Evaluating agreement between evidence from randomised controlled trials and cohort studies in nutrition: a meta-research replication study.

This meta-research study aims to evaluate the agreement of effect estimates between bodies of evidence (BoE) from RCTs and cohort studies included in the same nutrition evidence synthesis, to identify factors associated with disagreement, and to replicate the findings of a previous study. We searched Medline, Epistemonikos and the Cochrane Database of Systematic Reviews for nutrition systematic reviews that included both RCTs and cohort studies for the same patient-relevant outcome or intermediate-disease marker. We rated similarity of PI/ECO (population, intervention/exposure, comparison, outcome) between BoE from RCTs and cohort studies. Agreement of effect estimates across BoE was analysed by pooling ratio of risk ratios (RRR) for binary outcomes and difference of standardised mean differences (DSMD) for continuous outcomes. We performed subgroup and sensitivity analyses to explore determinants associated with disagreements. We included 82 BoE-pairs from 51 systematic reviews. For binary outcomes, the RRR was 1.04 (95% confidence interval (CI) 0.99 to 1.10, I2 = 59%, τ2 = 0.02, prediction interval (PI) 0.77 to 1.41). For continuous outcomes, the pooled DSMD was - 0.09 (95% CI - 0.26 to 0.09, PI - 0.55 to 0.38). Subgroup analyses yielded that differences in type of intake/exposure were drivers towards disagreement. We replicated the findings of a previous study, where on average RCTs and cohort studies had similar effect estimates. Disagreement and wide prediction intervals were mainly driven by PI/ECO-dissimilarities. More research is needed to explore other potentially influencing factors (e.g. risk of bias) on the disagreement between effect estimates of both BoE.Trial registration: CRD42021278908.

CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct.

Enlargement of the endolymphatic sac, duct, and vestibular aqueduct (EVA) is the most common inner ear malformation identified in patients with sensorineural hearing loss. EVA is associated with pathogenic variants in SLC26A4. However, in European-Caucasian populations, about 50% of patients with EVA carry no pathogenic alleles of SLC26A4. We tested for the presence of variants in CHD7, a gene known to be associated with CHARGE syndrome, Kallmann syndrome, and hypogonadotropic hypogonadism, in a cohort of 34 families with EVA subjects without pathogenic alleles of SLC26A4. In two families, NM_017780.4: c.3553A > G [p.(Met1185Val)] and c.5390G > C [p.(Gly1797Ala)] were detected as monoallelic CHD7 variants in patients with EVA. At least one subject from each family had additional signs or potential signs of CHARGE syndrome but did not meet diagnostic criteria for CHARGE. In silico modeling of these two missense substitutions predicted detrimental effects upon CHD7 protein structure. Consistent with a role of CHD7 in this tissue, Chd7 transcript and protein were detected in all epithelial cells of the endolymphatic duct and sac of the developing mouse inner ear. These results suggest that some CHD7 variants can cause nonsyndromic hearing loss and EVA. CHD7 should be included in DNA sequence analyses to detect pathogenic variants in EVA patients. Chd7 expression and mutant phenotype data in mice suggest that CHD7 contributes to the formation or function of the endolymphatic sac and duct.

Hippocampal and neocortical BRAF mutant non-expansive lesions in focal epilepsies.

OBJECTIVE: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAFV600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAFV600E oncogenic variants and characterise the CD34+ cells. METHODS: We analysed BRAFV600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAFwildtype MTLE-HS and BRAFV600E mutant non-expansive lesion of hippocampus and/or neocortex. RESULTS: We identified a BRAFV600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAFV600E mutant samples. The co-expression of the oncogene-induced senescence marker p16INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. INTERPRETATION: BRAFV600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.

Asymmetric cell divisions and asymmetric cell fates.

The regulation of self-renewal, cell diversity, and differentiation can occur by modulating symmetric and asymmetric cell divisions. Remarkably, asymmetric cell divisions can arise through multiple processes in which molecules in the cytoplasm and nucleus, as well as template "immortal" DNA strands, can segregate to one daughter cell during cell division. Explaining how these events direct distinct daughter cell fates is a major challenge to understanding how the organism is assembled and maintained for a lifetime. Numerous technical issues that are associated with assessing how distinct cell fates are executed in vivo have resulted in divergent interpretations of experimental findings. This review addresses some of these points and considers different developmental model systems that attempt to investigate how cell fate decisions are determined, as well as the molecules that guide these choices.

Empirical evidence of study design biases in nutrition randomised controlled trials: a meta-epidemiological study.

BACKGROUND: Instruments to critically appraise randomised controlled trials (RCTs) are based on evidence from meta-epidemiological studies. We aim to conduct a meta-epidemiological study on the average bias associated with reported methodological trial characteristics such as random sequence generation, allocation concealment, blinding, incomplete outcome data, selective reporting, and compliance of RCTs in nutrition research. METHODS: We searched the Cochrane Database of Systematic Reviews, for systematic reviews of RCTs, published between 01 January 2010 and 31 December 2019. We combined the estimates of the average bias (e.g. ratio of risk ratios [RRR] or differences in standardised mean differences) in meta-analyses using the random-effects model. Subgroup analyses were conducted to investigate the potential differences among the RCTs with low versus high/unclear risk of bias with respect to the different types of interventions (e.g. micronutrients, fatty acids, dietary approach), outcomes (e.g. mortality, pregnancy outcomes), and type of outcome (objective, subjective). Heterogeneity was assessed through I2 and τ2, and prediction intervals were calculated. RESULTS: We included 27 Cochrane nutrition reviews with 77 meta-analyses (n = 927 RCTs). The available evidence suggests that intervention effect estimates may not be exaggerated in RCTs with high/unclear risk of bias (versus low) judgement for sequence generation (RRR 0.97, 95% CI 0.93 to 1.02; I2 = 28%; τ2 = 0.002), allocation concealment (RRR 1.00, 95% CI 0.96 to 1.04; I2 = 27%; τ2 = 0.001), blinding of participants and personnel (RRR 0.95, 95% CI 0.91 to 1.00; I2 = 23%; τ2 = 0), selective reporting (RRR 0.97, 95% CI 0.92 to 1.02; I2 = 24%; τ2 = 0), and compliance (RRR 0.95, 95% CI 0.89 to 1.02; I2 = 0%; τ2 = 0). Intervention effect estimates seemed to be exaggerated in RCTs with a high/unclear risk of bias judgement for blinding of outcome assessment (RRR 0.81, 95% CI 0.70 to 0.94; I2 = 26%; τ2 = 0.03), which was predominately driven by subjective outcomes, and incomplete outcome data (RRR 0.92, 95% CI 0.88 to 0.97; I2 = 22%; τ2 = 0.001). For continuous outcomes, no differences were observed, except for selective reporting. CONCLUSIONS: On average, most characteristics of nutrition RCTs may not exaggerate intervention effect estimates, but the average bias appears to be greatest in trials of subjective outcomes. Replication of this study is suggested in this field to keep this conclusion updated.

A common SLC26A4-linked haplotype underlying non-syndromic hearing loss with enlargement of the vestibular aqueduct.

BACKGROUND: Enlargement of the vestibular aqueduct (EVA) is the most common radiological abnormality in children with sensorineural hearing loss. Mutations in coding regions and splice sites of the SLC26A4 gene are often detected in Caucasians with EVA. Approximately one-fourth of patients with EVA have two mutant alleles (M2), one-fourth have one mutant allele (M1) and one-half have no mutant alleles (M0). The M2 genotype is correlated with a more severe phenotype. METHODS: We performed genotype-haplotype analysis and massively parallel sequencing of the SLC26A4 region in patients with M1 EVA and their families. RESULTS: We identified a shared novel haplotype, termed CEVA (Caucasian EVA), composed of 12 uncommon variants upstream of SLC26A4. The presence of the CEVA haplotype on seven of ten 'mutation-negative' chromosomes in a National Institutes of Health M1 EVA discovery cohort and six of six mutation-negative chromosomes in a Danish M1 EVA replication cohort is higher than the observed prevalence of 28 of 1006 Caucasian control chromosomes (p<0.0001 for each EVA cohort). The corresponding heterozygous carrier rate is 28/503 (5.6%). The prevalence of CEVA (11 of 126) is also increased among M0 EVA chromosomes (p=0.0042). CONCLUSIONS: The CEVA haplotype causally contributes to most cases of Caucasian M1 EVA and, possibly, some cases of M0 EVA. The CEVA haplotype of SLC26A4 defines the most common allele associated with hereditary hearing loss in Caucasians. The diagnostic yield and prognostic utility of sequence analysis of SLC26A4 exons and splice sites will be markedly increased by addition of testing for the CEVA haplotype.

Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea.

Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the "muscle-type" nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870-5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651-2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (-)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs.

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4.

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4Cre and Mrf4nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.

Targeting pathological cells with senolytic drugs reduces seizures in neurodevelopmental mTOR-related epilepsy.

Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated ß-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.

Transgenic Tg(Kcnj10-ZsGreen) fluorescent reporter mice allow visualization of intermediate cells in the stria vascularis.

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP. These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Pregnancy after endometrial ablation. A report of three cases.

BACKGROUND: Pregnancies after endometrial ablation (EA) are rare and are associated with high morbidity, especially in the second and third trimesters. CASE: We report 3 cases of pregnancy after EA in which severe complications occurred during the first trimester. The first case concerns an unexpected pregnancy after EA in a 50-year-old woman. Hysterectomy via laparotomy was needed because of heavy bleeding and severe anemia. Pathology showed a complete hydatidiform mole without invasion. Ultrasound-guided in situ methotrexate injection could be a nonsurgical alternative to terminate an intrauterine pregnancy after EA when the pregnancy is located within uterine synechiae, as shown by the second case. The third case presented as an accreta placentation mimicking hyperplastic myometrial invasion in a 46-year-old patient. CONCLUSION: Because of the high morbidity of these pregnancies that could still occur after EA, patients need contraception after EA, and concomitant hysteroscopic sterilization should be proposed at the same time.

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, yet it remains refractory to systemic therapy. Elimination of senescent cells has emerged as a promising new treatment approach against cancer. Here, we investigated the contribution of senescent cells to GBM progression. Senescent cells are identified in patient and mouse GBMs. Partial removal of p16Ink4a-expressing malignant senescent cells, which make up less than 7 % of the tumor, modifies the tumor ecosystem and improves the survival of GBM-bearing female mice. By combining single cell and bulk RNA sequencing, immunohistochemistry and genetic knockdowns, we identify the NRF2 transcription factor as a determinant of the senescent phenotype. Remarkably, our mouse senescent transcriptional signature and underlying mechanisms of senescence are conserved in patient GBMs, in whom higher senescence scores correlate with shorter survival times. These findings suggest that senolytic drug therapy may be a beneficial adjuvant therapy for patients with GBM.

Effects of Dairy Intake on Markers of Cardiometabolic Health in Adults: A Systematic Review with Network Meta-Analysis.

The health effects of dairy products are still a matter of scientific debate owing to inconsistent findings across trials. Therefore, this systematic review and network meta-analysis (NMA) aimed to compare the effects of different dairy products on markers of cardiometabolic health. A systematic search was conducted in 3 electronic databases [MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL), and Web of Science; search date: 23 September 2022]. This study included randomized controlled trials (RCTs) with a ≥12-wk intervention comparing any 2 of the eligible interventions [e.g., high dairy (≥3 servings/d or equal amount in grams per day), full-fat dairy, low-fat dairy, naturally fermented milk products, and low dairy/control (0-2 servings/d or usual diet)]. A pairwise meta-analysis and NMA using random-effects model was performed in the frequentist framework for 10 outcomes [body weight, BMI, fat mass, waist circumference, low-density lipoprotein cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, fasting glucose, glycated hemoglobin, and systolic blood pressure]. Continuous outcome data were pooled using mean differences (MDs) and dairy interventions ranked using the surface under the cumulative ranking curve. Nineteen RCTs with 1427 participants were included. High-dairy intake (irrespective of fat content) showed no detrimental effects on anthropometric outcomes, blood lipids, and blood pressure. Both low-fat and full-fat dairy improved systolic blood pressure (MD: -5.22 to -7.60 mm Hg; low certainty) but, concomitantly, may impair glycemic control (fasting glucose-MD: 0.31-0.43 mmol/L; glycated hemoglobin-MD: 0.37%-0.47%). Full-fat dairy may increase HDL cholesterol compared with a control diet (MD: 0.26 mmol/L; 95% CI: 0.03, 0.49 mmol/L). Yogurt improved waist circumference (MD: -3.47 cm; 95% CI: -6.92, -0.02 cm; low certainty), triglycerides (MD: -0.38 mmol/L; 95% CI: -0.73, -0.03 mmol/L; low certainty), and HDL cholesterol (MD: 0.19 mmol/L; 95% CI: 0.00, 0.38 mmol/L) compared with milk. In conclusion, our findings indicate that there is little robust evidence that a higher dairy intake has detrimental effects on markers of cardiometabolic health. This review was registered at PROSPERO as CRD42022303198.

Transgenic Tg(Kcnj10-ZsGreen) Fluorescent Reporter Mice Allow Visualization of Intermediate Cells in the Stria Vascularis.

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Onset of cholinergic efferent synaptic function in sensory hair cells of the rat cochlea.

In the developing mammalian cochlea, the sensory hair cells receive efferent innervation originating in the superior olivary complex. This input is mediated by α9/α10 nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent SK2 potassium channels. We examined the acquisition of this cholinergic efferent input using whole-cell voltage-clamp recordings from inner hair cells (IHCs) in acutely excised apical turns of the rat cochlea from embryonic day 21 to postnatal day 8 (P8). Responses to 1 mm acetylcholine (ACh) were detected from P0 on in almost every IHC. The ACh-activated current amplitude increased with age and demonstrated the same pharmacology as α9-containing nAChRs. Interestingly, at P0, the ACh response was not coupled to SK2 channels, so that the initial cholinergic response was excitatory and could trigger action potentials in IHCs. Coupling to SK current was detected earliest at P1 in a subset of IHCs and by P3 in every IHC studied. Clustered nAChRs and SK2 channels were found on IHCs from P1 on using Alexa Fluor 488 conjugated α-bungarotoxin and SK2 immunohistochemistry. The number of nAChRs clusters increased with age to 16 per IHC at P8. Cholinergic efferent synaptic currents first appeared in a subset of IHCs at P1 and by P3 in every IHC studied, contemporaneously with ACh-evoked SK currents, suggesting that SK2 channels may be necessary at onset of synaptic function. An analogous pattern of development was observed for the efferent synapses that form later (P6-P8) on outer hair cells in the basal cochlea.

Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.

Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.

AAV8BP2 and AAV8 transduce the mammalian cochlear lateral wall and endolymphatic sac with high efficiency.

Inner ear gene therapy using adeno-associated viruses (AAVs) has been successfully applied to several mouse models of hereditary hearing loss to improve their auditory function. While most inner ear gene therapy studies have focused on the mechanosensory hair cells and supporting cells in the organ of Corti, the cochlear lateral wall and the endolymphatic sac have not garnered much attention. The cochlear lateral wall and the endolymphatic sac play critical roles in inner ear ionic and fluid homeostasis. Mutations in genes expressed in the cochlear lateral wall and the endolymphatic sac are present in a large percentage of patients with hereditary hearing loss. In this study, we examine the transduction patterns and efficiencies of conventional (AAV2 and AAV8) and synthetic (AAV2.7m8, AAV8BP2, and Anc80L65) AAVs in the mouse inner ear. We found that AAV8BP2 and AAV8 are capable of transducing the marginal cells and intermediate cells in the stria vascularis. These two AAVs can also transduce the epithelial cells of the endolymphatic sac. Our data suggest that AAV8BP2 and AAV8 are highly useful viral vectors for gene therapy studies targeting the cochlear lateral wall and the endolymphatic sac.

Genomic analysis of childhood hearing loss in the Yoruba population of Nigeria.

Although variant alleles of hundreds of genes are associated with sensorineural deafness in children, the genes and alleles involved remain largely unknown in the Sub-Saharan regions of Africa. We ascertained 56 small families mainly of Yoruba ethno-lingual ancestry in or near Ibadan, Nigeria, that had at least one individual with nonsyndromic, severe-to-profound, prelingual-onset, bilateral hearing loss not attributed to nongenetic factors. We performed a combination of exome and Sanger sequencing analyses to evaluate both nuclear and mitochondrial genomes. No biallelic pathogenic variants were identified in GJB2, a common cause of deafness in many populations. Potential causative variants were identified in genes associated with nonsyndromic hearing loss (CIB2, COL11A1, ILDR1, MYO15A, TMPRSS3, and WFS1), nonsyndromic hearing loss or Usher syndrome (CDH23, MYO7A, PCDH15, and USH2A), and other syndromic forms of hearing loss (CHD7, OPA1, and SPTLC1). Several rare mitochondrial variants, including m.1555A>G, were detected in the gene MT-RNR1 but not in control Yoruba samples. Overall, 20 (33%) of 60 independent cases of hearing loss in this cohort of families were associated with likely causal variants in genes reported to underlie deafness in other populations. None of these likely causal variants were present in more than one family, most were detected as compound heterozygotes, and 77% had not been previously associated with hearing loss. These results indicate an unusually high level of genetic heterogeneity of hearing loss in Ibadan, Nigeria and point to challenges for molecular genetic screening, counseling, and early intervention in this population.

Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses.

The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

Dissection of the Endolymphatic Sac from Mice.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.