Cholesteatoma Is Associated With Pediatric Progressive Sensorineural Hearing Loss.

OBJECTIVES: This study identified an association between cholesteatoma and progressive sensorineural hearing loss using a large pediatric longitudinal audiologic dataset. Cholesteatoma is a potential sequela of chronic otitis media with effusion, a commonly observed auditory pathology that can contribute to hearing loss in children. The purpose of this report is to (i) describe the process of identifying the association between cholesteatoma and progressive sensorineural hearing loss in a large pediatric dataset and (ii) describe the audiologic data acquired over time in patients identified with cholesteatoma-associated progressive sensorineural hearing loss. DESIGN: Records of patients included in the Audiologic and Genetics Database (n = 175,215 patients) were examined using specified criteria defining progressive hearing loss. A linear regression model examined the log frequency of all diagnostic codes in the electronic health record assigned to patients for a progressive hearing loss cohort compared with a stable hearing loss group. Based on findings from the linear regression analysis, longitudinal audiometric air (AC) and bone conduction (BC) thresholds were extracted for groups of subjects with cholesteatoma-associated progressive (n = 58 subjects) and stable (n = 55 subjects) hearing loss to further analyze changes in hearing over time. RESULTS: The linear regression analyses identified that diagnostic codes for cholesteatoma were associated with progressive sensorineural hearing loss in children. The longitudinal audiometric data demonstrated within-subject changes in masked BC sensitivity consistent with progressive sensorineural hearing loss in children diagnosed with cholesteatoma. Additional analyses showed that mastoidectomy surgeries did not appear to contribute to the observed progressive hearing loss and that a high number of cholesteatoma patients with progressive hearing loss had normal-hearing thresholds at their first test. CONCLUSIONS: The statistical analyses demonstrated an association between cholesteatoma and pediatric progressive sensorineural hearing loss. These findings inform clinical management by suggesting that children with cholesteatoma diagnoses may be at increased risk for progressive sensorineural hearing loss and should receive continued monitoring even after a normal masked BC baseline has been established.

Perspective on the Development of a Large-Scale Clinical Data Repository for Pediatric Hearing Research.

The use of "big data" for pediatric hearing research requires new approaches to both data collection and research methods. The widespread deployment of electronic health record systems creates new opportunities and corresponding challenges in the secondary use of large volumes of audiological and medical data. Opportunities include cost-effective hypothesis generation, rapid cohort expansion for rare conditions, and observational studies based on sample sizes in the thousands to tens of thousands. Challenges include finding and forming appropriately skilled teams, access to data, data quality assessment, and engagement with a research community new to big data. The authors share their experience and perspective on the work required to build and validate a pediatric hearing research database that integrates clinical data for over 185,000 patients from the electronic health record systems of three major academic medical centers.

Analyses with double knockouts of the Bmpr1a and Bmpr1b genes demonstrate that BMP signaling is involved in the formation of precerebellar mossy fiber nuclei derived from the rhombic lip.

Bone morphogenetic proteins (BMPs) have been hypothesized to specify distinct dorsal neural fates. During neural development, BMPs are expressed in the roof plate and adjacent neuroepithelium. Because several hindbrain nuclei that form the proprioceptive/vestibular/auditory sensory network originate from the rhombic lip, near the roof plate, BMP signaling may regulate the development of these nuclei. To test this hypothesis genetically, we have examined the development of the hindbrain in BMP type I receptor knockout mice. Our results demonstrate that BMP signaling is involved in the formation of precerebellar mossy fiber nuclei, which give rise to cerebellar mossy fibers, but is not required for the development of the inferior olivary nucleus, which gives rise to cerebellar climbing fibers.

Outcomes of evaluation and testing of 660 individuals with hearing loss in a pediatric genetics of hearing loss clinic.

Hearing loss is a relatively common condition in children, occurring in approximately 2 out of every 1,000 births with approximately 50% of reported diagnoses having a primary genetic etiology. Given the prevalence and genetic component of hearing loss, coupled with a trend toward early diagnosis with the institution of universal newborn hearing screening, The Genetics of Hearing Loss Clinic was established at The Children's Hospital of Philadelphia to manage the diagnosis, testing, and genetic counseling for individuals and families. This paper described a cohort of 660 individuals with a diagnosis of hearing loss evaluated between July 2008 and July 2015 in the Genetics of Hearing Loss Clinic. To elucidate the cause of hearing loss in this cohort for better management and prognostication, testing included single nucleotide polymorphism chromosomal microarray, hearing loss next generation sequencing panel, and additional clinical tests inclusive of thyroid and renal function studies, temporal bone magnetic resonance imaging, and electrocardiogram. Of those evaluated, most had bilateral sensorineural hearing loss, occurring in 489/660 (74%). Additionally, 612/660 (93%) of patients presented with a nonsyndromic form of hearing loss (no other observed clinical findings at the time of exam), of which pathogenic mutations in GJB2 were most prevalent. Of the individuals with syndromic manifestations (48/660), Usher and Waardenburg syndrome were most commonly observed. A family history of hearing loss (first degree relative) was present in 12.6% of families with available information. Through molecular analyses, clinical examination, and laboratory testing, a definitive etiologic diagnosis was established in 157/660 (23.8%) of individuals. © 2016 Wiley Periodicals, Inc.

Temporal bone radiology report classification using open source machine learning and natural langue processing libraries.

BACKGROUND: Radiology reports are a rich resource for biomedical research. Prior to utilization, trained experts must manually review reports to identify discrete outcomes. The Audiological and Genetic Database (AudGenDB) is a public, de-identified research database that contains over 16,000 radiology reports. Because the reports are unlabeled, it is difficult to select those with specific abnormalities. We implemented a classification pipeline using a human-in-the-loop machine learning approach and open source libraries to label the reports with one or more of four abnormality region labels: inner, middle, outer, and mastoid, indicating the presence of an abnormality in the specified ear region. METHODS: Trained abstractors labeled radiology reports taken from AudGenDB to form a gold standard. These were split into training (80 %) and test (20 %) sets. We applied open source libraries to normalize and convert every report to an n-gram feature vector. We trained logistic regression, support vector machine (linear and Gaussian), decision tree, random forest, and naïve Bayes models for each ear region. The models were evaluated on the hold-out test set. RESULTS: Our gold-standard data set contained 726 reports. The best classifiers were linear support vector machine for inner and outer ear, logistic regression for middle ear, and decision tree for mastoid. Classifier test set accuracy was 90 %, 90 %, 93 %, and 82 % for the inner, middle, outer and mastoid regions, respectively. The logistic regression method was very consistent, achieving accuracy scores within 2.75 % of the best classifier across regions and a receiver operator characteristic area under the curve of 0.92 or greater across all regions. CONCLUSIONS: Our results indicate that the applied methods achieve accuracy scores sufficient to support our objective of extracting discrete features from radiology reports to enhance cohort identification in AudGenDB. The models described here are available in several free, open source libraries that make them more accessible and simplify their utilization as demonstrated in this work. We additionally implemented the models as a web service that accepts radiology report text in an HTTP request and provides the predicted region labels. This service has been used to label the reports in AudGenDB and is freely available.

Pou3f4-mediated regulation of ephrin-b2 controls temporal bone development in the mouse.

The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. Ephrin-B2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development.

Harvest: an open platform for developing web-based biomedical data discovery and reporting applications.

Biomedical researchers share a common challenge of making complex data understandable and accessible as they seek inherent relationships between attributes in disparate data types. Data discovery in this context is limited by a lack of query systems that efficiently show relationships between individual variables, but without the need to navigate underlying data models. We have addressed this need by developing Harvest, an open-source framework of modular components, and using it for the rapid development and deployment of custom data discovery software applications. Harvest incorporates visualizations of highly dimensional data in a web-based interface that promotes rapid exploration and export of any type of biomedical information, without exposing researchers to underlying data models. We evaluated Harvest with two cases: clinical data from pediatric cardiology and demonstration data from the OpenMRS project. Harvest's architecture and public open-source code offer a set of rapid application development tools to build data discovery applications for domain-specific biomedical data repositories. All resources, including the OpenMRS demonstration, can be found at http://harvest.research.chop.edu.

Harvest: a web-based biomedical data discovery and reporting application development platform.

Biomedical researchers share a common challenge of making complex data understandable and accessible. This need is increasingly acute as investigators seek opportunities for discovery amidst an exponential growth in the volume and complexity of laboratory and clinical data. To address this need, we developed Harvest, an open source framework that provides a set of modular components to aid the rapid development and deployment of custom data discovery software applications. Harvest incorporates visual representations of multidimensional data types in an intuitive, web-based interface that promotes a real-time, iterative approach to exploring complex clinical and experimental data. The Harvest architecture capitalizes on standards-based, open source technologies to address multiple functional needs critical to a research and development environment, including domain-specific data modeling, abstraction of complex data models, and a customizable web client.

Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development.

Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C(2)H(2) zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.

Otic mesenchyme cells regulate spiral ganglion axon fasciculation through a Pou3f4/EphA4 signaling pathway.

Peripheral axons from auditory spiral ganglion neurons (SGNs) form an elaborate series of radially and spirally oriented projections that interpret complex aspects of the auditory environment. However, the developmental processes that shape these axon tracts are largely unknown. Radial bundles are comprised of dense SGN fascicles that project through otic mesenchyme to form synapses within the cochlea. Here, we show that radial bundle fasciculation and synapse formation are disrupted when Pou3f4 (DFNX2) is deleted from otic mesenchyme. Further, we demonstrate that Pou3f4 binds to and directly regulates expression of Epha4, Epha4⁻/⁻ mice present similar SGN defects, and exogenous EphA4 promotes SGN fasciculation in the absence of Pou3f4. Finally, Efnb2 deletion in SGNs leads to similar fasciculation defects, suggesting that ephrin-B2/EphA4 interactions are critical during this process. These results indicate a model whereby Pou3f4 in the otic mesenchyme establishes an Eph/ephrin-mediated fasciculation signal that promotes inner radial bundle formation.

The closely related transcription factors Sox4 and Sox11 function as survival factors during spinal cord development.

Development of the mouse CNS was reported to be normal in the absence of either Sox4 or its close relative Sox11 despite strong and widespread expression of both transcription factors. In this study, we show that combined absence of both Sox proteins in the mouse leads to severe hypoplasia of the developing spinal cord. Proliferation of neuroepithelial precursor cells in the ventricular zone was unaffected. These cells also acquired their correct positional identity. Both glial and neuronal progenitors were generated and neurons appeared in a similar spatiotemporal pattern as in the wild-type. Rates of cell death were however dramatically increased throughout embryogenesis in the double deficient spinal cord arguing that Sox4 and Sox11 are jointly and redundantly required for cell survival. The absence of pronounced proliferation, patterning, specification, and maturation defects furthermore indicates that the decreased cell survival is not a secondary effect of one of these events. We therefore conclude that the two Sox proteins directly function as pro-survival factors during spinal cord development in neural cell types.

Wnt signaling is sufficient to perturb oligodendrocyte maturation.

The development of oligodendrocytes, the myelinating cells of the central nervous system, is temporally and spatially controlled by local signaling factors acting as inducers or inhibitors. Dorsal spinal cord tissue has been shown to contain inhibitors of oligodendrogliogenesis, although their identity is not completely known. We have studied the actions of one family of dorsal signaling molecules, the Wnts, on oligodendrocyte development. Using tissue culture models, we have shown that canonical Wnt activity through beta-catenin activation inhibits oligodendrocyte maturation, independently of precursor proliferation, cell death, or diversion to an alternate cell fate. Mice in which Wnt/beta-catenin signaling was constitutively activated in cells of the oligodendrocyte lineage had equal numbers of oligodendrocyte precursors relative to control littermates, but delayed appearance of mature oligodendrocytes, myelin protein, and myelinated axons during development, although these differences largely disappeared by adulthood. These results indicate that activating the Wnt/beta-catenin pathway delays the development of myelinating oligodendrocytes.

Otic mesenchyme expression of Cre recombinase directed by the inner ear enhancer of the Brn4/Pou3f4 gene.

Brn4/Pou3f4 is a POU-domain transcription factor expressed in the otic mesenchyme that is required for the normal development of the inner ear. In this report, we describe the isolation of an otic mesenchyme enhancer in the Brn4 gene. Subsequently, this enhancer was used to drive the expression of Cre recombinase in the otic mesenchyme of transgenic mice. When intercrossed with the ROSA reporter strain, R26R, ss-galactosidase expression is detected in several inner ear structures derived from otic mesenchyme, including the temporal bone, spiral ligament, spiral limbus, and mesenchyme underlying sensory epithelium of the utricle, saccule and semicircular canals. Thus, this Cre pedigree can induce conditional rearrangement of genes in the otic mesenchyme, and will serve as a powerful genetic tool to characterize the function of genes in the mesenchymal tissues of the inner ear.

Cooperative function of Tbx1 and Brn4 in the periotic mesenchyme is necessary for cochlea formation.

The T-box transcription factor TBX1 has been identified as the major gene responsible for the etiology of velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS). Conductive hearing loss occurs in a majority of patients with this syndrome, while sensorineural deafness has also been reported in some cases. Mutations in POU3F4/BRN4, a POU domain transcription factor, cause DFN3, an X-linked nonsyndromic form of deafness characterized by mixed conductive and sensorineural hearing loss. Inactivation of the murine orthologues of these genes causes similar defects to those seen in humans and has provided excellent models for the study of inner ear development. Tbx1 and Brn4 are expressed in the mesenchymal cells surrounding the otic vesicle and have been shown to play roles in cochlear outgrowth. Furthermore, expression of Brn4 is reduced in Tbx1 null mutants, suggesting a possible genetic interaction between these genes. To test whether Tbx1 and Brn4 function in a common pathway, mice mutant for both genes were generated and analyzed for inner ear defects. Brn4-;Tbx1+/- mutants displayed a significant reduction in the number of turns of the cochlea compared to Brn4- or Tbx1+/- mice. In addition, Brn4-;Tbx1+/- mice displayed structural defects in the apical cochlea indicative of Mondini dysplasia found in patients with either VCFS/DGS or DFN3. These data establish a genetic interaction between Tbx1 and Brn4 relevant to human disease and indicate a function of these genes in signaling from the periotic mesenchyme to the otic vesicle to direct proper coiling of the cochlear duct.

BMP signaling mutant mice exhibit glial cell maturation defects.

Bone morphogenetic proteins have been implicated in the development of oligodendrocytes and astrocytes, however, a role for endogenous BMP signaling in glial development has not been demonstrated in a genetic model. Using mice in which signaling via type I BMP receptors Bmpr1a and Bmpr1b have been inactivated in the neural tube, we demonstrate that BMP signaling contributes to the maturation of glial cells in vivo. At P0, mutant mice exhibited a 25-40% decrease in GFAP+ or S100beta+ astrocytes in the cervical spinal cord. The number of oligodendrocyte precursors and the timing of their emergence was unchanged in the mutant mice compared to the normals, however myelin protein expression and mature oligodendrocyte numbers were significantly reduced. These data indicate that BMP signaling promotes the generation of astrocytes and mature, myelinating oligodendrocytes in vivo but does not affect oligodendrocyte precursor development, thus suggesting tight regulation of BMP signaling to ensure proper gliogenesis.

Bmp and Wnt/beta-catenin signals control expression of the transcription factor Olig3 and the specification of spinal cord neurons.

In the developing spinal cord, signals of the roof plate pattern the dorsal progenitor domain and control the specification of three neuron types, dorsal interneurons dI1, dI2, and dI3. Bmp and Wnt/beta-catenin signals as well as transcription factors like Olig3 or Ngn1/2 are essential in this process. We have studied the epistatic relationship between Bmp and Wnt/beta-catenin signals and the transcription factor Olig3 in dorsal spinal cord patterning. Using beta-catenin gain-of-function and compound beta-catenin gain-of-function/Olig3 loss-of-function mutations in mice, we could show that Wnt/beta-catenin signals act upstream of Olig3 in the specification of dI2 and dI3 neurons. The analysis of such compound mutant mice allowed us to distinguish between the two functions of Wnt/beta-catenin signaling in proliferation and patterning of dorsal progenitors. Using electroporation of chick spinal cords, we further demonstrate that Bmp signals act upstream of Wnt/beta-catenin in the regulation of Olig3 and that Wnt/beta-catenin signals play an instructive role in controlling Olig3 expression. We conclude that Wnt/beta-catenin and BMP signals coordinately control the specification of dorsal neurons in the spinal cord.

Noncanonical Wnt signaling and neural polarity.

The Wnt signaling pathway regulates multiple events in development and disease in both vertebrates and invertebrates. Recently, the noncanonical Wnt signaling cascades, those that do not signal through beta-catenin, have gained prominence for their role in the regulation of cellular polarity. It is not surprising that cellular polarization influences a number of different developmental events within the nervous system, including neurulation and neural tube closure, cellular migration, and uniform orientation of cells within an epithelial plane (planar cell polarity). In this review, we describe the differences between the canonical and noncanonical pathways, summarize recent data illustrating the roles of the noncanonical Wnt pathway in different polarizing events during neural development, and discuss the potential molecular mechanisms that underlie the generation of cellular asymmetry and polarity.

Genetic analyses demonstrate that bone morphogenetic protein signaling is required for embryonic cerebellar development.

The cerebellum has been a useful model for studying many aspects of neural development because of its relatively simple cytoarchitecture and developmental program. Yet, the genetic mechanisms underlying early differentiation and patterning of the cerebellum are still poorly characterized. Cell expression studies and culture experiments have suggested the importance of bone morphogenetic proteins (BMPs) in development of specific populations of cerebellar neurons. Here, we examined mice with targeted mutations in the BMP type I receptor genes Bmpr1a and Bmpr1b, to genetically test the hypothesis that BMPs play an inductive role in the embryogenesis of cerebellar granule cells. In Bmpr1a;Bmpr1b double knock-out mice, severe cerebellar patterning defects are observed resulting in smaller cerebella that are devoid of foliation. In mutants containing either single BMP receptor gene mutation alone, cerebellar histogenesis appears normal, thereby demonstrating functional redundancy of type I BMP receptors during cerebellar development. Loss of BMP signaling in double mutant animals leads to a dramatic reduction in the number of cerebellar granule cells and ectopic location of many of those that remain. Molecular markers of granule cell specification, including Math1 and Zic1, are drastically downregulated. In addition, Purkinje cells are disorganized and ectopically located, but they appear to be correctly specified. Consistent with the interpretation that granule cells alone are affected, phosphorylated Smad1/5/8 is immunolocalized predominantly to granule cell precursors and not appreciably detected in Purkinje cell precursors. This study demonstrates that BMP signaling plays a crucial role in the specification of granule cells during cerebellar development.

Abnormal mesenchymal differentiation in the superior semicircular canal of brn4/pou3f4 knockout mice.

OBJECTIVE: To examine the developmental time course of the mutant phenotype and cellular mechanisms that result in malformations of the superior semicircular canal (SSCC) in Brn4 knockout mice. Mutations in the Brn4/Pou3f4 gene result in characteristic inner ear abnormalities in mutant mouse pedigrees, and the findings in these mice are similar to those in human X-linked deafness type III. DESIGN: Mutant and control mice were killed at various neonatal time points to assess the development of the SSCC. Measurements of SSCC diameter were made on paint-perfused specimens at postnatal day (P) 0, P7, P10, and P14. Histologic evaluation of the SSCC was made on hematoxylin-eosin-stained sections at P10. RESULTS: A dysmorphic constriction of the superior arc of the SSCC in Brn4 knockout mice was initially detectable at P14. Interestingly, the mutant SSCC is indistinguishable from control mice at earlier neonatal time points. In mutant neonates, there is persistence of immature woven bone with high cellularity surrounding the perilymphatic space of the SSCC. These findings are not present in control animal specimens, which demonstrate appropriate lamellar bony architecture. CONCLUSIONS: In Brn4 knockout mice, constriction of the SSCC with narrowing of the bony labyrinth develops in the postnatal period at approximately P14. The persistence of immature bone in affected mice indicates that signaling abnormalities disrupt normal mesenchymal differentiation in the SSCC.

Mutation of the POU-domain gene Brn4/Pou3f4 affects middle-ear sound conduction in the mouse.

Mutagenesis of the POU-domain gene Brn4/Pou3f4 causes defects in the cochlear duct, semicircular canal, temporal bone and stapes footplate. The footplate defect suggested a middle-ear conductive component to the hearing loss associated with this mutation. This was examined by measuring velocity transfer functions at the umbo of wild type and knockout mice during sound stimulation of the tympanic membrane. When the median umbo velocity of test frequencies in the two groups were compared, the mid-range frequencies of the knockout mice showed a statistically reliable reduction in velocity (maximum of 13 dB) and high variability among animals. These results indicated that mutation of the POU-domain gene, Brn4, changed middle-ear sound conduction when measured at the umbo. The origin of the abnormal velocity response was sought by puncturing a hole in the pars flaccida (PF), and subsequently, measuring movements at the umbo and the head of the long arm of the incus. This hole permitted us to measure velocity at the tip of the incus long arm, just above the incudostapedial joint. The comparison of umbo behavior in both groups with PF perforated showed a loss of sensitivity in the mid-range frequencies of the knockout animals. A comparison of incus velocity in the two groups also exhibited a velocity reduction in the mid-range frequencies of the knockout animals. The reduction at the incus, however, was milder than observed at the umbo. The effect of the perforation in, and variability of, the knockout incus responses may have masked a more potent mid-range frequency effect. Nevertheless, evaluation of the stapes and oval window in knockout mice showed variable pathology from ear to ear. The presence of this pathology, the mid-frequency loss in incus sensitivity and the variability in incus velocity among animals suggested that abnormal stapes behavior in Brn4 deficient mice may determine the response of the ossicles, and thus account for the abnormal mid-frequency umbo behavior seen in knockout animals.