The role of CDHR3 in susceptibility to otitis media.

Otitis media (OM) is common in young children and can cause hearing loss and speech, language, and developmental delays. OM has high heritability; however, little is known about OM-related molecular and genetic processes. CDHR3 was previously identified as a locus for OM susceptibility, but to date, studies have focused on how the CDHR3 p.Cys529Tyr variant increases epithelial binding of rhinovirus-C and risk for lung or sinus pathology. In order to further delineate a role for CDHR3 in OM, we performed the following: exome sequencing using DNA samples from OM-affected individuals from 257 multi-ethnic families; Sanger sequencing, logistic regression and transmission disequilibrium tests for 407 US trios or probands with OM; 16S rRNA sequencing and analysis for middle ear and nasopharyngeal samples; and single-cell RNA sequencing and differential expression analyses for mouse middle ear. From exome sequence data, we identified a novel pathogenic CDHR3 splice variant that co-segregates with OM in US and Finnish families. Additionally, a frameshift and six missense rare or low-frequency variants were identified in Finnish probands. In US probands, the CDHR3 p.Cys529Tyr variant was associated with the absence of middle ear fluid at surgery and also with increased relative abundance of Lysobacter in the nasopharynx and Streptomyces in the middle ear. Consistent with published data on airway epithelial cells and our RNA-sequence data from human middle ear tissues, Cdhr3 expression is restricted to ciliated epithelial cells of the middle ear and is downregulated after acute OM. Overall, these findings suggest a critical role for CDHR3 in OM susceptibility. KEY MESSAGES: • Novel rare or low-frequency CDHR3 variants putatively confer risk for otitis media. • Pathogenic variant CDHR3 c.1653 + 3G > A was found in nine families with otitis media. • CDHR3 p.Cys529Tyr was associated with lack of effusion and bacterial otopathogens. • Cdhr3 expression was limited to ciliated epithelial cells in mouse middle ear. • Cdhr3 was downregulated 3 h after infection of mouse middle ear.

The FUT2 Variant c.461G>A (p.Trp154*) Is Associated With Differentially Expressed Genes and Nasopharyngeal Microbiota Shifts in Patients With Otitis Media.

Otitis media (OM) is a leading cause of childhood hearing loss. Variants in FUT2, which encodes alpha-(1,2)-fucosyltransferase, were identified to increase susceptibility to OM, potentially through shifts in the middle ear (ME) or nasopharyngeal (NP) microbiotas as mediated by transcriptional changes. Greater knowledge of differences in relative abundance of otopathogens in carriers of pathogenic variants can help determine risk for OM in patients. In order to determine the downstream effects of FUT2 variation, we examined gene expression in relation to carriage of a common pathogenic FUT2 c.461G>A (p.Trp154*) variant using RNA-sequence data from saliva samples from 28 patients with OM. Differential gene expression was also examined in bulk mRNA and single-cell RNA-sequence data from wildtype mouse ME mucosa after inoculation with non-typeable Haemophilus influenzae (NTHi). In addition, microbiotas were profiled from ME and NP samples of 65 OM patients using 16S rRNA gene sequencing. In human carriers of the FUT2 variant, FN1, KMT2D, MUC16 and NBPF20 were downregulated while MTAP was upregulated. Post-infectious expression in the mouse ME recapitulated these transcriptional differences, with the exception of Fn1 upregulation after NTHi-inoculation. In the NP, Candidate Division TM7 was associated with wildtype genotype (FDR-adj-p=0.009). Overall, the FUT2 c.461G>A variant was associated with transcriptional changes in processes related to response to infection and with increased load of potential otopathogens in the ME and decreased commensals in the NP. These findings provide increased understanding of how FUT2 variants influence gene transcription and the mucosal microbiota, and thus contribute to the pathology of OM.

Multi-omic studies on missense PLG variants in families with otitis media.

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045 T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045 T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

A2ML1 and otitis media: novel variants, differential expression, and relevant pathways.

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.

FUT2 Variants Confer Susceptibility to Familial Otitis Media.

Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154∗) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202∗) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2 × 10-5) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154∗) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10-7) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154∗, and p.Arg202∗-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.

Dornase Alfa Ototoxic Effects in Animals and Efficacy in the Treatment of Clogged Tympanostomy Tubes in Children: A Preclinical Study and a Randomized Clinical Trial.

Importance: Many treatments for clogged tympanostomy tubes (TTs) have been proposed, but none have met scientific rigor for safety and efficacy, including the popular empirical use of ototopical antibiotic drops. Dornase alfa, a recombinant molecule with the unique property of cleaving DNA, may be ideal in treating clogged TTs because both middle-ear effusion and the plug are abundant with DNA. Objective: To investigate the ototoxic effects of dornase alfa in a chinchilla model and its efficacy in a clinical trial in children with clogged TTs. Design, Setting, and Participants: The safety profiles of dornase alfa (full-strength and 1:10 strength) were evaluated in chinchilla middle ears using serial auditory brainstem response. The efficacy of ototopical dornase alfa (full-strength) was evaluated in children with clogged TTs in a prospective, single-blind randomized clinical trial. The animal study included 21 chinchillas and was conducted at Loma Linda University, Loma Linda, California, and the clinical trial was conducted at Children's Hospital Colorado, Aurora. A total of 40 children (50 ears with tubes) were enrolled. Interventions: In the animal study, chinchillas were assigned to 3 groups: controls (saline), full-strength dornase alfa, or 1:10 dornase alfa dilution. Children were randomly assigned to receive either topical dornase alfa or ofloxacin for clogged TT, 5 drops each ear twice a day for 7 days. Main Outcomes and Measures: Animal study: Auditory brainstem responses. Randomized trial of children participants: The primary outcome was patency of TT at day 14 assessed by otoscopy and tympanometry. Results: The chinchilla study showed similar auditory brainstem response degradation during a 6-hour period between the control (n = 5) and treatment groups (n = 21). In the clinical trial, a total of 40 clogged TTs (in 33 children, including 25 boys [76%]; mean age, 4.3 years; median [range] age, 3.4 [1.0-14.3] years) were analyzed. The number of unclogged TTs was higher in the dornase alfa group (13 [59%]) compared with the ofloxacin group (8 [44%]), with a difference of 15% (odds ratio, 1.8; 95% CI, 0.54-6.72). Conclusions and Relevance: The chinchilla model suggests that dornase alfa is likely nonototoxic. The pilot clinical trial failed to show efficacy of dornase alfa to unclog TTs. With the difference seen between the treatment groups, a sample size estimate could be calculated for a future large-scale trial. Trial Registration: ClinicalTrials.gov identifier: NCT00419380.

Craniomaxillofacial Trauma.

Facial trauma causes significant of morbidity in the United States. With injuries varying widely, the clinical benefits of antibiotics use in facial fracture treatment are not easily determined. The pediatric population is more predisposed to craniofacial trauma secondary to their increased cranial mass to body ratio. All patients with traumatic injury should be assessed according to the Advanced Trauma Life Support protocol. This article discusses the types and prevalence of injuries and approaches to management.

Odontogenic myxoma with orbital involvement.

A 15-month-old female child presented with progressive right-sided facial swelling, nasal obstruction, and deviation of the nose to the left. Computed tomography revealed a cystic mass in the maxillary sinus with disruption of the medial orbital floor. Incisional biopsy of this mass was nondiagnostic and the mass continued to grow rapidly. The mass was removed by excisional biopsy and curettage with conservative margins via combined lateral rhinotomy and sublabial approaches. Intraoperatively, a large defect of the floor that extended to the orbital apex was noted. Histopathology revealed an odontogenic myxoma. Odontogenic myxomas are uncommon tumors that are usually seen in adults. Our case is unique because to the best of our knowledge, it is the first reported case with orbital involvement in the pediatric population.

Synthetic hydrogel scaffold is an effective vehicle for delivery of INFUSE (rhBMP2) to critical-sized calvaria bone defects in rats.

Medtronic's INFUSE Bone Graft provides surgeons with a potent tool for stimulating bone formation. Current delivery vehicles that rely on Absorbable Collagen Sponges (ACS) require excessive quantities of the active ingredient in INFUSE, recombinant human Bone Morphogenic Protein-2 (rhBMP2), to achieve physiologically relevant concentrations of the growth factor, driving up the cost of the product and increasing the likelihood of undesirable side effects in neighboring tissues. We demonstrate that a simple light-mediated, thiol-ene chemistry can be used to create an effective polymer delivery vehicle for rhBMP2, eliminating the use of xenographic materials and reducing the dose of rhBMP2 required to achieve therapeutic effects. Comprised entirely of synthetic components, this system entraps rhBMP2 within a biocompatible hydrogel scaffold that is degraded by naturally occurring remodeling enzymes, clearing the way for new tissue formation. When tested side-by-side with ACS in a critical-sized bone defect model in rats, this polymeric delivery system significantly increased bone formation over ACS controls.

Standardized letter of recommendation for otolaryngology residency selection.

OBJECTIVES/HYPOTHESIS: Develop a standardized letter of recommendation (SLOR) for otolaryngology residency application that investigates the qualities desired in residents and the letter writer's experience. Compare this SLOR to narrative letters of recommendation (NLORs). STUDY DESIGN: Prospective SLOR/NLOR comparison. METHODS: The SLOR was sent to an NLOR writer for each applicant. The applicant's NLOR/SLOR pair was blinded and ranked in seven categories by three reviewers. Inter-rater reliability and NLOR/SLOR rankings were compared. Means of cumulative NLOR and SLOR scores were compared to our departmental rank list. RESULTS: Thirty-one SLORs (66%) were collected. The SLORs had higher inter-rater reliability for applicant's qualifications for otolaryngology, global assessment, summary statement, and overall letter ranking. Writer's background, comparison to contemporaries/predecessors, and letter review ease had higher inter-rater reliability on the NLORs. Mean SLOR rankings were higher for writer's background (P = .0007), comparison of applicant to contemporaries/predecessors (P = .0031), and letter review ease (P < .0001). Mean SLOR writing time was 4.17 ± 2.18 minutes. Mean ranking time was significantly lower (P < .0001) for the SLORs (39.24 ± 23.45 seconds) compared to the NLORs (70.95 ± 40.14 seconds). Means of cumulative SLOR scores correlated with our rank list (P = .004), whereas means of cumulative NLOR scores did not (P = .18). Means of cumulative NLOR and SLOR scores did not correlate (P = .26). CONCLUSIONS: SLORs require little writing time, save reviewing time, and are easier to review compared to NLORs. Our SLOR had higher inter-rater reliability in four of seven categories and was correlated with our rank list. This tool conveys standardized information in an efficient manner.

Repair of a calvarial defect with biofactor and stem cell-embedded polyethylene glycol scaffold.

OBJECTIVE: Segmental bony defects resulting from congenital facial anomalies, facial trauma, infection, or oncologic surgical resection represent a common and significant clinical problem. Currently, these defects are reconstructed with autologous or allogeneic bone grafts or prosthetic devices. These options are limited by bone supply for grafting, donor site morbidity, risk of infection, and extrusion. This study investigated the in vivo osteogenic capability of polyethylene glycol-diacrylate (PEG-DA) and a protease-sensitive PEG matrix metalloproteinases (PEG-MMP), photoencapsulated with mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP)-2, in healing a critical-size rat calvarial defect. METHODS: Both PEG-DA and PEG-MMP scaffolds photoencapsulated with rat MSCs (rMSCs) and/or BMP-2 were implanted into a critical-size defect. Microcomputed-tomographic (micro-CT) analysis was completed 1, 4, and 8 weeks after implantation. Bone growth was histologically evaluated. The micro-CT data were analyzed using ASPIProVM software to calculate the percentage of closure of cranial defects. RESULTS: Both PEG-MMP and PEG-MMP + BMP2 showed significantly enhanced bone compared with controls. Polyethylene glycol-diacrylate seemed to inhibit bone growth regardless of biofactor and rMSCs. The addition of rMSCs did not enhance bone regeneration. CONCLUSION: Polyethylene glycol sensitive to proteolysis significantly improved bone repair in a critical-size calvarial defect.

Cochlear implantation in patients with osteogenesis imperfecta.

OBJECTIVE: Hearing loss has been shown to occur in 42% to 58% of patients with osteogenesis imperfecta (OI), with deafness arising in 25% to 60% of the patients. Implantation in patients with OI is relatively rare, with only 4 prior single case reports published in the English-language literature. The goal of this study was to evaluate the feasibility and functional outcome of cochlear implantation in 2 patients with OI tarda type I with profound sensorineural hearing loss. STUDY DESIGN: Case series. SETTING: The implantations were performed in a tertiary academic referral center (Johns Hopkins University). RESULTS: Though promontory vascularity was encountered, full insertion of a normal cochlear implant array could be achieved in both cases. One-year postimplant scores demonstrated 20 to 40 dB hearing thresholds, Consonant-Nucleus-Consonant Test word scores of 54% and 70%, Consonant-Nucleus-Consonant Test phoneme scores of 75% and 83%, Hearing in Noise Test scores of 76% and 99%, and Central Institute of the Deaf Sentence Score sentence scores of 99% and 100%, for patients 1 and 2, respectively. CONCLUSIONS: Cochlear implantation in patients with OI is not only technically possible but the results are similar to implant outcomes for patients with sensorineural hearing loss from a variety of other causes. EBM RATING: C.

Dehiscence of bone overlying the superior canal as a cause of apparent conductive hearing loss.

OBJECTIVE: To identify patients with superior semicircular canal dehiscence and apparent conductive hearing loss and to define the cause of the air-bone gap. STUDY DESIGN: Prospective study of patients with superior canal dehiscence. SETTING Tertiary referral center. PATIENTS: Vestibular and/or auditory findings indicative of canal dehiscence and demonstration of superior canal dehiscence on computed tomography of the temporal bone. INTERVENTION: Vestibular-evoked myogenic potentials, three-dimensional eye movement recordings, and surgical resurfacing of the superior canal. OUTCOME MEASURE: Association of superior canal dehiscence with an air-bone gap on audiometry. RESULTS: Four patients with dehiscence of bone overlying the superior canal were found to have air-bone gaps in the affected ears that were greatest at lower frequencies and averaged 24 +/- 7 dB over the frequency range of 250 to 4,000 Hz. Three of these patients had undergone stapedectomy before the identification of superior canal dehiscence. The air-bone gap was unchanged postoperatively. Each patient had an intact vestibular-evoked myogenic potential (VEMP) response from the affected ear, a finding that would not have been expected based on a middle ear cause of conductive hearing loss. One patient underwent resurfacing of the superior canal through a middle fossa approach. Postoperatively, his vestibular symptoms were relieved, and his air conduction thresholds were improved by 20 dB. CONCLUSIONS: Superior canal dehiscence can result in apparent conductive hearing loss. The third mobile window created by the dehiscent superior canal results in dissipation of acoustic energy and is a cause of inner ear conductive hearing loss.