Using multi-scale genomics to associate poorly annotated genes with rare diseases.

BACKGROUND: Next-generation sequencing (NGS) has significantly transformed the landscape of identifying disease-causing genes associated with genetic disorders. However, a substantial portion of sequenced patients remains undiagnosed. This may be attributed not only to the challenges posed by harder-to-detect variants, such as non-coding and structural variations but also to the existence of variants in genes not previously associated with the patient's clinical phenotype. This study introduces EvORanker, an algorithm that integrates unbiased data from 1,028 eukaryotic genomes to link mutated genes to clinical phenotypes. METHODS: EvORanker utilizes clinical data, multi-scale phylogenetic profiling, and other omics data to prioritize disease-associated genes. It was evaluated on solved exomes and simulated genomes, compared with existing methods, and applied to 6260 knockout genes with mouse phenotypes lacking human associations. Additionally, EvORanker was made accessible as a user-friendly web tool. RESULTS: In the analyzed exomic cohort, EvORanker accurately identified the "true" disease gene as the top candidate in 69% of cases and within the top 5 candidates in 95% of cases, consistent with results from the simulated dataset. Notably, EvORanker outperformed existing methods, particularly for poorly annotated genes. In the case of the 6260 knockout genes with mouse phenotypes, EvORanker linked 41% of these genes to observed human disease phenotypes. Furthermore, in two unsolved cases, EvORanker successfully identified DLGAP2 and LPCAT3 as disease candidates for previously uncharacterized genetic syndromes. CONCLUSIONS: We highlight clade-based phylogenetic profiling as a powerful systematic approach for prioritizing potential disease genes. Our study showcases the efficacy of EvORanker in associating poorly annotated genes to disease phenotypes observed in patients. The EvORanker server is freely available at https://ccanavati.shinyapps.io/EvORanker/ .

SMARCA4 mutation causes human otosclerosis and a similar phenotype in mice.

BACKGROUND: Otosclerosis is a common cause of adult-onset progressive hearing loss, affecting 0.3%-0.4% of the population. It results from dysregulation of bone homeostasis in the otic capsule, most commonly leading to fixation of the stapes bone, impairing sound conduction through the middle ear. Otosclerosis has a well-known genetic predisposition including familial cases with apparent autosomal dominant mode of inheritance. While linkage analysis and genome-wide association studies suggested an association with several genomic loci and with genes encoding structural proteins involved in bone formation or metabolism, the molecular genetic pathophysiology of human otosclerosis is yet mostly unknown. METHODS: Whole-exome sequencing, linkage analysis, generation of CRISPR mutant mice, hearing tests and micro-CT. RESULTS: Through genetic studies of kindred with seven individuals affected by apparent autosomal dominant otosclerosis, we identified a disease-causing variant in SMARCA4, encoding a key component of the PBAF chromatin remodelling complex. We generated CRISPR-Cas9 transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue. Mutant Smarca4+/E1548K mice exhibited marked hearing impairment demonstrated through acoustic startle response and auditory brainstem response tests. Isolated ossicles of the auditory bullae of mutant mice exhibited a highly irregular structure of the incus bone, and their in situ micro-CT studies demonstrated the anomalous structure of the incus bone, causing disruption in the ossicular chain. CONCLUSION: We demonstrate that otosclerosis can be caused by a variant in SMARCA4, with a similar phenotype of hearing impairment and abnormal bone formation in the auditory bullae in transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue.

Gene Therapy for Inherited Hearing Loss: Updates and Remaining Challenges.

Hearing loss stands as the most prevalent sensory deficit among humans, posing a significant global health challenge. Projections indicate that by 2050, approximately 10% of the world's population will grapple with disabling hearing impairment. While approximately half of congenital hearing loss cases have a genetic etiology, traditional interventions such as hearing aids and cochlear implants do not completely restore normal hearing. The absence of biological treatment has prompted significant efforts in recent years, with a strong focus on gene therapy to address hereditary hearing loss. Although several studies have exhibited promising recovery from common forms of genetic deafness in mouse models, existing challenges must be overcome to make gene therapy applicable in the near future. Herein, we summarize the primary gene therapy strategies employed over past years, provide an overview of the recent achievements in preclinical studies for genetic hearing loss, and outline the current key obstacles to cochlear gene therapy.

Shared and organ-specific gene-expression programs during the development of the cochlea and the superior olivary complex.

The peripheral and central auditory subsystems together form a complex sensory network that allows an organism to hear. The genetic programs of the two subsystems must therefore be tightly coordinated during development. Yet, their interactions and common expression pathways have never been systematically explored. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and are essential for normal development of the auditory system. We performed mRNA and small-RNA sequencing of organs from both auditory subsystems at three critical developmental timepoints (E16, P0, P16) to obtain a comprehensive and unbiased insight of their expression profiles. Our analysis reveals common and organ-specific expression patterns for differentially regulated mRNAs and miRNAs, which could be clustered with a particular selection of functions such as inner ear development, Wnt signalling, K+ transport, and axon guidance, based on gene ontology. Bioinformatics detected enrichment of predicted targets of specific miRNAs in the clusters and predicted regulatory interactions by monitoring opposite trends of expression of miRNAs and their targets. This approach identified six miRNAs as strong regulatory candidates for both subsystems. Among them was miR-96, an established critical factor for proper development in both subsystems, demonstrating the strength of our approach. We suggest that other miRNAs identified by this analysis are also common effectors of proper hearing acquirement. This first combined comprehensive analysis of the developmental program of the peripheral and central auditory systems provides important data and bioinformatics insights into the shared genetic program of the two sensory subsystems and their regulation by miRNAs.

Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain.

Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as 'gaps' in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.

Bats experience age-related hearing loss (presbycusis).

Hearing loss is a hallmark of aging, typically initially affecting the higher frequencies. In echolocating bats, the ability to discern high frequencies is essential. However, nothing is known about age-related hearing loss in bats, and they are often assumed to be immune to it. We tested the hearing of 47 wild Egyptian fruit bats by recording their auditory brainstem response and cochlear microphonics, and we also assessed the cochlear histology in four of these bats. We used the bats' DNA methylation profile to evaluate their age and found that bats exhibit age-related hearing loss, with more prominent deterioration at the higher frequencies. The rate of the deterioration was ∼1 dB per year, comparable to the hearing loss observed in humans. Assessing the noise in the fruit bat roost revealed that these bats are exposed to continuous immense noise-mostly of social vocalizations-supporting the assumption that bats might be partially resistant to loud noise. Thus, in contrast to previous assumptions, our results suggest that bats constitute a model animal for the study of age-related hearing loss.

A homozygous AP3D1 missense variant in patients with sensorineural hearing loss as the leading manifestation.

Loss-of-function variants in AP3D1 have been linked to Hermansky-Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein-protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

The long and short: Non-coding RNAs in the mammalian inner ear.

Non-coding RNAs (ncRNAs) play a critical role in the entire body, and their mis-regulation is often associated with disease. In parallel with the advances in high-throughput sequencing technologies, there is a great deal of focus on this broad class of RNAs. Although these molecules are not translated into proteins, they are now well established as significant regulatory components in many biological pathways and pathological conditions. ncRNAs can be roughly divided into two main sub-groups based on the length of the transcript, with both the small and long non-coding RNAs having diverse regulatory functions. The smaller length group includes ribosomal RNAs (rRNA), transfer RNAs (tRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), microRNAs (miRNA), small interfering RNAs (siRNA), and PIWI-associated RNAs (piRNA). The longer length group includes linear long non-coding RNAs (lncRNA) and circular RNAs (circRNA). This review is designed to present the different classes of small and long ncRNA molecules and describe some of their known roles in physiological and pathological conditions, as well as methods used to assess the validity and function of miRNAs and lncRNAs, with a focus on their role and functions in the inner ear, hearing and deafness.

A Nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Molecular Features of SLC26A4 Common Variant p.L117F.

The SLC26A4 gene, which encodes the anion exchanger pendrin, is involved in determining syndromic (Pendred syndrome) and non-syndromic (DFNB4) autosomal recessive hearing loss. SLC26A4 c.349C>T, p.L117F is a relatively common allele in the Ashkenazi Jewish community, where its minor allele frequency is increased compared to other populations. Although segregation and allelic data support the pathogenicity of this variant, former functional tests showed characteristics that were indistinguishable from those of the wild-type protein. Here, we applied a triad of cell-based assays, i.e., measurement of the ion transport activity by a fluorometric method, determination of the subcellular localization by confocal microscopy, and assessment of protein expression levels, to conclusively assign or exclude the pathogenicity of SLC26A4 p.L117F. This protein variant showed a moderate, but significant, reduction in ion transport function, a partial retention in the endoplasmic reticulum, and a strong reduction in expression levels as a consequence of an accelerated degradation by the Ubiquitin Proteasome System, all supporting pathogenicity. The functional and molecular features of human pendrin p.L117F were recapitulated by the mouse ortholog, thus indicating that a mouse carrying this variant might represent a good model of Pendred syndrome/DFNB4.

The Genomics of Auditory Function and Disease.

Current estimates suggest that nearly half a billion people worldwide are affected by hearing loss. Because of the major psychological, social, economic, and health ramifications, considerable efforts have been invested in identifying the genes and molecular pathways involved in hearing loss, whether genetic or environmental, to promote prevention, improve rehabilitation, and develop therapeutics. Genomic sequencing technologies have led to the discovery of genes associated with hearing loss. Studies of the transcriptome and epigenome of the inner ear have characterized key regulators and pathways involved in the development of the inner ear and have paved the way for their use in regenerative medicine. In parallel, the immense preclinical success of using viral vectors for gene delivery in animal models of hearing loss has motivated the industry to work on translating such approaches into the clinic. Here, we review the recent advances in the genomics of auditory function and dysfunction, from patient diagnostics to epigenetics and gene therapy.

Autosomal dominant non-syndromic hearing loss maps to DFNA33 (13q34) and co-segregates with splice and frameshift variants in ATP11A, a phospholipid flippase gene.

Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

PNPT1, MYO15A, PTPRQ, and SLC12A2-associated genetic and phenotypic heterogeneity among hearing impaired assortative mating families in Southern India.

The study was conducted between 2018 and 2020. From a cohort of 113 hearing impaired (HI), five non-DFNB12 probands identified with heterozygous CDH23 variants were subjected to exome analysis. This resolved the etiology of hearing loss (HL) in four South Indian assortative mating families. Six variants, including three novel ones, were identified in four genes: PNPT1 p.(Ala46Gly) and p.(Asn540Ser), MYO15A p.(Leu1485Pro) and p.(Tyr1891Ter), PTPRQ p.(Gln1336Ter), and SLC12A2 p.(Pro988Ser). Compound heterozygous PNPT1 variants were associated with DFNB70 causing prelingual profound sensorineural hearing loss (SNHL), vestibular dysfunction, and unilateral progressive vision loss in one family. In the second family, MYO15A variants in the myosin motor domain, including a novel variant, causing DFNB3, were found to be associated with prelingual profound SNHL. A novel PTPRQ variant was associated with postlingual progressive sensorineural/mixed HL and vestibular dysfunction in the third family with DFNB84A. In the fourth family, the SLC12A2 novel variant was found to segregate with severe-to-profound HL causing DFNA78, across three generations. Our results suggest a high level of allelic, genotypic, and phenotypic heterogeneity of HL in these families. This study is the first to report the association of PNPT1, PTPRQ, and SLC12A2 variants with HL in the Indian population.

The noncoding genome and hearing loss.

The age of sequencing has provided unprecedented insights into the human genome. The coding region of the genome comprises nearly 20,000 genes, of which approximately 4000 are associated with human disease. Beyond the protein-coding genome, which accounts for only 3% of the genome, lies a vast pool of regulatory elements in the form of promoters, enhancers, RNA species, and other intricate elements. These features undoubtably influence human health and disease, and as a result, a great deal of effort is currently being invested in deciphering their identity and mechanism. While a paucity of material has caused a lag in identifying these elements in the inner ear, the emergence of technologies for dealing with a minimal number of cells now has the field working overtime to catch up. Studies on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), methylation, histone modifications, and more are ongoing. A number of microRNAs and other noncoding elements are known to be associated with hearing impairment and there is promise that regulatory elements will serve as future tools and targets of therapeutics and diagnostics. This review covers the current state of the field and considers future directions for the noncoding genome and implications for hearing loss.

Disease-specific ACMG/AMP guidelines improve sequence variant interpretation for hearing loss.

PURPOSE: The ClinGen Variant Curation Expert Panels (VCEPs) provide disease-specific rules for accurate variant interpretation. Using the hearing loss-specific American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, the Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in variant interpretation. METHODS: A total of 157 variants across nine HL genes, previously submitted to ClinVar, were curated by the HL VCEP. The curation process involved collecting published and unpublished data for each variant by biocurators, followed by bimonthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification. RESULTS: Before expert curation, 75% (117/157) of variants had single or multiple variants of uncertain significance (VUS) submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS and 69% (69/100) of discordant variants were resolved into benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant classification. CONCLUSION: Expert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study highlights the utility of ClinGen VCEPs in supporting more consistent clinical variant interpretation.

Genetic Heterogeneity and Core Clinical Features of NOG-Related-Symphalangism Spectrum Disorder.

OBJECTIVES: To better distinguish NOG-related-symphalangism spectrum disorder (NOG-SSD) from chromosomal 17q22 microdeletion syndromes and to inform surgical considerations in stapes surgery for patients with NOG-SSD. BACKGROUND: Mutations in NOG cause a variety of skeletal syndromes that often include conductive hearing loss. Several microdeletions of chromosome 17q22 lead to severe syndromes with clinical characteristics that overlap NOG-SSD. Isolated deletion of NOG has not been described, and therefore the contribution of NOG deletion in these syndromes is unknown. METHODS: Two families with autosomal dominant NOG-SSD exhibited stapes ankylosis, facial dysmorphisms, and skeletal and joint anomalies. In each family, NOG was evaluated by genomic sequencing and candidate mutations confirmed as damaging by in vitro assays. Temporal bone histology of a patient with NOG-SSD was compared with temporal bones of 40 patients diagnosed with otosclerosis. RESULTS: Family 1 harbors a 555 kb chromosomal deletion encompassing only NOG and ANKFN1. Family 2 harbors a missense mutation in NOG leading to absence of noggin protein. The incus-footplate distance of the temporal bone was significantly longer in a patient with NOG-SSD than in patients with otosclerosis. CONCLUSION: The chromosomal microdeletion of family 1 led to a phenotype comparable to that due to a NOG point mutation and much milder than the phenotypes due to other chromosome 17q22 microdeletions. Severe clinical findings in other microdeletion cases are likely due to deletion of genes other than NOG. Based on temporal bone findings, we recommend that surgeons obtain longer stapes prostheses before stapes surgery in individuals with NOG-SSD stapes ankylosis.

Auditory Performance in Recovered SARS-COV-2 Patients.

OBJECTIVE: While COVID-19 symptoms impact rhinology (anosmia) and laryngology (airways), two major disciplines of the otolaryngology armamentarium, the virus has seemed to spare the auditory system. A recent study, however, reported changes in otoacoustic emission (OAE) signals measured in SARS-COV-2 positive patients. We sought to assess the effect of COVID-19 infection on auditory performance in a cohort of recovered SARS-COV-2 patients and controls. To avoid a potential bias of previous audiological dysfunction not related to SARS-COV-2 infection, the study encompasses patients with normal auditory history. We hypothesized that if SARS-COV-2 infection predisposes to hearing loss, we would observe subtle and early audiometric deficits in our cohort in the form of subclinical auditory changes. STUDY DESIGN: Cross-sectional study. SETTING: Tertiary referral center. PATIENTS: The Institutional Review Board approved the study and we recruited participants who had been positive for SARS-COV-2 infection, according to an Reverse Transcription Polymerase Chain Reaction (RT-PCR) test on two nasopharyngeal swabs. The patients included in this study were asymptomatic for the SARS-COV-2 infection and were evaluated following recovery, confirmed by repeated swab testing. The control group comprised healthy individuals matched for age and sex, and with a normal auditory and otologic history. INTERVENTIONS: The eligibility to participate in this study included a normal audiogram, no previous auditory symptoms, normal otoscopy examination with an intact tympanic membrane, and bilateral tympanometry type A. None of our volunteers reported any new auditory symptoms following SARS-COV-2 infection. Ototacoustic emissions (OAE) and auditory brainstem response (ABR) measurements were used to evaluate the auditory function. MAIN OUTCOME MEASURES: OAE and ABR measurements. RESULTS: We have found no significant differences between recovered asymptomatic SARS-COV-2 patients and controls in any of transitory evoked otoacoustic emission (TEOAE), distortion product otoacoustic emissions (DPOAE), or ABR responses. CONCLUSIONS: There is no cochlear dysfunction represented by ABR, TEOAE, and DPOAE responses in recovered COVID-19 asymptomatic patients. Retrocochlear function was also preserved as evident by the ABR responses. A long-term evaluation of a larger cohort of SARS-COV-2 patients will help to identify a possible contribution of SARS-COV-2 infection to recently published anecdotal auditory symptoms associated with COVID-19.

Reprogramming of two induced pluripotent stem cell lines from a heterozygous GRIN2D developmental and epileptic encephalopathy (DEE) patient (BGUi011-A) and from a healthy family relative (BGUi012-A).

The GLUN2D subunit of the N-methylD-aspartate receptor (NMDAR) is encoded by the GRIN2D gene. Mutations in GRIN2D have been associated with neurodevelopmental and epileptic encephalopathies. Access to patient samples harboring mutations in GRIN2D can contribute to understanding the role of NMDAR in neuronal development and function. We report the generation of induced pluripotent stem cell (iPSC) lines from a GRIN2D-developmental and epileptic encephalopathy (DEE) patient, carrying a de novo c.1999G>A heterozygous pathogenic variant, and his healthy parent. Generated lines highly expressed pluripotency markers, spontaneously differentiated into the three germ layers, retained the deficiency-causing mutation, and displayed normal karyotypes.