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.

Methylation changes in the peripheral blood of Filipinos with type 2 diabetes suggest spurious transcription initiation at TXNIP.

While much work has been done in associating differentially methylated positions (DMPs) to type 2 diabetes (T2D) across different populations, not much attention has been placed on identifying its possible functional consequences. We explored methylation changes in the peripheral blood of Filipinos with T2D and identified 177 associated DMPs. Most of these DMPs were associated with genes involved in metabolism, inflammation and the cell cycle. Three of these DMPs map to the TXNIP gene body, replicating previous findings from epigenome-wide association studies (EWAS) of T2D. The TXNIP downmethylation coincided with increased transcription at the 3' UTR, H3K36me3 histone markings and Sp1 binding, suggesting spurious transcription initiation at the TXNIP 3' UTR as a functional consequence of T2D methylation changes. We also explored potential epigenetic determinants to increased incidence of T2D in Filipino immigrants in the USA and found three DMPs associated with the interaction of T2D and immigration. Two of these DMPs were located near MAP2K7 and PRMT1, which may point towards dysregulated stress response and inflammation as a contributing factor to T2D among Filipino immigrants.

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.

Training in clinical genetics and genetic counseling in Asia.

The status of training in clinical genetics and genetic counseling in Asia is at diverse stages of development and maturity. Most of the training programs are in academic training centers where exposure to patients in the clinics or in the hospital is a major component. This setting provides trainees with knowledge and skills to be competent geneticists and genetic counselors in a variety of patient care interactions. Majority of the training programs combine clinical and research training which provide trainees a broad and integrated approach in the diagnosis and management of patients while providing opportunities for research discoveries that can be translated to better patient care. The background on how the training programs in clinical genetics and genetic counseling in Asia evolved to their current status are described. Each of these countries can learn from each other through sharing of best practices and resources.

A Genome-wide Association Study of Nonsyndromic Cleft Palate Identifies an Etiologic Missense Variant in GRHL3.

Cleft palate (CP) is a common birth defect occurring in 1 in 2,500 live births. Approximately half of infants with CP have a syndromic form, exhibiting other physical and cognitive disabilities. The other half have nonsyndromic CP, and to date, few genes associated with risk for nonsyndromic CP have been characterized. To identify such risk factors, we performed a genome-wide association study of this disorder. We discovered a genome-wide significant association with a missense variant in GRHL3 (p.Thr454Met [c.1361C>T]; rs41268753; p = 4.08 × 10(-9)) and replicated the result in an independent sample of case and control subjects. In both the discovery and replication samples, rs41268753 conferred increased risk for CP (OR = 8.3, 95% CI 4.1-16.8; OR = 2.16, 95% CI 1.43-3.27, respectively). In luciferase transactivation assays, p.Thr454Met had about one-third of the activity of wild-type GRHL3, and in zebrafish embryos, perturbed periderm development. We conclude that this mutation is an etiologic variant for nonsyndromic CP and is one of few functional variants identified to date for nonsyndromic orofacial clefting. This finding advances our understanding of the genetic basis of craniofacial development and might ultimately lead to improvements in recurrence risk prediction, treatment, and prognosis.

A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13.

Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10-8), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10-8). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10-8) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Transcriptional profiles associated with coronary artery disease in type 2 diabetes mellitus.

Background: Coronary artery disease (CAD) is a common complication of Type 2 diabetes mellitus (T2DM). Understanding the pathogenesis of this complication is essential in both diagnosis and management. Thus, this study aimed to characterize the presence of CAD in T2DM using molecular markers and pathway analyses. Methods: The study is a sex- and age-frequency matched case-control design comparing 23 unrelated adult Filipinos with T2DM-CAD to 23 controls (DM with CAD). Healthy controls served as a reference. Total RNA from peripheral blood mononuclear cells (PBMCs) underwent whole transcriptomic profiling using the Illumina HumanHT-12 v4.0 expression beadchip. Differential gene expression with gene ontogeny analyses was performed, with supporting correlational analyses using weighted correlation network analysis (WGCNA). Results: The study observed that 458 genes were differentially expressed between T2DM with and without CAD (FDR<0.05). The 5 top genes the transcription factor 3 (TCF3), allograft inflammatory factor 1 (AIF1), nuclear factor, interleukin 3 regulated (NFIL3), paired immunoglobulin-like type 2 receptor alpha (PILRA), and cytoskeleton-associated protein 4 (CKAP4) with AUCs >89%. Pathway analyses show differences in innate immunity activity, which centers on the myelocytic (neutrophilic/monocytic) theme. SNP-module analyses point to a possible causal dysfunction in innate immunity that triggers the CAD injury in T2DM. Conclusion: The study findings indicate the involvement of innate immunity in the development of T2DM-CAD, and potential immunity markers can reflect the occurrence of this injury. Further studies can verify the mechanistic hypothesis and use of the markers.

Genetic counseling/consultation in South-East Asia: a report from the workshop at the 10th Asia pacific conference on human genetics.

This paper reports on the workshop 'Genetic Counseling/Consultations in South-East Asia' at the 10(th) Asia Pacific Conference on Human Genetics in Kuala Lumpur, Malaysia, in December 2012. The workshop brought together professionals and language/communication scholars from South-East Asia, and the UK. The workshop aimed at addressing culture- and context-specific genetic counseling/consultation practices in South-East Asia. As a way of contextualizing genetic counseling/consultation in South-East Asia, we first offer an overview of communication-oriented research generally, drawing attention to consultation and counseling as part of a communicative continuum with distinctive interactional features. We then provide examples of genetic counseling/consultation research in Hong Kong. As other countries in South-East Asia have not yet embarked on communication-oriented empirical research, we report on the current practices of genetic counseling/consultation in these countries in order to identify similarities and differences as well as key obstacles that could be addressed through future research. Three issues emerged as 'problematic': language, religion and culture. We suggest that communication-oriented research can provide a starting point for evidence-based reflections on how to incorporate a counseling mentality in genetic consultation. To conclude, we discuss the need for creating a platform for targeted training of genetic counselors based on communication-oriented research findings.

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.

Unmet needs in countries participating in the undiagnosed diseases network international: an international survey considering national health care and economic indicators.

Background: Patients, families, the healthcare system, and society as a whole are all significantly impacted by rare diseases (RDs). According to various classifications, there are currently up to 9,000 different rare diseases that have been recognized, and new diseases are discovered every month. Although very few people are affected by each uncommon disease individually, millions of people are thought to be impacted globally when all these conditions are considered. Therefore, RDs represent an important public health concern. Although crucial for clinical care, early and correct diagnosis is still difficult to achieve in many nations, especially those with low and middle incomes. Consequently, a sizeable amount of the overall burden of RD is attributable to undiagnosed RD (URD). Existing barriers and policy aspects impacting the care of patients with RD and URD remain to be investigated. Methods: To identify unmet needs and opportunities for patients with URD, the Developing Nations Working Group of the Undiagnosed Diseases Network International (DNWG-UDNI) conducted a survey among its members, who were from 20 different nations. The survey used a mix of multiple choice and dedicated open questions covering a variety of topics. To explore reported needs and analyze them in relation to national healthcare economical aspects, publicly available data on (a) World Bank ranking; (b) Current health expenditure per capita; (c) GDP per capita; (d) Domestic general government health expenditure (% of GDP); and (e) Life expectancy at birth, total (years) were incorporated in our study. Results: This study provides an in-depth evaluation of the unmet needs for 20 countries: low-income (3), middle-income (10), and high-income (7). When analyzing reported unmet needs, almost all countries (N = 19) indicated that major barriers still exist when attempting to improve the care of patients with UR and/or URD; most countries report unmet needs related to the availability of specialized care and dedicated facilities. However, while the countries ranked as low income by the World Bank showed the highest prevalence of referred unmet needs across the different domains, no specific trend appeared when comparing the high, upper, and low-middle income nations. No overt trend was observed when separating countries by current health expenditure per capita, GDP per capita, domestic general government health expenditure (% of GDP) and life expectancy at birth, total (years). Conversely, both the GDP and domestic general government health expenditure for each country impacted the presence of ongoing research. Conclusion: We found that policy characteristics varied greatly with the type of health system and country. No overall pattern in terms of referral for unmet needs when separating countries by main economic or health indicators were observed. Our findings highlight the importance of identifying actionable points (e.g., implemented orphan drug acts or registries where not available) in order to improve the care and diagnosis of RDs and URDs on a global scale.

Undiagnosed diseases: Needs and opportunities in 20 countries participating in the Undiagnosed Diseases Network International.

Introduction: Rare diseases (RD) are a health priority worldwide, overall affecting hundreds of millions of people globally. Early and accurate diagnosis is essential to support clinical care but remains challenging in many countries, especially the low- and medium-income ones. Hence, undiagnosed RD (URD) account for a significant portion of the overall RD burden. Methods: In October 2020, the Developing Nations Working Group of the Undiagnosed Diseases Network International (DNWG-UDNI) launched a survey among its members, belonging to 20 countries across all continents, to map unmet needs and opportunities for patients with URD. The survey was based on questions with open answers and included eight different domains. Conflicting interpretations were resolved in contact with the partners involved. Results: All members responded to the survey. The results indicated that the scientific and medical centers make substantial efforts to respond to the unmet needs of patients. In most countries, there is a high awareness of RD issues. Scarcity of resources was highlighted as a major problem, leading to reduced availability of diagnostic expertise and research. Serious equity in accessibility to services were highlighted both within and between participating countries. Regulatory problems, including securing informed consent, difficulties in sending DNA to foreign laboratories, protection of intellectual property, and conflicts of interest on the part of service providers, remain issues of concern. Finally, most respondents stressed the need to strengthen international cooperation in terms of data sharing, clinical research, and diagnostic expertise for URD patients in low and medium income countries. Discussion: The survey highlighted that many countries experienced a discrepancy between the growing expertise and scientific value, the level of awareness and commitment on the part of relevant parties, and funding bodies. Country-tailored public health actions, including general syllabus of medical schools and of the education of other health professionals, are needed to reduce such gaps.

The rs1458038 variant near FGF5 is associated with poor response to calcium channel blockers among Filipinos.

ABSTRACT: Genetic variation is known to affect response to calcium channel blockers (CCBs) among different populations. This study aimed to determine the genetic variations associated with poor response to this class of antihypertensive drugs among Filipinos.One hundred eighty one hypertensive participants on CCBs therapy were included in an unmatched case-control study. Genomic deoxyribonucleic acid were extracted and genotyped for selected genetic variants. Regression analysis was used to determine the association of genetic and clinical variables with poor response to medication.The variant rs1458038 near fibroblast growth factor 5 gene showed significant association with poor blood pressure-lowering response based on additive effect (CT genotype: adjusted OR 3.41, P = .001; TT genotype: adjusted OR 6.72, P < .001).These findings suggest that blood pressure response to calcium channels blockers among Filipinos with hypertension is associated with gene variant rs1458038 near fibroblast growth factor 5 gene. Further studies are recommended to validate such relationship of the variant to the CCB response.

Variants of SLC2A10 may be Linked to Poor Response to Metformin.

Purpose: A study among Filipinos revealed that only 15% of patients with diabetes achieved glycemic control, and poor response to metformin could be one of the possible reasons. Recent studies demonstrate how genetic variations influence response to metformin. Hence, the present study aimed to determine genetic variants associated with poor response to metformin. Methods: Using a candidate variant approach, 195 adult Filipino participants with newly diagnosed type 2 diabetes mellitus (T2DM) were enrolled in a case-control study. Genomic DNA from blood samples were collected. Allelic and genotypic associations of variants with poor response to metformin were determined using exact statistical methods. Results: Several polymorphisms were nominally associated with poor response to metformin (P uncorr < 0.05). The most notable is the association of multiple variants in the SLC2A10 gene-rs2425911, rs3092412, and rs2425904-with common additive genetic mode of inheritance. Other variants that have possible associations with poor drug response include rs340874 (PROX-AS1), rs815815 (CALM2), rs1333049 (CDKN2B-AS1), rs2010963 (VEGFA), rs1535435 and rs9494266 (AHI1), rs11128347 (PDZRN3), rs1805081 (NPC1), and rs13266634 (SLC30A8). Conclusion: In Filipinos, a trend for the association for several variants was noted, with further observation that several mechanisms may be involved. The results may serve as pilot data for further validation of candidate variants for T2DM pharmacotherapy.

The Novel Phosphatase Domain Mutations Q171R and Y65S Switch PTEN from Tumor Suppressor to Oncogene.

Phosphatase and tensin homolog deleted on chromosome 10, or PTEN, is a well-characterized tumor suppressor with both lipid and protein phosphatase activities. PTEN is often downregulated by epigenetic mechanisms such as hypermethylation, which leads to constitutive activation of the PI3K-Akt pathway. Large datasets from next-generation sequencing, however, revealed that mutations in PTEN may not only hamper protein function but may also affect interactions with downstream effectors, leading to variable oncogenic readouts. Here, two novel PTEN mutations, Q171R and Y65S, identified in Filipino colorectal cancer patients, were phenotypically characterized in NIH3T3 and HCT116 cells, alongside the C124S canonical mutant and wild-type controls. The novel mutants increased cellular proliferation, resistance to apoptosis and migratory capacity. They induced gross morphological changes including cytoplasmic shrinkage, increased cellular protrusions and extensive cytoskeletal reorganization. The mutants also induced a modest increase in Akt phosphorylation. Further mechanistic studies will help determine the differential oncogenic potencies of these mutants, and resolve whether the structural constraints imposed by the mutations may have altered associations with downstream effectors.

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.

Detection and Genome Sequencing of SARS-CoV-2 Variants Belonging to the B.1.1.7 Lineage in the Philippines.

We report the sequencing and detection of 36 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples containing lineage-defining mutations specific to viruses belonging to the B.1.1.7 lineage in the Philippines.

The Klotho Variant rs36217263 Is Associated With Poor Response to Cardioselective Beta-Blocker Therapy Among Filipinos.

A common drug used for hypertension among Filipinos is beta-blockers. Variable responses to beta-blockers are observed, and genetic predisposition is suggested. This study investigated the association of genetic variants with poor response to beta-blockers among Filipinos. A total of 76 Filipino adult hypertensive participants on beta-blockers were enrolled in an unmatched case-control study. Genotyping was done using DNA from blood samples. Candidate variants were correlated with clinical data using χ2 and logistic regression analysis. The deletion of at least one copy of allele A of rs36217263 near Klotho showed statistically significant association with poor response to beta-blockers (dominant; odds ratio (OR) = 3.89; P = 0.017), adjusted for diabetes and dyslipidemia. This association is observed among participants using cardioselective beta-blockers (crude OR = 5.60; P = 0.008) but not carvedilol (crude OR = 2.56; P = 0.67). The genetic variant rs36217263 is associated with poor response to cardioselective beta-blockers, which may become a potential marker to aid in the management of hypertension.