Otitis media susceptibility and shifts in the head and neck microbiome due to SPINK5 variants.

BACKGROUND: Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility. METHODS: We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues. RESULTS: A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma. CONCLUSION: SPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.

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.

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.

Design of a Blended Learning Course for Training Community Healthcare Providers on eHealth-enabled Newborn Hearing Screening.

Objectives: We present in this article the design and evaluation of a blended learning approach for training community healthcare providers in performing newborn hearing screening (NHS). Methods: We developed a blended learning course for training community healthcare providers on eHealth-enabled NHS, following Bloom's revised taxonomy of educational objectives. The training involved three components: computer-based training (CBT), face-to-face (FTF) training, and on-site coaching. We used surveys and post-training interviews following Level 1 Kirkpatrick's training evaluation model to get initial feedback on the training program. Results: Thirty-one community healthcare providers from five rural health units and a private hearing screening center, with a mean age of 42.2 ± 12.0 years, participated in the pilot. 93.5% of the participants agreed that the program content met stated objectives and was relevant to their practice. The length of the course was perceived to be adequate. Overall satisfaction with the program was rated at 8.5 ± 0.9 (with ten as the highest). The majority expressed that the CBT and FTF course were satisfactory at 93.5% and 100%, respectively. All participants agreed that the course enhanced their knowledge of newborn hearing screening and telehealth. Positive reviews were received from participants on the use of CBT to improve theoretical knowledge before FTF training. Participants declared that FTF training and on-site coaching helped improved NHS skills and implementation. Conclusion: Competent community healthcare providers are critical to strengthening the performance of the health system, and advances in the education and technology sectors offer promising potential in upskilling local healthcare providers. The increasing access of Filipino healthcare providers to improved information and communications technology (ICT) is a significant catalyst for pedagogical innovation, like the use of blended learning in the continuous professional development of health practitioners. As ICTs gradually penetrate the health sector, the challenge we now face is not whether but how we can use innovations in education strategies to benefit healthcare providers.

Pilot Implementation of a Community-based, eHealth-enabled Service Delivery Model for Newborn Hearing Screening and Intervention in the Philippines.

Objectives: This study explores the potential of the HeLe Service Delivery Model, a community-based newborn hearing screening (NHS) program supported by a web-based referral system, in improving provision of hearing care services. Methods: This prospective observational study evaluated the HeLe Service Delivery Model based on records review and user perspectives. We collected system usage logs from July to October 2018 and data on patient outcomes. Semi-structured interviews and review of field reports were conducted to identify implementation challenges and facilitating factors. Descriptive statistics and content analysis were used to analyze quantitative and qualitative data, respectively. Results: Six hundred ninety-two (692) babies were screened: 110 in the RHUs and 582 in the Category A NHS hospital. Mean age at screening was 1.4±1.05 months for those screened in the RHU and 0.46±0.74 month for those in the Category A site. 47.3% of babies screened at the RHU were ≤1 month old in contrast to 86.6% in the Category A hospital. A total of 10 babies (1.4%) received a positive NHS result. Eight of these ten patients were referred via the NHS Appointment and Referral System; seven were confirmed to have bilateral profound hearing loss, while one patient missed his confirmatory testing appointment. The average wait time between screening and confirmatory testing was 17.1±14.5 days. Facilitating factors for NHS implementation include the presence of champions, early technology adopters, legislations, and capacity-building programs. Challenges identified include perceived inconvenience in using information systems, cost concerns for the patients, costly hearing screening equipment, and unstable internet connectivity. The lack of nearby facilities providing NHS diagnostic and intervention services remains a major block in ensuring early diagnosis and management of hearing loss in the community. Conclusion: The eHealth-enabled HeLe Service Delivery Model for NHS is promising. It addresses the challenges and needs of community-based NHS by establishing a healthcare provider network for NHS in the locale, providing a capacity-building program to train NHS screeners, and deploying health information systems that allows for documentation, web-based referral and tracking of NHS patients. The model has the potential to be implemented on a larger scale - a deliberate step towards universal hearing health for all Filipinos.

Pilot Implementation of a Computer-based Training Course on Newborn Hearing Screening and Teleaudiology among Primary Healthcare Providers in Low Resource Settings.

Objective: Newborn hearing screening (NHS) in the Philippines has been mandated by law since 2009. However, lack of awareness and knowledge about NHS remains a challenge, especially among healthcare providers. This paper describes the pilot implementation of a computer-based training (CBT) course on NHS and teleaudiology among primary healthcare providers (PHCPs) in rural Philippines. Methods: A four-module web-based training course on newborn hearing screening and teleaudiology in an online learning management system (LMS) was field-tested among PHCPs from eight rural communities in the Philippines. Participants were given four weeks to complete the course. Results: Forty-two PHCPs participated in the CBT. Thirty-four (81%) completed the whole course (mean attrition rate of 4.8% per module) at a mean duration of 10.2 days. Baseline data shows that participants had no NHS training, although the majority (83%) had information and communications technology (ICT) training. Comparison of pre- and post-test mean scores showed a 24.0% (p<0.001) significant increase in the post-test in all four modules. Passing rates (i.e., score ≥70%) from pre- to post-test increased by 54.6% (range: 38-80% increase). Usability of the CBT was rated high with a mean score of 4.32 out of 5 (range: 4.13 to 4.47), covering all eight parameters. Participants expressed general satisfaction and a positive attitude on CBT to improve knowledge on NHS and teleaudiology. Conclsion: Even in low resource settings where gaps in ICT infrastructure exist, eLearning can be used as an alternative approach to increase awareness and support training of healthcare providers on newborn hearing screening.

Audiologic Measures in an Indigenous Community with A2ML1- and FUT2-Related Otitis Media.

Background: Many indigenous peoples are at elevated risk for otitis media, however there is limited information on hearing loss due to OM in these communities. An Indigenous Filipino community that has previously been described with an elevated prevalence of OM that is due to rare A2ML1 variants and a common FUT2 variant underwent additional phenological testing. In this study, we describe the audiologic profiles in A2ML1- and FUT2-related otitis media and the validity of otoscopy and genotyping for A2ML1 and FUT2 variants in screening for otitis media and hearing loss. Method: We analyzed A2ML1 and FUT2 genotypes together with demographic, otologic and audiologic data from tympanometry and hearing level assessments of 109 indigenous individuals. Results: We confirmed previous findings of a spectrum of nonsyndromic otitis media as associated with A2ML1 variants. A2ML1 and FUT2 variants were associated with high-frequency hearing loss at 4000 Hz. As expected, young age was associated with flat tympanograms, and eardrum perforations due to chronic otitis media were associated with severe-to-profound hearing loss across frequencies. Adding A2ML1 or FUT2 genotypes improved the validity of otoscopy as a screening test to rule out moderate-to-profound hearing loss. Conclusion: Continued multi-disciplinary management and audiologic follow-up using tympanometry and screening audiometry are needed to document and treat otitis media and prevent permanent hearing loss in the indigenous community.

Lack of Methylation Changes in GJB2 and RB1 Non-coding Regions of Cochlear Implant Patients with Sensorineural Hearing Loss.

Objective: Recent advances in epigenetic studies continue to reveal novel mechanisms of gene regulation and control, however little is known on the role of epigenetics in sensorineural hearing loss (SNHL) in humans. We aimed to investigate the methylation patterns of two regions, one in RB1 and another in GJB2 in Filipino patients with SNHL compared to hearing control individuals. Methods: We investigated an RB1 promoter region that was previously identified as differentially methylated in children with SNHL and lead exposure. Additionally, we investigated a sequence in an enhancer-like region within GJB2 that contains four CpGs in close proximity. Bisulfite conversion was performed on salivary DNA samples from 15 children with SNHL and 45 unrelated ethnically-matched individuals. We then performed methylation-specific real-time PCR analysis (qMSP) using TaqMan® probes to determine percentage methylation of the two regions. Results: Using qMSP, both our cases and controls had zero methylation at the targeted GJB2 and RB1 regions. Conclusion: Our study showed no changes in methylation at the selected CpG regions in RB1 and GJB2 in the two comparison groups with or without SNHL. This may be due to a lack of environmental exposures to these target regions. Other epigenetic marks may be present around these regions as well as those of other HL-associated genes.

Microbiota Associated With Cholesteatoma Tissue in Chronic Suppurative Otitis Media.

Otitis media (OM), defined as infection or inflammation of the middle ear (ME), remains a major public health problem worldwide. Cholesteatoma is a non-cancerous, cyst-like lesion in the ME that may be acquired due to chronic OM and cause disabling complications. Surgery is required for treatment, with high rates of recurrence. Current antibiotic treatments have been largely targeted to previous culturable bacteria, which may lead to antibiotic resistance or treatment failures. For this study, our goal was to determine the microbiota of cholesteatoma tissue in comparison with other ME tissues in patients with long-standing chronic OM. ME samples including cholesteatoma, granulation tissue, ME mucosa and discharge were collected from patients undergoing tympanomastoidectomy surgery for chronic OM. Bacteria were profiled by 16S rRNA gene sequencing in 103 ME samples from 53 patients. Respiratory viruses were also screened in 115 specimens from 45 patients. Differences in bacterial profiles (beta-diversity) and the relative abundances of individual taxa were observed between cholesteatoma and ME sample-types. Additionally, patient age was associated with differences in overall microbiota composition while numerous individual taxa were differentially abundant across age quartiles. No viruses were identified in screened ME samples. Biodiversity was moderately lower in cholesteatoma and ME discharge compared to ME mucosal tissues. We also present overall bacterial profiles of ME tissues by sample-type, age, cholesteatoma diagnosis and quinolone use, including prevalent bacterial taxa. Our findings will be useful for fine-tuning treatment protocols for cholesteatoma and chronic OM in settings with limited health care resources.

Identification of Novel Candidate Genes and Variants for Hearing Loss and Temporal Bone Anomalies.

Background: Hearing loss remains an important global health problem that is potentially addressed through early identification of a genetic etiology, which helps to predict outcomes of hearing rehabilitation such as cochlear implantation and also to mitigate the long-term effects of comorbidities. The identification of variants for hearing loss and detailed descriptions of clinical phenotypes in patients from various populations are needed to improve the utility of clinical genetic screening for hearing loss. Methods: Clinical and exome data from 15 children with hearing loss were reviewed. Standard tools for annotating variants were used and rare, putatively deleterious variants were selected from the exome data. Results: In 15 children, 21 rare damaging variants in 17 genes were identified, including: 14 known hearing loss or neurodevelopmental genes, 11 of which had novel variants; and three candidate genes IST1, CBLN3 and GDPD5, two of which were identified in children with both hearing loss and enlarged vestibular aqueducts. Patients with variants within IST1 and MYO18B had poorer outcomes after cochlear implantation. Conclusion: Our findings highlight the importance of identifying novel variants and genes in ethnic groups that are understudied for hearing loss.

Genetic counseling in an indigenous Filipino community with a high prevalence of A2ML1-related otitis media.

In this report, we describe the knowledge and beliefs on causes and management of otitis media of an indigenous Filipino community with a high prevalence of otitis media that is associated with an A2ML1 variant. Community lectures and individual genetic counseling were provided as intervention. Knowledge, beliefs, and health care-seeking behavior pertaining to otitis media were assessed pre- and post-genetic counseling. Twenty-five heads of households were interviewed. Beliefs regarding etiology of ear discharge varied widely, with swimming in the sea as the most commonly cited cause of ear discharge. During the post-counseling session, poor personal hygiene, dirty environment, and familial inheritance were mentioned as risk factors for otitis media or ear discharge. Knowledge about the genotypes for the A2ML1 variant and otitis media diagnoses within the household influenced beliefs on the role of hygiene and genetic susceptibility to otitis media and attitudes towards health care-seeking behavior. Genetic counseling was associated with variable improvement in knowledge on otitis media and in their understanding of genetic susceptibility to otitis media due to the A2ML1 variant. Language barriers, literacy level, cultural factors, and the complex nature of genetic information made genetic counseling in the particular population a challenge. Insights drawn from this experience recommend follow-up visits in the community.

The SLC26A4 c.706C>G (p.Leu236Val) Variant is a Frequent Cause of Hearing Impairment in Filipino Cochlear Implantees.

HYPOTHESIS: Variants in SLC26A4 are an important cause of congenital hearing impairment in the Philippines. BACKGROUND: Cochlear implantation is a standard rehabilitation option for congenital hearing impairment worldwide, but places a huge cost burden in lower-income countries. The study of risk factors such as genetic variants that may help determine genetic etiology of hearing loss and also predict cochlear implant outcomes is therefore beneficial. METHODS: DNA samples from 29 GJB2-negative Filipino cochlear implantees were Sanger-sequenced for the coding exons of SLC26A4. Exome sequencing was performed to confirm results. RESULTS: Four cochlear implantees with bilaterally enlarged vestibular aqueducts (EVA) were homozygous for the pathogenic SLC26A4 c.706C>G (p.Leu236Val) variant, which has a minor allele frequency of 0.0015 in Filipino controls. In patients with the SLC26A4 variant there was no association between cochlear implant outcome and age at implantation or duration of implant. There was also no association between the occurrence of the SLC26A4 variant and postsurgical audiometric thresholds and parents' evaluation of aural/oral performance of children (PEACH) scores. On the other hand, the SLC26A4 variant increased presurgical median audiometric thresholds (p = 0.01), particularly at 500 to 2000 Hz. CONCLUSION: The SLC26A4 c.706C>G (p.Leu236Val) variant is a frequent cause of congenital hearing impairment in Filipinos and is associated with bilateral EVA and increased presurgical audiometric thresholds, but does not adversely affect post-implant outcomes.