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Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: ZP is employed by Ambry Genetics, and exome sequencing is among the commercially available tests. The other authors have declared that no competing interests exist. Hearing loss is a very common disorder with a significant social impact, including delayed speech and language development, reduced academic achievement, increased social isolation, and risk of depression, and has recently been reported to be a major risk factor for dementia [ 1 ], adding new impetus to the need to develop therapies.

Environmental factors including noise or drug exposure play an important role in its etiology, but there is also a strong genetic contribution. Over genes are known to be involved in human or mouse deafness, but ascertainment bias has led to many of these having early developmental effects, and little is known about the genetic contribution to adult-onset hearing loss. We set out to identify further genes underlying deafness, including those with mild effects, using a physiological screen based on the auditory brainstem response ABR in a large cohort of newly generated targeted mouse mutants.

In this report, we review the findings from this screen and present the new data; both mice and ABR waveform data are available for further analysis. From the unbiased sample of 1, genes tested, we found 38 unexpected genes to be involved in hearing impairment. This indicates that around additional genes remain to be found see later , making deafness an extremely heterogeneous condition, with around 1, genes that may contribute.

The observed impairments ranged from mild to profound, including several with progressive hearing loss, and with a wide range of underlying pathological mechanisms. The 38 genes represent a range of functions from transcription factors and a microRNA to enzymes involved in lipid metabolism.

Eleven were found to be significantly associated with auditory function in the human population, and one gene, SPNS2 , was associated with childhood deafness, emphasising the value of the mouse for identifying genes and mechanisms underlying complex processes like hearing. We used a rapid minute , noninvasive electrophysiological test, the ABR [ 4 ] S1 Fig ; S1 Data in anaesthetised mice aged 14 weeks old as part of an extensive pipeline of phenotyping tests on a set of new mouse mutants generated from targeted embryonic stem ES cells [ 5 , 6 , 7 ].

The allele design was mostly the knockout first, conditional-ready tm1a ; targeted mutation, first allele with design type a [ 6 ] allele, which reduced or eliminated expression of the targeted gene by inclusion of a large cassette designed to interfere with transcription, but a few were the derived tm1b allele with an exon deleted or were edited alleles S1 and S2 Tables.

A total of 1, genes were tested. Of these, 38 genes with no prior association with deafness had raised thresholds for detecting a response to sounds Fig 1 ; a small number of these have been published recently, after their discovery in the screen. Using objective criteria see Materials and methods we classified these 38 genes into five main groups based on thresholds: 5 showed severe or profound deafness, 10 had raised thresholds at high frequencies only, 2 showed raised thresholds at low frequencies only, 7 had moderately raised thresholds across frequencies, and 14 had a mild hearing impairment Fig 1.

Thresholds for individual mutants are shown by open grey circles and lines. Gene symbols are given on each plot, and when both the tm1a and tm1b alleles were screened, both sets of data are presented, indicated by a and b suffixes. Mice screened were homozygous mutants except for Brd2 , Srsf7 , and Setd5 , which were screened as heterozygotes due to reduced viability of homozygotes.

The number of mutant mice screened of each line is given in column E of S1 Table and on each panel. Plotted data points are given S5 Data. Of the set of 1, genes, 3. As the genes targeted were an unbiased set showing no significant enrichment for any functional class compared with the total set of mouse genes see Materials and methods , we can extrapolate to estimate that over further genes required for normal auditory thresholds remain to be found.

Added to the human and mouse genes already known and 38 reported here, this indicates that there may be as many as 1, genes involved in deafness, a very high level of genetic heterogeneity. The lack of raised thresholds could be due to incomplete knockdown of targeted gene expression in the tm1a allele e. Alternatively, the original allele might have led to deafness via a long-range cis effect on a nearby gene, as in the Slc25a21 tm1a KOMP Wtsi targeted mutation, which causes deafness by reducing expression of Pax9 [ 8 ].

Thus, we probably missed additional genes involved in hearing loss, so our calculation of more genes awaiting association with deafness may be an underestimate. As mouse and human inner ears are very similar in structure and function e. A child from a United States clinic detected through clinical whole exome sequencing inherited a frameshift mutation of SPNS2 from her father c.

Serdel; CADD phred score Visual reinforcement audiometry at two years old revealed moderate to moderately severe hearing loss between Hz and 4 kHz with no response at 8 kHz in the right ear, and severe hearing loss sloping to profound deafness from Hz to 8 kHz with no response at 4 and 8 kHz in the left ear. Bone conduction testing indicated moderate-severe hearing loss at 2 kHz in the right ear, suggesting a sensorineural not conductive impairment, and acoustic reflexes were absent. However, the child had surprisingly good sound localisation performance. The severe level of hearing impairment associated with predicted damaging SPNS2 variants is similar to our findings in the mouse Spns2 mutant Fig 1 in [ 11 ].

Furthermore, we have previously reported that deaf children from two families in a Chinese cohort carried recessive WBP2 mutations [ 10 ]. We asked if these new candidates had any role in hearing ability in the general population by a candidate gene association analysis. We tested genomic markers within 0. Eleven of the thirty-seven candidate genes tested including SPNS2 showed a significant association of markers with threshold at either 1 or 4 kHz or both frequencies Table 1 , indicating that these 11 genes may play a role in normal variation of hearing ability in the human population.

Several of the new genes that we found to be involved in deafness in the mouse had human orthologues close to or within unidentified non-syndromic deafness loci S4 Table , column J and so are good candidates for further exploration. The 38 genes newly associated with hearing are involved in a broad range of functions, including transcriptional and translational regulation, chromatin modification, splicing factors, cytoskeletal proteins, membrane trafficking, calcium buffering, peroxisome biosynthesis, thyroid hormone generation, ubiquitination and deubiquitination, kinases, signaling molecules including Wnt signaling , and proteins with no known or predicted function S4 Table.

A microRNA gene, Mir , was one of the new genes underlying hearing impairment. Seven of the gene products have a role in lipid metabolism: Fads3 is a fatty acid desaturase; Agap1 and Zcchc14 bind phospholipids; Klc2 transports phosphatidylinositol 3-kinase, which is required for phospholipid processing; Pex3 is involved in biosynthesis of peroxisomes, which are involved in lipid processing; Acsl4 is a long-chain fatty acid coenzyme A ligase converting free long-chain fatty acids into fatty acyl-CoA esters; and Spns2 is a transporter of sphingosinephosphate, a key intermediate in sphingolipid metabolism with a role in signalling.

The novel genes showed nothing notably different to the full set of genes screened Fig 2. This finding suggests that our unbiased screen for new genes involved in hearing impairment at all levels of severity has revealed a fundamentally different class of genes compared with previously known genes underlying deafness. The proportion of genes in each group with the high-level GO terms listed are plotted for the three categories: Cellular component, Molecular function, and Biological process. Plotted data points are given in S6 Data.

Some of the 38 genes had links to existing pathways involved in deafness. For example, Duoxa2 is required for maturation and transport from the endoplasmic reticulum ER to the plasma membrane of Duox2, also known to underlie deafness through its role in hypothyroidism, leading to retarded cochlear development and impaired hearing [ 18 ]. Spns2 is a sphingosinephosphate S1P transporter, and our discovery of its involvement in deafness supports the role of the S1P signaling pathway in hearing loss, alongside reports of S1PR2 and Sgms1 mutations causing deafness [ 19 , 20 , 21 , 22 , 23 ].

In contrast, many of the other genes discovered in this screen, such as AC20Rik Minar2 , have no demonstrated role in a biological process and no a priori reason to predict they might be involved in deafness. As our goal in carrying out the screen was to identify new genes involved in adult-onset hearing loss, we carried out recurrent ABR recordings usually at 4, 8, and 14 weeks old, plus shortly after the normal onset of hearing at 2 and 3 weeks old if thresholds were raised at 4 weeks on some of the mutant lines.

Remarkably, several of the mutants we studied showed relatively normal early development of ABRs followed by progressive increase in thresholds. Several new mutant lines showed normal or near-normal ABR thresholds at young ages followed by progressive increases in thresholds with age. Srsf7 heterozygous mutants. Gpr homozygous mutants.

In all panels, means with standard deviations are plotted in red for mutants and green for wild-type littermate controls. Arrows indicate that there was no response, so the maximum sound level used was plotted. Plotted data points are given in S7 Data. Scanning electron microscopy revealed extensive loss of OHC hair bundles at P28 in the cochlear regions, corresponding to the worst thresholds in mutants 12 kHz; Gi. Remaining hair bundles had a normal appearance Gv. Quantification of OHC nuclei from confocal images demonstrated significant reduction in mutants red at best-frequency regions from 6 to 24 kHz and no significant difference with controls green at the kHz region.

Black line represents ABR threshold elevation in mutants compared with littermate controls. EPs in wild types green and homozygotes red show no significant difference in mutants homozygotes Representation of the allele, with exons in grey, FRT sites in green, loxP sites in red, and lacZ and neo components of the inserted construct labelled.

Blue-labelled areas show expression in cells surrounding the cochlear duct and spiral ganglion. Kiii shows a higher magnification of the organ of Corti. Confocal images of the organ of Corti in a wild type left and homozygote right at P28 labelled with Myo7a antibody false-coloured green showing hair cell bodies and DAPI false-coloured red showing nuclei. Plotted data points are given in S8 Data. The mouse alleles studied here are relatively severe in their effect on protein expression, but variants in the human population may have milder effects on protein function and lead to later onset of hearing loss.

Importantly, the finding of genes involved in normal development but later deterioration of hearing identifies molecular pathways likely to underlie adult-onset progressive hearing loss in humans. We analysed further a subset of mutant lines and revealed a wide range of pathological conditions underlying hearing impairment. Two examples of contrasting phenotypes are the Klc2 and the Ywhae mutant lines. Klc2 mutants showed a progressive increase in ABR thresholds with age, mostly affecting low frequencies Fig 4A—4F , with a sensorineural not conductive pathology.

Klc2 encodes kinesin light chain 2, which, together with kinesin heavy chains encoded by Kif5 , forms the kinesin-1 motor complex, a microtubule-associated anterograde transporter. The middle ear and gross structure of the inner ear appeared normal. The endocochlear potential EP was maintained at a normal level even up to 6 months of age, but the anoxia potential in scala media was significantly less negative in these mutants, consistent with loss of hair cell conductance Fig 4I. There were few signs of inner hair cell IHC degeneration, but the increase in threshold was larger than would be expected if only OHCs were affected, suggesting IHC dysfunction.

Klc2 also interacts with Kcnma1, the calcium-activated potassium channel BK channel that underlies the I K,f current required for very rapid responses of IHCs and contributes to protective efferent suppression of OHCs [ 27 , 28 , 29 , 30 ]. We found that labelling of Kcnma1 in IHCs was less extensive in mutants compared with wild-type controls at 12kHz; Fig 4Ji-vi , implicating Kcnma1 in the pathological mechanism; however, knockout of Kcnma1 leads to less severe loss of thresholds [ 31 ], so this alone cannot explain the extent of dysfunction in the Klc2 mutants.

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Finally, kinesin-1 has been implicated in maintenance of the hair cell nucleus in its correct position by interacting with Nesp4 in the outer nuclear membrane [ 32 , 33 ], and Nesp4 mutations lead to location of the OHC nucleus at the top of the cell and subsequent degeneration [ 34 ]. We did not find mislocalisation of OHC or IHC nuclei Fig 4Li-ii , suggesting this was not the mechanism underlying hearing loss in the Klc2 mutants, and that redundancy between kinesin light chains may compensate for loss of Klc2 in nuclear localisation.

However, a human gain-of-function KLC2 mutation bp deletion upstream of the coding region leading to increased KLC2 expression causes spastic paraplegia, optic atrophy and neuropathy SPOAN , a neurodegenerative disorder involving progressive axonal neuropathy [ 35 ]. In contrast, the Ywhae mutants showed increased thresholds across all frequencies associated with variable amounts of accumulated fluid and exudate containing inflammatory cells in the middle ear, suggesting predisposition to otitis media Fig 5A—5M.

The middle ear mucosa appeared thickened with granulation tissue in sections Fig 5L , and scanning electron microscopy of the luminal surface showed an open Eustachian tube in mutants, but abundant clusters of goblet cells presumed to produce mucus with fewer ciliated epithelial cells, which would normally contribute to the clearing of excess mucus Fig 5M and 5N. The variability in thresholds between individual Ywhae mutants, relatively flat increase across all frequencies, and near-normal ABR waveform support a conductive hearing loss Fig 5A—5H.

The surface of the organ of Corti looked normal Fig 5O and 5P but we cannot exclude a sensorineural component in some of the more severely affected mutants, possibly due to the impact of S5-derived alleles in the mixed genetic background, or an effect of persistent inflammation of the middle ear [ 36 ].

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Ywhae is normally widely and strongly expressed throughout the body [ 39 ] and within the cochlea [ 40 ], and the mutation led to an absence of detectable Ywhae protein in homozygotes Fig 5R. Craniofacial malformations may affect Eustachian tube structure and function, leading to otitis media, but there are many other possible pathological mechanisms not yet explored. Similar fluid-filled middle ear and conductive hearing loss phenotypes were found in the Mcph1 mutant [ 12 ] and Slc25a21 mutants with reduced Pax9 expression [ 8 ].

Heterozygotes have normal ABR thresholds. The tympanic membrane is retracted in the mutant, and the middle ear contains inflammatory debris. Black rectangles in I and K indicate the areas enlarged in J and L. Scanning electron micrographs of the middle ear epithelium near the opening of the Eustachian tube in wild-type M and homozygous Ywhae mutant N , showing widespread goblet cells and fewer ciliated epithelial cells in the mutant compared with the wild type, which is rich in ciliated cells.

Scanning electron micrographs of the organ of Corti in a heterozygote O and homozygous mutant P showing normal appearance. Vinculin was used as a loading control. Plotted data points are given in S9 Data. A third distinct pathology found was a reduction in EP. Normally, a high resting potential in the cochlear endolymph is generated by the stria vascularis. This is necessary for normal sensory hair cell function.

Progressive disorganisation of the stria vascularis accompanies the reduced EP in Spns2 mutants [ 11 ]. A fourth example is the Wbp2 mutant, in which abnormal structure of synapses between IHCs and cochlear neurons and swelling of nerve terminals leads to progressive increase in ABR thresholds [ 10 ]. The finding of a wide range of primary pathological processes in these mouse mutants as outlined above suggests that the pathogenesis of hearing loss in the human population may be equally heterogeneous.

The limited information gleaned from human temporal bone studies supports the suggestion of heterogeneous pathophysiology underlying progressive hearing loss [ 43 ]. It is notable that none of the 38 new mutant genes we report here showed any sign of leading to a balance defect S1 Table. Nine lines had reduced viability assessed at postnatal day 14, with three of these lines producing so few homozygotes that heterozygotes were passed through the phenotyping pipelines instead Brd2 , Srsf7 , and Setd5.

Occurrence of anomalous features in other systems tested were generally scattered across mutant lines and phenotypes, with Duoxa2 and Ywhae mutants showing the largest number of other abnormalities S1 Table. By analysis of click-evoked ABR waveforms, we identified 27 additional mutant lines with normal hearing sensitivity, but which had abnormal patterns of neural responses, such as smaller ABR wave amplitudes or prolonged latencies, determined using objective criteria S1 Table ; Fig 6 ; S3 Fig ; S3 Data.

The ABR waveform is a complex mixture of voltage changes reflecting the sum of excitatory and inhibitory activity at different times after stimulus onset and different physical locations within the brain relative to the position of the recording electrodes. Wave 1 reflects auditory nerve activity, and later waves reflect activity higher up the central auditory pathways. Other mutants showed abnormal amplitudes or latencies of later waves, suggesting auditory processing anomalies in the central auditory system Fig 6 ; S3 and S4 Figs; S3 and S4 Data.

These changes could reflect abnormal inherent excitability of auditory neurons or an alteration in the balance of excitatory and inhibitory inputs onto these neurons, resulting in increased or decreased discharge or synchrony.

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Mutants showing changes in latency are most easily explained by changes in neural conduction speed, alterations in synaptic delays, or changes in the relative contributions of different components in this complex neuronal pathway. Finally, in the most extreme example Bai1 , also known as Adgrb1 , the mice exhibited clear auditory-evoked responses and measurable thresholds, but the ABRs were so abnormal that it was not possible to determine the equivalent peaks to quantify and compare with control mice Fig 6. This set of 27 mutants with waveform anomalies will be an interesting group to analyse further, because central auditory function is critical for normal sound perception.

Such deficits may translate in humans to altered performance in sound localisation, ability to follow salient acoustic stimuli in background noise, discrimination of specific speech features, and other auditory processing disorders e. Examples of three mutant mouse lines with altered ABR waveforms are shown. Responses from individual mutants are shown by grey lines and open circles.

The Famb mutants produced a mild increase in thresholds but also have ABR waveforms with significantly reduced wave 1 amplitude. Sesn3 mutants showed normal thresholds but prolonged P3 latency. Plotted data points are given in S10 Data. We used a rapid ABR protocol to carry out a high-throughput screen of 1, new mouse mutants and revealed a new spectrum of functional deficits in hearing that would not have been detected using simpler screens, such as the startle response.

These include mild-moderate degrees of hearing impairment, frequency-specific impairments low or high frequencies , and a group with abnormal ABR waveforms that likely have deficits in central auditory pathways.


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In a subset of the new mutant lines, we have examined other ages to establish the time course of hearing loss and investigate the pathophysiological mechanisms underlying the raised ABR thresholds. A broad range of pathologies was found, and many mutants showed normal development followed by progressive hearing loss. We have shown that some of the genes highlighted by this study play a role in human hearing, including 2 genes with mutations that can account for recessive deafness in families and 11 genes that are associated with variation in auditory thresholds in the UK British Birth Cohort cross-sectional population.

Thus, mouse mutants can be an effective means to identify candidate genes for human deafness. This project has provided insights into the wide range of pathological processes involved in hearing impairment and has revealed a surprising number of unexpected genes involved in deafness, suggesting extreme genetic heterogeneity. For this reason, it is likely that therapies will need to be directed at common molecular pathways involved in deafness rather than individual genes or mutations.

Each new gene identified gives insight into the metabolic pathways and regulatory processes involved in hearing and thus provides a rich source of targets for development of therapies for the restoration of hearing. Mice were culled using methods approved under these licences to minimise any possibility of suffering.

For human studies, informed consent was obtained from the adult participants and the parents or guardians of children prior to participation, and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. The US patient provided consent for clinical whole exome analysis and written consent for inclusion as a case report.

Testing was conducted during the routine clinical care of a patient in the US; thus, in accordance with US law, this study is exempt from Institutional Research Board approval.

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Mutant mouse lines were generated using targeted mutations in mouse ES cells [ 5 , 6 ]. The viability of new mutants was determined by genotype distribution at weaning. When possible, mice homozygous for the targeted mutation were used for screening. If a mutation proved to be embryonic lethal or had significantly reduced viability at weaning, heterozygous mice were used instead genes out of 1,; In most cases, the knockout first conditional-ready tm1a allele for each gene was used, but a subset of the genes were tested using the derived tm1b allele, which had deletion of a critical exon s , or other mutations S2 Table , column B.

The tm1a allele is designed to knock down transcription by introducing a large cassette into the gene, but not all genes were completely inactivated see S2 Table and column X in [ 5 ], for some examples. Mutant data were compared with a large set of wild-type data on the same genetic background. When mice were screened on mixed genetic backgrounds for example, Ywhae tm1e , age and strain-matched wild-type mice were used alongside the mutants.

Positive control lines known to have a hearing impairment were compared with their littermate controls on the same, varied genetic backgrounds S1 Table. The Cdh23 ahl allele may have interacted with any of the new mutations to exacerbate their effect, such that the phenotype was easier to detect at 14 weeks, so this screen could be regarded as a sensitised screen [ 47 ].

Balance was assessed by observation of gait, head bobbing or circling, the rotarod test, or contact righting test. ABRs were recorded using the methods described in detail in [ 4 ]. Mice were placed on a heating blanket inside a sound-attenuating booth. Subcutaneous needle electrodes were inserted in the skin on the vertex active and overlying the ventral region of the left reference and right ground bullae. The sound delivery system was calibrated using an ACO Pacific microphone.

For threshold determination, custom software and Tucker Davis Technologies hardware were used to deliver click 0. Averaged responses to stimuli, presented at Tone-evoked ABRs were recorded for a fixed set of frequencies 6, 12, 18, 24, and 32 kHz over sound levels ranging from 0 to 85 dB SPL in 5-db intervals. Responses were recorded in an array, beginning with the lowest stimulus level, in decreasing frequency order before stepping up to the next 5 dB higher stimulus level. If mice appeared to have hearing impairment, the upper limit of SPLs was extended to 95 dB for each test frequency and for clicks representing the upper limit of the linear range of our sound system at these frequencies.

For the ABR screen, we aimed to test a minimum of four mutant mice per line of either sex. For other tests on the pipeline, 14 mutant mice 7 males and 7 females were required. Phenotyping cohorts were issued as mice became available, such that several partial cohorts were issued, to achieve the required number of 14 mice for each single line.

This allowed the ABR assay to pick up further mice from any lines that exhibited any features of interest to extend the number tested beyond the target of four. In addition to mutant mice, at least four wild-type mice from the same matings used to generate the mutants were tested each week from each core genetic background of the mutants tested.

These wild-type results formed a local control group for comparison with the mutant lines and also contributed to a large reference range of control data that were used to determine if ABR results from a particular mutant line were significantly abnormal. ABR threshold and hearing sensitivity.

Threshold dB SPL was estimated by visual inspection of the stacked ABR traces as the lowest sound level at which any component of the ABR waveform was recognisable and consistent with responses recorded at higher sound levels, taking into account the characteristic lengthening of peak latency as threshold is approached. Thresholds for each stimulus were plotted to give a profile of the hearing sensitivity of each mouse. Waveform shape comparisons. Through consistent, reproducible electrode placements, it was possible to compare, qualitatively and quantitatively, the waveform shapes of click-evoked ABRs.

Wave 1 is understood to reflect auditory nerve activity, but as the responses represent a complex mixture of responses detected at a single point, there is some uncertainty in ascribing specific brain locations to specific features of the ABR waveform. The free-field binaural stimulation conditions we used complicates interpretation further, because there are binaural interactions even within the cochlear nucleus, e.

The ABR represents the summed electrical vectors detected by the electrodes as synchronised action potential volleys particularly from onset-responding neurons traverse the central auditory pathways. As these pathways can be both excitatory and inhibitory, as well as both ascending and descending, and are distributed in a 3D volume, interpretation is complex. Waveforms recorded to clicks at 20 dB and 50 dB above threshold sensation level [SL] were plotted for mutant and control mice, along with an average of the ABR amplitude over time across mice for each genotype.

In these responses, we could identify four waves positive to negative deflections; S1 Fig ; S1 Data. We also compared individual mutant responses with the reference range. If both comparisons were in agreement between at least two of three experienced observers, a quantitative analysis was carried out of the peak amplitude, latencies, and intervals of these waveforms, by measurement of input-output functions IOFs. Using click-evoked ABRs, waveforms were analysed in detail to determine the amplitude and latency of positive and negative peaks of the waveform at each stimulus level recorded S1 Fig ; S1 Data.

This was performed using software routines developed by Brad Buran and kindly donated for our use by M. Liberman Harvard University. We found wave 1 and wave 3 were highly consistent in control mice. However, whilst wave 2 was clearly present as a single peak at low sound levels, it often split into two components at higher sound levels, making analysis complicated, so we did not include it.

From these measures, we calculated the peak-peak amplitude of waves 1, 3, and 4, the amplitude of the N2-P3 component, and the intervals from P1 to P3 and N1 to N3. IOF curves were plotted relative to click threshold for each mouse i. Wild-type control mice of the same genetic background tested in the same week as mutants were used as a local control population. As mutant mice were often tested in separate weeks to obtain the required numbers, the local control population for each mutant line varied in numbers.

Furthermore, the large disparity in population sizes of the groups invalidates the use of traditional statistical tests giving p -values e. Thus, we used the following criteria to define parameters considered to be abnormal compared with controls. ABR thresholds. Thus, thresholds could be considered abnormal if they were elevated above controls lower sensitivity or reduced below controls enhanced sensitivity. We did not find any mutants with enhanced sensitivity. Waveform shape. These comparisons were used as a subjective triage step in the assessment of whether waveform shapes were normal or perturbed in responses from mutant mice.

A dataset was considered potentially interesting if two experienced observers considered the waveforms to be perturbed. In these cases, peak amplitudes and latencies were determined and IOFs plotted. IOFs were plotted for peak amplitude, latency, and also for wave 1—3 inter-peak interval as a function of dB SL. Due to the dependency of amplitude and latency on SPL, it is important to plot IOFs relative to stimulus threshold, so that any changes seen are not a result of variation in response threshold. All mice issued for phenotyping were genotyped [ 49 ] at least twice; once was prior to cohort generation from a clip of pinna skin and a second time was at the end of the pipeline from postmortem skin tissue.

ABR results were also subject to a quality control process. Visual inspection of the ABR traces recorded was used to look for significant noise or artefact on the recordings, which was accounted for when allocating threshold and other parameters. Thresholds were allocated by experienced operators using the criteria outlined above.

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Data from random mice were checked by a second operator. Our aim was to report robust effects on hearing that are likely to be reproducible in other laboratories, so we were cautious about calling the positive hits. All threshold calls made according to the two criteria detailed above were assessed by two experienced auditory scientists one of whom was blinded to the genotype to identify any false positive hits.


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  • A small number of false positive hits were discounted based on a number of principles, including excessive variability between individual mice in some cases due to segregation of an independent new mutation, e. Of the 1, genes tested, this represents a false discovery rate of 0.

    We carried out this additional statistical test for information about which threshold calls might be significant if the data had been normally distributed see above for reasons why this was not our primary test. A two-way contingency table using the number of threshold observations that were inside or outside the reference range was generated for each mutant group compared with the wild-type control group, and this was used to carry out a Fisher exact test, giving a p -value indicating the likelihood of the two sets of data belonging to the same population see [ 7 ] for details.

    Methods used for histology, immunolabelling and confocal analysis, EP recording, and associated statistical tests used have been published elsewhere [ 10 , 11 , 12 , 20 , 42 ]. ABR thresholds were compared using the Mann-Whitney test. ABRs were recorded in new cohorts of Srsf7 , Gpr , Klc2 , and Ywhae mutants along with littermate controls at 4 weeks, 8 weeks, 14 weeks, and 6 months old, as indicated in Figs 3 , 4 and 5 , and waveforms were analysed as described previously [ 10 ]. EPs were recorded in Klc2 mutants as described previously [ 10 , 11 ].

    The 2f1-f2 distortion product otoacoustic emission DPOAE was recorded in Klc2 mutants in response to f2 frequencies ranging from 6, to 30, Hz in Hz steps, where the f2:f1 frequency ratio was 1. Scanning electron microscopy was used to assess the organ of Corti in Klc2 mutants at 4 weeks old and Ywhae mutants at 8—9 weeks old, along with their littermate controls, and the middle ear mucosa of Ywhae mutants at 8—9 weeks of age.

    Inner ears were isolated 7 Ywhae homozygotes, 5 Ywhae heterozygotes, 2 wild-type littermates; 11 Klc2 homozygotes, 4 Klc2 heterozygotes, 2 wild-type littermates and fixed in 2. Specimens were imaged using a Zeiss Imager confocal microscope interfaced with ZEN software. The cell-counter plugin in Fiji ImageJ software was used for counting. Three-dimensional reconstruction of the hair cell confocal stacks was performed using Fiji software 3D project function.

    Myo7a was labelled with the secondary antibody Alexa Fluor and the nuclei with DAPI; however, false colours were used in the 3D reconstruction. X-gal Promega, cat. The samples were gradually dehydrated in ethanol Leica TP tissue processor and embedded in paraffin using xylene as clearing agent Leica EGH tissue embedder.

    Specimens were imaged using a Zeiss Axioskop microscope connected to an AxioCam camera and interfaced with Axiovision 3. External, middle, and inner ear regions were isolated from Ywhae mutants aged 16 weeks 4 homozygotes, 3 heterozygotes, 4 wild types. Left sides were examined by dissection for any signs of malformation or inflammation, including excessive cerumen in the external ear canal; thickening, whitening, sponginess, or vascularisation of the bulla wall; clarity of the tympanic membrane; presence of fluid or inflammatory debris in the middle ear cavity; and ossicle malformation.

    Sections were stained with haemotoxylin and eosin, scanned using a Hamamatsu NanoZoomer Hamamatsu City, Japan , and examined. Examiners of middle ears and sections were blinded to mouse genotype. Giles, UK. GO term enrichment was analysed using FuncAssociate v3. Only 0. The over- and under-represented GO attributes were not significantly clustered into distinct subsets visualised as TreeMaps. We also analysed this list of genes with the Reactome pathway database using Reactome V58 www. Of the 1, genes tested, were not listed in Reactome Of the 38 new genes underlying increased ABR thresholds, only 12 were included in Reactome.

    Of the 27 genes associated with abnormal ABR waveforms, only 17 were included in Reactome. Of deafness genes already known, were found in the Reactome databases. The Comprehensive online chemical technology encyclopedia with more than 1, articles written by a panel of prominent scholars from industry, academia, and research institutions from around the world. Provides excellent overviews on materials used in manufacturing ionic liquids, amino acids, silylating agents , or processes used in manufacturing e. Springer eBooks includes more than 60, titles in English and selectively in German from every scientific discipline and many social sciences.

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    Access for eBooks begins primarily with Those items available are marked with an unlocked lock icon. The full-text is searchable across titles and searches can be limited to code fragments. Books can also be browsed by topic. Books can be read online, or individual chapter sections can be printed out or emailed. Access is limited to 28 simultaneous UC connections. Searching in a library catalog requires looking for broader topics such as population genetics or molecular chaperones rather than the more specific metapopulation gene flow or Chaperonin GroEL. Learn about how books are arranged in the UCD libraries with this guide.

    Indexes over journals, serials, books and book chapters, conference proceedings and patents. Provides citations and abstracts to the international agricultural literature, including veterinary medicine, human and animal nutrition, forestry, rural development, as well as other related topics such as tourism and human ecology.

    More than 28 million citations and abstracts for articles published in approximately 8, journals in medicine, life sciences, health administration, veterinary medicine, nursing, molecular biology and genetics. PubMed is a service of the U. Embase is a biomedical and pharmacological database containing bibliographic records with citations, abstracts and indexing derived from biomedical articles in peer reviewed journals, and is especially strong in its coverage of drug and pharmaceutical research and conference abstracts.

    Embase contains over 22 million records spanning present, with over 1 million records added annually. The Embase journal collection is international with over 7, active peer-reviewed journals more than 90 countries. Broad biomedical scope covering the following areas:. Published by the National Agricultural Library, the Agricola database describes publications and resources encompassing all aspects of agriculture and allied disciplines including: animal science; veterinary science; entomology; plant science; forestry; aquaculture and fisheries; farming, farming systems and crops; agricultural economics; extension and education; food and human nutrition; and earth sciences and environmental sciences.

    The Bibliography of Agriculture is the print index to the agricultural literature going back to located on the Shields Library, Third Floor, at call number Z Journal Citation Reports is an annual publication by Thomson Reuters with each yearly data analysis being completed in the following year. The most recent edition is always published in the summer following the year analyzed. Journal Citation Reports indicates the most frequently cited journals in a field, the highest impact journals in a field, and the largest journals in a field. JCR Science Edition contains data from over 7, journals in science and technology.

    NOTE : For disciplines that have longer timespans for citation, consider sorting for ranking by using the 5-year Impact Factor sort. Includes over 40 Faculties Broad subject areas such as biochemistry, ecology or plant biology , which are further subdivided into over Sections.

    On average, 1, new evaluations are published each month. Faculty members are more than 5, experts worldwide assisted by 5, associates and assess articles from more than 3, peer-review journals. The DMPTool provides a guide for drafting the document that you must submit with your grant application.

    Users of the DMPTool can view sample plans, preview funder requirements, and view the latest changes to their plans. The tool also permits the user to create an editable document for submission to a funding agency, and can accommodate different versions as funding requirements change. It supports data management plans and funder requirements across the disciplines and includes multiple agency requirements.

    Use EZID to: create identifiers for anything, texts, data, bones, terms, etc. Merritt is a cost-effective repository service from the University of California Curation Center UC3 that lets the UC community manage, archive, and share its valuable digital content. Merritt features: an easy-to-use interface for deposit and update; access via persistent URLs; tools for long-term management; and permanent storage.

    Dash is a self-service tool for researchers to describe, upload, and share their research data. It is designed to be a simple self-service curation tool for researchers, and helps them perform the following tasks:. Need help accessing an Elsevier journal article? Click here. Jump to Section Library Catalogs Databases Citation Tools.

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    Current Protocols Series [via Wiley]. Springer Nature Experiments. Cold Spring Harbor Protocols. Methods in Enzymology [via ScienceDirect]. Coverage: Tags: Biology , Life Science. Springer Protocols. Nature Protocols.