Xue Liu - US grants

Hearing loss is the most common sensorineural disorder affecting at least 5 percent of population. Genetic factors are one of the most important causes for profound hearing impairment. About 1 in 2000 children are born with hereditary deafness. The bulk (around 80 percent) of genetic deafness is non-syndromic form. Studies on molecular basis of non-syndromic hearing loss (NSHL) are not only important for improving our understanding of normal hearing and hearing loss, but also for developing more precise genetic counseling and specific treatments for both genetic and environment-related hearing impairment. Because of genetic heterogeneity, large families usually from isolated communities have been selected for study that are independently capable of yielding evidence for linkage. We have collected more than 130 DNA samples from three large dominantly inherited multigenerational families in China with non-syndromic dominant deafness and have access to four additional pedigrees with NSHL that would be suitable for linkage analysis. The proposed research will exploit this unique source for large multigenerational families. We propose (1) to continue collecting samples from all available members of relevant sibships in these families, (2) to type the samples for polymorphic markers in order to exclude linkage to known loci, (3) to subject the remaining pedigrees to genome wide marker screening for linkage analysis. For the largest family NSHL.O1, linkage has been excluded to any of known loci for dominant NSHL and DNA samples from this family have already been submitted for a genome wide marker screen. We will then seek to identify and ultimately clone the relevant genes by the positional candidate gene approach. The goal of the proposed studies is to map at least one new gene for non-syndromic deafness. The proposal will provide the preliminary data required for further localization and positional cloning of the gene. This strategy has been successfully employed to map 50 loci for NSHL. This knowledge is an essential prerequisite to the development and provision of molecular diagnostic services for families with NSHL, as well as the further delineation of the functional genomics of the cochlea.

DESCRIPTION (Investigator's abstract): The goal of this proposal is to identify molecular basis of non-syndromic deafness by using a multidisciplinary Approach in several unique resources of patients/families from Far Eastern populations. We have a large and expanding collection of genomic DNA from patients/families with non-syndromic deafness from China and Japan First, because of genetic heterogeneity, large multigenerational families and consanguineous families have been selected for study that are independently capable of yielding evidence of for linkage. We will then seek to identify and ultimately clone the relevant genes by the positional candidate gene approach. Second, a novel sequential screening strategy will be used to identify new genes for deafness in probands from multiplex families. Candidate genes to be screened will include the human orthologs of murine genes for deafness and other members of gene families in which some are known to be the cause of deafness. The research will yield important information o ethnic differences in the frequency and distribution of mutations at currently recognized genes for non-syndromic deafness. Using available information on outcome variables such as age of onset, audiologic findings, and the mating structure of the deaf population, these data will permit a search for clinically relevant genotype-phenotype correlations and a clearer understanding of the cause for secular changes in the frequency of specific forms o genetic deafness. Finally, we will use yeast 2-hybrid systems and the immunoaffinity chromatography to search for proteins that interact with the products of the MYO7A and USH1C genes. The identification of such interactions could lead to identifying potential modifiers and exciting new therapeutic approaches to attenuate the specific effects of mutations on the cochlear. The proposed work will identify the new gene (s) for non-syndromic deafness and enable us to understand better cellular and molecular basis of genetic deafness. Our studies will develop a more comprehensive picture of the involvement of genes in non-syndromic deafness to gain further insight into the function of these genes in the inner ear. This knowledge is an essential prerequisite to the development and provision of molecular diagnostic services for families with non-syndromic deafness.

Data-intensive real-time applications, such as transportation management, military surveillance, and network monitoring, need to handle massive amounts of stream data in a timely fashion. It is challenging to support real-time stream data services (RTSDS) due to stringent timing constraints, potentially unbounded continuous stream data, bursty stream data arrivals, and workload variations due to data value changes. This project will develop cost-effective methods and a runtime system for RTSDS. The project will systematically investigate methods and tools to support real-time continuous queries for RTSDS even in the presence of dynamic workloads. Specifically, the project will study a) real-time continuous query modeling, b) new performance metric design c) adaptive query scheduling design, d) tardiness control and load shedding, for both single node and clustered RTSDS. The project will also have prototype implementation and testbed evaluations. The results and findings of this project will advance and seamlessly integrate real-time computing and stream data management.

Real-time stream data services (RTSDS) play an important role in many emerging application including intelligent transportation, green buildings, smart grid management, military surveillance, and network monitoring. Our everyday lives are highly dependent on these applications. RTSDS is a fundamental technology for developing critical data-intensive real-time applications with great socio-economic impacts. The project will develop the scientific foundations and associated engineering principles for building RTSDS.

The project will provide excellent opportunities for undergraduate and graduate students to acquire hands-on experience as well as theoretical backgrounds in RTSDS. The PIs will work closely with students to carry out the research in this project. Through this project, the PIs will train students to become strong in both analytical and implementation skills, and help them solve challenging problems in RTSDS.

DESCRIPTION (provided by applicant): The majority of inherited hearing loss (HL) is non-syndromic, and is often neuroepithelial in origin arising from defects in the function of the organ of Corti - the site of auditory transduction in the inner ear. Of these, up to 30% are autosomal dominant non-syndromic hearing loss (ADNSHL) (Liu and Xu, 1994). Although the past few years have witnessed a rapidly expanding list of HL genes using genomics based approaches, there is evidence that there are more HL genes and loci to be discovered. First, of the 65 mapped genes for ADSNHL, the gene has been identified for only 26. Second, many deaf pedigrees still fail to show linkage to any of these known loci, indicating that additional genes are involved. Third, despite recent progress in identifying genes underlying non syndromic HL (NSHL), there are still relatively few mouse models for progressive hearing loss. A large number of deaf mouse mutants also exist with no obvious human homologue, and human deafness genes localized or identified with no equivalent mouse model available, indicating that we still have much to learn about deafness from a genetic approach in humans. Therefore, there is an established need for mapping and identifying new genes for ADNSHL in order to provide accurate diagnosis of the genetic cause of deafness. Advances in DNA enrichment and Next Generation Sequencing (NGS) technology have made it possible to quickly and cost-effectively sequence all the genes of the genome, and then to rapidly identify variants responsible for Mendelian disorders. Our long-range goal is to better understand the genetic and molecular basis of hereditary deafness so that effective genetic counseling and successful treatment strategies can be developed. We have recently identified several new genes and mapped several novel loci for ADNSHL. We have successfully generated the animal models for some of these genes. In addition, we have collected 15 large multi-generational families with ADNSHL not linked to known ADNSHL loci, providing the basis for the present proposal for gene identification (Short-term objective) (Specific Aims 1 and 2) and for in vitro and in vivo function studies on both newly identified ADNSHL genes in the current proposal (Specific Aims 2 and 3). Our Specific Aims in this grant are: 1. Map new loci for ADNSHL. 2. Identify new genes for ADNSHL using traditional and innovative technologies. 3. Characterize the structural and functional consequences of the human S71L mutation of the SMAC gene in the smac knock-in mice. 4. Complete in vitro and in vivo functional studies of the P2XR2_V60L mutation. Completion of the proposed aims will not only increase our understanding of the biology of hearing and deafness, but will be highly translational by increasing availability of genetic testin, improving molecular diagnosis and, consequently, genetic counseling.

DESCRIPTION (provided by applicant): Abstract: Hearing loss (HL) is the most common sensory disorder affecting more than 28 million Americans. Despite the significant role of genetic factors in the etiology of HL, and astonishing success that has been achieved in the identification of approximately 70 genes for non-syndromic hearing loss (NSHL), much remains to be known about genes involved in the hearing process and the molecular mechanisms of disorders due to defects of these genes. Our long-range goal is to better understand the genetic and molecular basis of hereditary deafness so that effective genetic counseling and successful treatment strategies can be developed. Given the facts that many deafness pedigrees still fail to show linkage to any of the known loci and that mutation frequencies in all the known genes in persons with NSHL remains to be determined, it is therefore important for us to continue identifying new human deafness genes and to complete mutation screening of all known genes for NSHL. The recent technological advances in target- enrichment methods and next generation sequencing (NGS) offers a unique opportunity to break through the barriers of limitations imposed by gene arrays and now allows for the complete analysis of all known deafness-causing genes. The application of NGS will greatly accelerate the pace of disease gene discovery and is now making molecular epidemiological studies of genetic deafness possible for the first time. Interestingly, as shown in our preliminary studies, we have collected a unique group of large families segregating autosomal dominant or recessive deafness, confirmed further heterogeneity of NSHL in these families, successfully identified two new genes using NGS, mapped new loci, and established mutation screening protocol for known NSHL genes. These interesting preliminary results have thus led us to continue identification of novel genes for NSHL and to fully investigate the molecular mechanisms underlying NSHL. Overall, completion of the proposed aims will not only increase our understanding of the biology of hearing and deafness, but will be highly translational by improving the clinical diagnosis of NSHL and patient care. Our Specific Aims in this competitive renewal are: 1. Identify new genes for NSHL. 1a. Identify new genes for autosomal dominant NSHL (ADNSHL) using traditional and innovative technologies; 1b. Identify new genes for autosomal recessive NSHL (ARNSHL) using traditional and innovative technologies in the collected consanguineous families and in the selected probands from deaf x deaf mating families with extensive family histories of NSHL but known not to carry mutations in any known deafness gene. 2. Complete mutation screening of deafness-causing mutations in known NSHL genes. 2a. Determine the prevalence of deafness-causing mutations in known NSHL genes. 2b. Search for clinically relevant genotype- phenotype correlations in our large database.

Abstract: This continuing proposal will translate basic research utilizing high-throughput genomic approaches and functional genomics into routine diagnostic and therapeutic tools for non-syndromic hearing loss (NSHL), the most common type of hearing impairment in children and adults. We have developed a genomic variant detection platform MiamiOtogenomics - composed of MiamiCapitalArray/MiamiOtoGenes panels/exome (WES)/genome (WGS) and developed a genotype and phenotype database ? MiamiGeneHeal. As shown in the preliminary data, we have already collected approximately 3,000 DNA samples with phenotypic data from a large international cohort (Miami Otogenetic Repository) of families with NSHL. Moreover, we have excluded all known HL genes in over 200 families, successfully identified more than 18 potential new candidate genes, created animal models for human HL, and have generated human iPSCs from patients with genetic deafness. We will build on these accomplishments and preliminary data by proposing to complete the following specific aims: 1. Apply an innovative genomics-based MiamiOtogenomics pipeline for NSHL; 2. Identify factors influencing the decision to pursue and act on genomic testing; 3. Initiate preclinical therapeutic experiments as a proof-of-concept for potential treatments for HL. The foundation of the proposal will leverage the exceptional genomics capacity of collaborators at the University of Miami into a genomic-based, minority-focused, diagnostic and treatment pipeline for HL. The overarching purpose of this application is to transit discoveries made in laboratory to patient care. This study will translate genomic analysis into clinical hearing screening to elucidate the exact molecular etiology for HL, which will enable more accurate diagnoses, better quality of care, more effective genetic counseling, as well as improved cost-effectiveness in medical care. In addition, we expect to contribute significantly to genotype-phenotype studies and to establish a robust framework for assessing long-term clinical outcomes. Moreover, this study will contribute to our fundamental understanding of HL. Finally, our innovative preclinical therapeutic experiments in our knockin mouse and human iPSC models using CRISPR will potentially discover new treatments for HL. This study will inform two important clinical aspects of precision medicine in USA populations, especially in USA minorities: general acceptance in clinical practice and clinical utility. We will perform one of the largest and most integrated clinical/genomic/functional/novel therapeutic studies on NSHL to date. Our prior results, the interdisciplinary team's expertise and our established study infrastructure and population access support feasibility of our Aims.

Abstract: Hearing loss (HL) is the most common sensory disorder affecting more than 28 million Americans. Clinically significant HL is present in at least 1 per 500 infants at birth. Nearly 70% of HL expressed at birth has a genetic etiology. At least 80% o f n o n s y n d r o m i c deafness is autosomal recessive (ARNSHL). The identification of these genes has dramatically improved the clinical diagnosis and management of deaf and hard-of-hearing families. However, there is a pressing need to continue identifying new human HL genes and to determine causative variants in known NSHL genes for completing a genomic and phenotypic database. This is critical as we and others have provided direct evidence of further genetic heterogeneity of HL with a number of genes/mutations yet to be identified: causative variants are found in less than 4 0 % of cases and recent studies suggest that over a thousand genes are involved in deafness suggesting there are many deafness-causative genes still remaining to be identified in both human and mouse. Identifying all of the HL genes and causative mutation is imperative to our understanding of the biology of normal hearing and the disease etiology/processes, provides immediate benefit to the families involved for counseling and diagnosis, and, at the molecular level, allows for the development of novel gene-specific and even mutation-specific therapies to treat HL. The latter by using genome editing that can have much wider use than just in the families with mutations in that gene. Importantly, as shown in our preliminary studies, we have already collected DNA samples and phenotype data from a large international cohort (Miami Otogenetic Repository) of families with NSHL, well established the Miami Otogenetic Clinic and pipelines for the genetic and functional analysis of variants, excluded all known HL genes in a large cohort of multiplex families, successfully identified many potential new candidate genes, and generated several animal models with deafness phenotype for these human deafness genes using the CRISPR/Cas9 system and traditional targeted mutagenesis approach. In this proposal, we will build on our previous accomplishments and preliminary data by proposing to complete the following specific aims: 1) to determine causative variants in known NSHL genes for expanding a combined genomic and phenotypic deafness database; 2) to identify novel ARNSHL genes using targeted sequence capture/ whole exome (WES)/genome (WGS) analysis using a customized local pipeline platform; 3) to determine functional consequences of deafness genes with in vitro and in vivo models using innovative approaches. We will perform one of the largest and most integrated clinical/genomic/functional studies on ARNSHL to date. This innovative study will not only increase our understanding of the biology of hearing and deafness, but will be highly translational leading to improvements in the etiological diagnosis of NSHL and patient care as well as pave the wave for gene/variant specific treatments with animal models generated in the proposal.

ABSTRACT Mutations in the merlin (NF2) tumor suppressor gene cause the benign tumor disorder, Neurofibromatosis type 2 (NF2). This disorder predisposes individuals to develop bilateral vestibular schwannomas (VS) that cause progressive hearing loss and can cause life-threatening brainstem compression. Because surgical removal of a VS often causes deafness, facial paralysis, and imbalance, there is a need to develop drug therapies to slow or prevent VS growth and preserve nerve function. We have worked to establish an in vitro and in vivo drug screening platform to identify novel compounds as well as FDA-approved drugs that can be developed/repurposed for VS therapies. Toward this goal, we have created mouse and human merlin-deficient Schwann cell lines and optimized their use in 384-well high-throughput and high-content assays in order to screen large compound/drug libraries using robotic platforms. This approach identified several phosphoinositide- 3 kinase (PI3K) inhibitors that selectively reduce viability of mouse merlin-deficient compared to wild-type Schwann cells with nanomolar IG50. This initial finding was confirmed in human merlin-deficient Schwann cell lines for multiple PI3K, dual PI3K/mTOR and PI3K/HDAC inhibitors. Because PI3K plays a critical role in cell proliferation, survival, and invasion, there are currently 15 different PI3K inhibitors in clinical trials for various blood cancers and solid tumors. The first in class PI3K inhibitor (idelalisib) was approved in 2014 for leukemia. In this proposal, we advance our findings by conducting a systematic screen of PI3K pathway inhibitors in vitro and in vivo. The aims of this proposal are to: 1) profile a library of PI3K pathway inhibitors for efficacy in reducing viability of human merlin-deficient Schwann cell lines and primary human VS cells; 2) test efficacy of the advanced PI3K inhibitors to slow graft expansion and preserve hearing and balance in a novel rat xenograft model, and 3) conduct phenotypic, kinome, and transcriptome analysis to reveal the molecular signatures and adaptive changes of the cells and grafts to the inhibitors. We expect to obtain the necessary pre-clinical data to support the potential use of PI3K inhibitors in patients with NF2-associated VS.

Abstract: Health problems related to our subspecialty require multidisciplinary approaches for understanding and treating hearing and communication disorders. Advancing the scientific foundation of otolaryngology requires well-trained investigative teams with diverse skills and backgrounds in basic and clinical science. A new integrated training program for Otolaryngology-Head and Neck Surgery residents in basic, translational, and/or clinical sciences related to otolaryngology is proposed based in the Department of Otolaryngology-Head and Neck Surgery at the University Miami Miller School of Medicine (UMMSM). The training program's mission is to address these needs by supporting research opportunities in the disciplines related to otolaryngology, providing strong curricula in an integrative framework, with an interdisciplinary research culture emphasizing mentoring, academic advancement, grantsmanship, diversity outreach, responsible and ethical conduct of research, and productivity. The Specific Aims of the IRTO are to: 1) Provide research experience with mentors conducting basic, clinical, or translational research in diseases and conditions related to hearing and communication disorders; 2) Provide core and elective didactic opportunities to ensure trainees acquire in-depth knowledge of relevant basic, clinical, and translational research techniques; and 3) Provide curricula, seminars, workshops, and tutorials focusing on topics related to professional development skills. This program is supported by a highly collaborative group of mentors who direct active, NIDCD funded research programs; opportunities for interdisciplinary research collaboration and specialized training in a broad spectrum of research fields relevant to hearing and communication disorders; and an exceptional institutional infrastructure and research culture provided by multiple departments and colleges. A history and record of interdepartmental collaborations and collegiality among researchers in disciplines related to otolaryngology is a key factor for long-term success for our training program. Equally important is the long-standing culture of support, value, and enthusiasm for research training by the leadership in the Department and the institution. Moreover, we have successfully obtained a new ACGME approved 6-year research track position. We aim to provide the scientific training, as well as communication and administrative skills to enable our graduates to compete successfully for individual NIDCD-sponsored research awards and eventually become creative contributors to the future of otolaryngology.

Abstract: Neurosensory symptoms are a well-known but poorly characterized sequelae of SARS CoV-2 infection. In particular, there is evidence that smell and taste disturbances may sometimes be an early or singular marker of SARS-CoV-2 infection. ENTs and other clinicians involved in the diagnosis and treatment of sensory and communication disorders are by the nature of their work at high risk for exposure to respiratory pathogens. Furthermore, due to this high risk of disease exposure and infection, they are also particularly vulnerable to stress and emotional disturbances. Our team has been on the forefront to collect preliminary data on the presence of neurosensory disturbances in COVID-19 patients, the role of ENT in the treatment of these patients, and the protection of the physical and mental health of high risk health care workers. In this study we hope to further expand our work on these topics through the following specific aims: Aim 1 - Investigate the incidence and characteristics of sensory disorders in COVID-19 patients including anosmia, dysgeusia, dizziness, and hearing loss; Aim 2 - Assess the role of ENT in the treatment of COVID-19 and the effectiveness of implemented PPE measures; and Aim 3 - Evaluate mental health symptoms in high-risk healthcare workers during and after the COVID-19 pandemic. We have established an internationally interdisciplinary collaboration to pursue our work on these topics including experts from China, Germany, and France. In our hospitals, we have access to our local patients including over 462 patients with positive COVID-19, 1565 with confirmed negative, and 390 patients with pending testing results as well as a large cohort of COVID-19 patients from our international collaborators (see their LOS) for the current study. We have published or are working to publish a variety of preliminary data including the following studies: ?Approaching otolaryngology patients during the COVID-19 pandemic?, ?Olfactory or taste disorders as an early identifier of COVID-19 in adults and children: an international multicenter study?, ?Low Rate of Seroconversion in High Risk Medical Professionals Using a Novel Assay for COVID- 19 Exposure?, ?A Systematic Approach to Early Identification and Healthcare Worker Protection?, ?Otolaryngology providers must be alert for mild and asymptomatic COVID-19 patients? and ?COVID-19: Specific challenges faced by Individuals with Autism spectrum disorders and their family?. Innovations in our proposal include: 1. A multidisciplinary, international collaboration; 3. Our minority focused Miami sensory screening pipeline, and a database of genomic variation and phenotypes ? sensory disorders and COVID-19-positive people; and 3.Identifying a sensory impairment battery for the early detection of mild and asymptotic that can be incorporated into population-based screening studies.

Abstract entitled ?Genetic underpinnings of dysosmia in COVID-19?: There is evidence that smell and taste dysfunction is an early and often the only identifier in COVI-19 positive patients. We hypothesize that genetic variants in specific candidate genes associated with the development of unique sensory phenotypes of COVID-19 patients: In patients reported to the American Academy of Otolaryngology-Head and Neck Surgery using the COVID-19 Anosmia Reporting Tool for Clinicians, in the first 237 entries anosmia was noted in 73% of patients prior to COVID-19 diagnosis and was the initial symptom in 26.6%. The occurrence in asymptomatic individuals makes this finding a useful target for public health screening and would facilitate earlier diagnosis and treatment, as well as the identification of those individuals who are not ill but still capable of spreading the disease. The mechanism underlying sensory alteration is currently unknown. Infectious diseases may demonstrate a heritable component ? that is the propensity to contract and develop active infection and the severity of the immune response is influenced by host genetic factors to some extent and may reflect inter-individual variation in the host immune response. The genetic basis of this variability in response will provide important clues for therapeutics and lead to identification of groups at high risk of death. Public health measures to identify those at increased genetic risk of severe infection would be useful as a way of mitigating the economic effects of lockdown and social distancing policies. The genetic influence on COVID-19 symptoms may reflect genotype status of candidate genes such as ACE2 and TMPRSS2. Our team has been on the forefront to collect preliminary data on the presence of neurosensory disturbances in COVID-19 patients. We and others reported that anosmia is an important predictive symptom of COVID-19. Moreover, a UK twin study has shown that anosmia in COVID-19 patients has a high heritability (h2 = 0.48), suggesting that an individual's genotype plays a role in the presence or absence of this symptom. The goal of this study is to determine the susceptibility which may be influenced by host genotype to sensory disorders in COVID-19 patients to estimate the heritability of covid-19 sensory symptoms. In this study we will expand our work on following Specific Aim: Identify genetic variation associated with the development of dysosmia in COVID-19 patients. We hypothesize that there are genetic variants in the candidate genes that underlie smell and taste dysfunction in both symptomatic and otherwise asymptomatic COVID-19 patients. Preliminary data: We have established an international interdisciplinary collaboration team and have collected and published preliminary data on the role of ENT in COVID-19 and on the prevalence of sensory dysfunction in COVID-19 in our pilot studies. To determine if variants in specific candidate genes are associated with the development of anosmia in COVID-19 patients, we will collect DNA samples in our local COVID-19 patients and international patients from our collaborators for the study. In our hospitals, we have access to over 462 patients with confirmed positive COVID-19, 1565 with confirmed negative, and 390 patients with pending testing results. Over 10% of these patients report smell and taste dysfunction; our international collaborators have a large cohort of COVID-19 patients with smell and taste dysfunction available to us as well (see their LOS). We will perform WES on 120 samples from COVID-19 patients including 60 with smell and taste dysfunction and 60 without. We have established a COVID19 blood sample treatment process at our pathology lab for our ongoing study. Whole exome sequencing will be performed using established methods in the Center for Genome Technology in the Hussman Institute for Human Genomics (HIHG). Innovations in our proposal include: 1. Identify genetic markers for the early detection of mild and asymptotic COVID-19. 2. Our minority focused Miami sensory screening and genomic screening pipeline, and a database of genomic variation and phenotypes ? sensory disorders and COVID-19-positive people. 3. A multidisciplinary, international collaboration with samples for duplicating studies.