Shiming Yang

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9–guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9–guide RNA–lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein–RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

A novel DFNA5 mutation, IVS8+4 A>G, in the splice donor site of intron 8 causes late-onset non-syndromic hearing loss in a Chinese family.

We report here the clinical, genetic, and molecular characteristics of a large Chinese family exhibiting non‐syndromic, late‐onset autosomal dominant sensorineural hearing loss. Clinical evaluation revealed variable phenotypes of hearing loss in terms of severity and age‐at‐onset of disease in these subjects. Genome‐wide linkage analysis mapped the disease gene to the DFNA5 locus with a maximum two‐point log odds score of 5.39 at [theta] = 0 for marker D7S2457. DNA sequencing of DFNA5 revealed a novel heterozygous IVS8+4 A>G substitution in the splice donor site of intron 8. Reverse transcriptase–polymerase chain reaction (RT–PCR) showed skipping of exon 8 in the mutant transcript. This mutation faithfully cosegregated with hearing loss in the family. In addition, the mutation was absent in 100 unrelated control DNA samples of Chinese origin. The IVS8+4 A>G mutation is predicted to create a shift in the reading frame and introduce a stop codon at position 372, thereby resulting in a prematurely truncated DFNA5 protein. Up to date, a total of four mutations in DFNA5 have been reported to lead to hearing impairment, all of them result in skipping of exon 8 at the mRNA level. Our findings provide further support for the hypothesis that DFNA5‐associated hearing loss is caused by a very specific gain‐of‐function mutation.

The differentiation of mesenchymal stem cells into inner ear hair cell-like cells in vitro

Abstract Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

Novel mutations in the vWFA2 domain of COCH in two Chinese DFNA9 families.

To the Editor: The autosomal dominant sensorineural nonsyndromic hearing loss (HL) 9 (DFNA9) locus is known to be associated with vestibular dysfunction (1). The DFNA9 causative gene COCH localizes on chromosome 14q12-q13 (2) and encodes cochlin, an extracellular matrix protein. Cochlin contains a region homologous to a domain in factor C of Limulus, also known as LCCL domain, and two von Willebrand factor A-like domains (vWFA1 and 2). Interestingly, most of the previously described COCH mutations are missense point mutations located within exon 4 or 5 encoding the LCCL domain of cochlin (1, 3–10). In 2005, Street et al. (11) reported a p.C542F mutation in an American DFNA9 familywithHLaswell as vestibular andoculomotor disturbances. This mutation located within the second vWFA (vWFA2) domain of cochlin, representing the first reported DFNA9 mutation outside the LCCL domain. In the present study, we have ascertained a large Chinese family (SD-Z001) with late-onset autosomal dominant non-syndromic progressive sensorineural HL. The family included 113 members spanning six generations. Appropriate informed consent was obtained from participants in accordance with the Ethics Committee of Chinese PLA General Hospital. Thirty-six members from the family considered informative were selected for linkage analysis. A maximum two-point logarithm of odds ratio score of 6.69 at y 1⁄4 0 was obtained for marker D14S1040. Haplotype analysis placed the locus within a 7.6-cM genetic interval defined by markers D14S1021 and D14S70, overlapping with the DFNA9 locus on chromosome 14q12-q13. DNA sequencing of the coding regions as well as exon–intron boundaries of the COCH gene and subsequent confirmation by MfeI restriction endonuclease analysis revealed a c.1625G.A mutation (Fig. 1a,c) in exon 12 that cosegregates with auditory dysfunction in the pedigree. The mutation results in a predicted p.C542Y substitution at an evolutionarily conserved cysteine residue in the vWFA2 domain of cochlin (Fig. 1b,d). Furthermore, we screened COCH for mutations in 26 Chinese DFNA families as well as in 19 small families (the inheritance pattern could not be recognized) and 22 sporadic patients with lateonset progressive sensorineural HL. A heterozygous missense mutation (c.1535T.C) that converted an evolutionarily conserved methionine residue to a threonine residue (p.M512T) was also identified in the vWFA2 domain (Fig. 1 a,b,d) in a small family (HLJ-Z079), which had only two patients. Both mutations were absent in 100 unrelated control DNA samples of Chinese background, supporting the hypothesis that they represent a causative mutation and not a rare polymorphism. We did not detect any polymorphism in the COCH gene in the families during the sequence analysis. The age of onset of HL varied in the SD-Z001 family. It ranged from the second to the fifth decade of life. For generation III, the age at onset was at the fifth decade, generation IV at fourth, generation V at third and generation VI at second to third. The finding of a lower age at diagnosis among offspring compared with their parents suggests the presence of genetic anticipation. To the best of our knowledge, there are no earlier reports on the phenomenon in DFNA9 families. There are multiple mechanisms by which anticipation occurs including biological, environmental, and also statistical because of ascertainment or truncation bias (12). Therefore, in the absence of biological information, the intergenerational difference in age at diagnosis is difficult to interpret, and attention to observational biases is warranted. Tinnitus at the onset of HL was reported in 82% of the affected family members. The HL first affected the high frequencies and later involved all frequencies. Overall, the patients display a downward sloping audiogram contour. In the HLJ-Z079 family, the proband (III-5) began suffering bilateral HL at the age of 43 years. At the onset of HL, she

Noise induced reversible changes of cochlear ribbon synapses contribute to temporary hearing loss in mice.

Abstract Conclusion: Noise exposure can cause a decline in cochlear ribbon synapses and result in consequent hearing loss. The reduction of synaptic puncta appears reversible and may contribute to hearing restoration in mice after noise exposure. Objective: To detect whether noise induced reversible changes of cochlear ribbon synapses contribute to temporary hearing loss in C57BL/6J mice. Methods: The mice were assigned randomly to five groups and exposed to white noise at 110 dB SPL for 2 h except the control group. ABR thresholds were acquired before noise exposure (control), immediately following exposure (Day 0), or on Days 4, 7, or 14 after noise exposure. Light microscopy, scanning emission microscopy, and whole mounts examination was utilized to study whether there is morphology change of outer hair cells (OHC), inner hair cells (IHC), or spiral ganglion cells (SGN) due to the 110 dB white noise. Moreover, experimental approaches, including immunostaining and confocal microcopy, were used to detect whether ribbon synapses were the primary targets of noise-induced temporary hearing loss. Result: Exposure to 110 dB white noise for 2 h induced TTS in mice, with the maximal ABR threshold elevations seen on the 4th day after noise exposure. There were no significant morphological changes in the cochlea. Paralleled changes of pre-synaptic ribbons in both the number and post-synaptic density (PSDs) during this noise exposure were detected. The number of pre-synaptic ribbon, post-synaptic density (PSDs), and co-localized puncta correlated with the shifts of ABR thresholds. Moreover, a complete recovery of ABR thresholds and synaptic puncta was seen on the 14th day after the noise stimulations.

De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome

De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome

Primary tumours of the facial nerve: diagnostic and surgical treatment experience in Chinese PLA General Hospital.

Conclusions. The commonest manifestation of facial nerve tumours was facial paralysis, followed by hearing loss. During tumour resection facial nerve continuity should be maintained and reconstructed in one stage wherever possible. If this is not a viable option, second-stage surgery should be performed as soon as possible after surgery. Objective. To summarize the clinical characteristics of tumours of the facial nerve and discuss their diagnosis and treatment. Patients and methods. Twenty-two cases of primary facial nerve tumours were reviewed. These cases were confirmed pathologically and treated in the Chinese PLA General Hospital during the period 1986–2003, where the clinical manifestations, diagnosis and treatment of this series were analysed. Results. Among the 22 cases, 14 were facial neurilemmomas, 6 were facial neurofibromas and 2 were facial nerve haemangiomas. The commonest presenting symptom in all cases was facial paralysis (14/22) followed by hearing loss (10/22). Facial paralysis was also the commonest sign of a facial nerve tumour (18/22), followed by a swollen mass in the tympanic cavity (8/22) and a swollen mass in the external auditory canal (5/22). The 22 tumours were totally resected surgically. The function of the facial nerve was normal (grade I) in two cases where the integrity of the nerve was preserved during the operation, grade II in one case and grade III in another case where it was possible to maintain partial continuity of the facial nerve. The facial nerve was reconstructed in one stage when the tumours were resected, with facial–great auricular–facial nerve cable grafting (10 cases) and facial–lateral femoral cutaneous–facial nerve cable grafting (1 case). The facial nerve function consequently recovered to grade II–IV. The second stage facial–hypoglossal nerve anastomosis was carried out in two cases, and facial function consequently recovered to grade II in one case at 3 years and grade III in another with 2 years follow-up. In five cases, the facial nerve remained discontinuous and the facial nerve function showed no recovery (grade VI).

The Morphology and Electrophysiology of the Cochlea of the Miniature Pig

To report the cochlear morphology and electrophysiology of Chinese experimental miniature pigs. Twenty Chinese experimental miniature pigs were used in this study. Auditory brainstem responses (ABR), cochlear endolymphatic potentials (EP), and the potassium concentrations of cochlear endolymph were recorded. Hair cell morphology was examined using electron microscopy. The capsule of cochlea of the miniature pig has three and one‐half turns which contains a 39‐mm long membranous labyrinth. The organ of Corti in the labyrinth encompasses three rows of outer hair cells and one row of inner hair cells. The stereocilia of the hair cells in the apical turn of the cochlea were significantly longer than those in the basal turn. The vestibular apparatus consists of three semicircular canals and the otolith organs. The average threshold of the ABR was 35–45 dB SPL (n = 20) from 4 to 32 kHz. There was no significant difference in the threshold or latency of the ABR between 1‐day‐old and 30‐day‐old miniature pigs. The average EP value was 77.3 ± 14 mV (n = 9) and the average potassium concentration was 147.1 ± 13 mM (n = 5) recorded from the second turn of the cochlea. These studies on the cochlear morphology and electrophysiology of the miniature pigs help to establish the Chinese experimental miniature pig as an animal model for future studies in otology and audiology. Anat Rec, 298:494–500, 2015.

Genetic and Phenotypic Heterogeneity in Chinese Patients with Waardenburg Syndrome Type II

Waardenburg Syndrome (WS) is an autosomal-dominant disorder characterized by sensorineural hearing loss and pigmentary abnormalities of the eyes, hair, and skin. Microphthalmia-associated transcription factor (MITF) gene mutations account for about 15% of WS type II (WS2) cases. To date, fewer than 40 different MITF gene mutations have been identified in human WS2 patients, and few of these were of Chinese descent. In this study, we report clinical findings and mutation identification in the MITF gene of 20 Chinese WS2 patients from 14 families. A high level of clinical variability was identified. Sensorineural hearing loss (17/20, 85.0%) and heterochromia iridum (20/20, 100.0%) were the most commonly observed clinical features in Chinese WS2 patients. Five affected individuals (5/20, 25.0%) had numerous brown freckles on the face, trunk, and limb extremities. Mutation screening of the MITF gene identified five mutations: c.20A>G, c.332C>T, c.647_649delGAA, c.649A>G, and c.763C>T. The total mutational frequency of the MITF gene was 21.4% (3/14), which is significantly higher than the 15.0% observed in the fair-skinned WS2 population. Our results indicate that MITF mutations are relatively common among Chinese WS2 patients.