary widely, and interest in CPM method development remains strong. Here, we evaluated a novel method for assessing CPM in healthy controls and fibromyalgia (FM) patients using thumb pressure as both a test and conditioning stimulus. METHODS: Sixteen female FM patients and 14 matched healthy controls underwent CPM testing with thumbnail pressure as the test stimulus, and either cold water or noxious pressure as the conditioning stimulus. CPM magnitude was evaluated as the difference in pain rating of the test stimulus applied before and during the conditioning stimulus. RESULTS: In healthy controls, application of either pressure or cold water conditioning stimulation induced CPM as evidenced by a significant reduction in test stimulus pain rating during conditioning (P=0.007 and P=0.021, respectively). In contrast, in FM patients, neither conditioning stimulus induced a significant CPM effect (P>0.274). There was a significant difference in CPM magnitude for FM patients compared to healthy controls with noxious pressure conditioning stimulation (P=0.023); however, no significant difference in CPM was found between groups using cold water as a conditioning stimulus (P=0.269). CONCLUSION: The current study demonstrates that thumbnail pressure can be used as both a test and conditioning stimulus in the assessment of CPM. This study further confirms previous findings of attenuated CPM in FM patients compared with healthy controls.

Diaphanous homolog 3 (Diap3) overexpression causes progressive hearing loss and inner hair cell defects in a transgenic mouse model of human deafness.

We previously demonstrated that a mutation in the 5' untranslated region of Diaphanous homolog 3 (DIAPH3) results in 2 to 3-fold overexpression of the gene, leading to a form of delayed onset, progressive human deafness known as AUNA1 (auditory neuropathy, nonsyndromic, autosomal dominant, 1). To investigate the mechanism of deafness, we generated two lines of transgenic mice overexpressing Diap3, the murine ortholog of DIAPH3, on an FVB/NJ background. Line 771 exhibits a relatively mild 20 dB hearing loss at 12 kHz at 4 and 8 weeks of age, progressing to 40 dB and 60 dB losses at 16 and 24 weeks, respectively, at 12 and 24 kHz. Line 924 shows no hearing loss at 4 or 8 weeks, but manifests 35 and 50 dB threshold shifts at 16 and 24 weeks, respectively, at both 12 and 24 kHz. Notably, mice from the two transgenic lines retain distortion product otoacoustic emissions, indicative of normal cochlear outer hair cell (OHC) function despite elevation of auditory thresholds. Scanning electron microscopy of the organ of Corti demonstrates striking anomalies of the inner hair cell (IHC) stereocilia, while OHCs are essentially intact. Over time, IHCs of both lines develop elongated stereocilia that appear fused with neighboring stereocilia, in parallel to the time course of hearing loss in each line. Furthermore, we observe significant reduction in the number of IHC ribbon synapses over 24 weeks in both lines, although this reduction does not correlate temporally with onset and progression of hearing loss or stereociliary anomalies. In summary, overexpression of wild-type Diap3 in two lines of transgenic mice results in hearing loss that recapitulates human AUNA1 deafness. These findings suggest an essential role of Diap3 in regulating assembly and/or maintenance of actin filaments in IHC stereocilia, as well as a potential role at the IHC ribbon synapse.

GABAergic lineage differentiation of AF5 neural progenitor cells in vitro.

We have previously described an immortal rat central-nervous-system progenitor cell line, AF5, which is able to exit the cell cycle and assume a differentiated state with neuronal properties. The phenotypic specification of differentiated AF5 cells, however, is not known. In the present study, when induced to differentiate by serum starvation in Neurobasal medium, AF5 cells down-regulate glial fibrillary acidic protein and up-regulate expression of beta-III-tubulin, medium-molecular-weight neurofilament protein, and neuronal growth-associated protein 43. Expression of the gamma-aminobutyric acid (GABA) lineage marker, glutamic acid decarboxylase 67 (GAD67), increases during differentiation, suggesting that AF5 cells adopt a GABAergic lineage. Time-course analysis of the GABAergic neuron specification transcription factor, Pitx2, by reverse transcription/polymerase chain reaction, has shown an increase in the Pitx2 transcript 48 h after initiation of differentiation. In differentiated AF5 cells, expression of the Pitx2 target gene products GAD65 and GABA transporter-1 increases. Cellular GABA levels in differentiated AF5 cells increase by about 26-fold, and GABA release into the medium is 150-fold higher compared with that of undifferentiated cells. Therefore, AF5 cells can be induced to differentiate to a neuronal phenotype with a GABAergic lineage.