ProfessorJose Antonio Lopez-Escamez

Project Opportunities

1 Identification and validation of novel genes in Meniere disease by comprehensive analysis of coding and non-coding regions in the human genome.

Summary of opportunity:

Meniere's disease (MD) is an inner ear disorder, characterized by episodes of vertigo associated with sensorineural hearing loss, tinnitus with a multifactorial origin including genetic mutations. This research proposal plans to investigate the genetics underpinnings of Meniere disease in different ethnic groups and compare these data with genes involved in European descendent population. The PhD project goal is to identify new genes in familial MD cases and individuals with early onset by genome sequencing and segregation analysis or rare variants.

Opportunity synopsis: 
Background
Meniere's disease (MD) is an inflammatory inner ear disorder with a multifactorial origin, including a genetic contribution MD is associated with endolymph accumulation in the inner ear; however, this considered a late event in MD pathophysiology and is associated with hearing loss (4-6). Familial aggregation studies have shown that MD has a genetic contribution in European descendent population, however there are few studies supporting a familial clustering in East Asian or other ethnicities. Nine genes including FAM136A, DTNA, PRKCB, COCH, DPT, SEMA3D, TECTA, GUSB and SLC6A7 have been associated with autosomal dominant familial MD, whilst 4 genes that have been reported in recessive familial MD, including HMX2, LSAMP, OTOG and STRC. However, few genes have been reported in multiple families and they encode proteins that links the hair cell stereocilia in the sensory epithelia with the tectorial and otolithic membranes. In this sense, OTOG, MYO7A or TECTA are the main genes associated with familial MD in European population (9-11), but molecular epidemiology studies in other ethnicities are missing.

Aim of the study
The goal of this research proposal is to search for rare variation in coding and non-coding regions of the genome of patients with MD in different populations in Australia to define the genetic structure of the condition in each population.
Research Methods
Selection of individuals and Inclusion criteria
Patients will be recruited at the Kolling Institute, Sydney. We aim for a minimum of 20 probands and their parents (n (min) = 60 samples). We also plan to recruit families with 2-3 affected individuals for segregation analysis. We will include 10 familial cases and their parents/relatives in first degree (n= 40 samples). Population-specific reference datasets will be used as controls.

Inclusion criteria:

Individuals with any ancestry including European, South Asian and East Asian ancestry and criteria A or B.
A. Early-onset sporadic cases (individuals <30 years old) with definite MD diagnosed according to the Barany Society Classification Committee guidelines with a minimum follow-up of 2 years. Both parents must have no familial record of MD.
B. Familial cases (at least 2 individuals with definite MD) and 1 or more relatives without auditory or vestibular symptoms to perform a segregation analysis.

Genome Sequencing and whole enzymatic methylation sequencing
DNA will be isolated from blood samples and after quality controls, DNA libraries will be prepared by the Truseq DNA PCR free kit (350bp). Genome sequencing will be addressed using the Novaseq 6000 platform (Illumina) with a mean depth of 30X.

Bioinformatic analysis
Pre-process of sequencing data include alignment, sorting and joint calling of the samples to a reference genome. The fastq files will be analysed and quality valued through FastQC/MultiQC tools. Alignment will be done using BWA mem tool against the last version of the reference genome (GRCh38/hg38). Pre-process and variant calling will be performed using GATK suite, Samtools and Picard. Variant calling will be addressed with different tools: Manta, Delly2, CNVnator for CNV and structural variants, GATK HaplotypeCaller for SNV and indels. Annotation will be carried using Variant Effect Predictor from Ensembl consortium and snpEff tool. Allelic frequencies will be used for variant filtering to target candidate genes. We will phase genotypes of each trio through SHAPEIT2 for parent-of-origin different specific allele discovery. Non-coding region variation will be annotated for regulatory variants in open-chromatin regions for further analyses.

2. Role of mobile elements in the genetic architecture of Meniere disease and tinnitus disorder
Summary of opportunity:

Meniere's disease (MD) is an inner ear disorder, characterized by episodes of vertigo associated with sensorineural hearing loss, tinnitus with a multifactorial origin including genetic mutations. Tinnitus and tinnitus disorder are complex disorders with a genetic and environmental contribution This research proposal plans to investigate the role of structural variants and mobile elements insertions (i.e., L1 or Alu elements), on rare variation in existing datasets from patients with MD and tinnitus in European descendent population. The PhD project goal is to identify new genes and mechanisms driving de novo or ultrarare loss-of-function variants in MD or tinnitus patients.

Opportunity synopsis:

Background
Mobile elements (MEs) are DNA segments that can propagate through the genome using an RNA intermediate. In humans, three groups of MEs are still active: Long Interspersed Nuclear Elements 1(L1), Alu and SINE-VNTR-Alu (SVA). During retrotransposition, MEs duplicate their targeted region while inserting, creating repeated sequences in the genome. Retrotransposition can affect gene structure and/or expression, leading to rare genetic diseases. It can potentially involve exonic, intronic, splicing or UTR regions, and can cause deletions, leading to gene loss of function or changes in gene expression.
Meniere's disease (MD) is an inflammatory inner ear disorder with a multifactorial origin, including a genetic contribution
Familial aggregation studies have shown that MD has a genetic contribution in European population (10), with missense variants in coding regions. Few genes have been reported in multiple families and they encode proteins that links the hair cell stereocilia in the sensory epithelia with the tectorial and otolithic membranes. In this sense, OTOG, MYO7A or TECTA are the main genes associated with familial MD, but the genetic architecture of sporadic MD and the role of mobile elements in loss-of-function variants is largely unknown.
Severe tinnitus is considered a disorder in around 1% of the population, and it is associated with emotional distress, cognitive dysfunction, and/or autonomic arousal, leading to behavioural changes and functional disability. By selecting individuals with extreme phenotype, we have found a burden or rare missense variation in ANK2, TSC2, AKAP9 and CACNA1E and large structural variants in CACNA1E, NAV2 and TMEM132D (15-16); however, the role of mobile element insertion in splice-site variant and structural variants has not been investigated.
Aim of the study
The goal of this research proposal is to search for ME insertion leading to loss-of-function and structural variants in patients with MD or tinnitus to define their contribution to the genetic architecture of these conditions.Research Methods
Genomic datasets
Existing genomic datasets from > 600 individuals with MD and tinnitus will be used. The capture and sequencing steps were performed with several different kits and sequencing technologies. Informed written consent was obtained from individuals and the University of Sydney Human Research Ethical Committee has approved the data transfer for the analysis.
Bioinformatic analysis
The following tools will be used will be used to detect MEs: MELT tool (17), Tangram (18), Mobster (19) and SCRAMble (20). First, sequencing depth will be determined using SAMtools. Three main VCF files will be obtained, one each for Alu, L1 and SVA. A file report will be generated by Python2 script for each individual. MEs will be filtered to retain only those located in non-intronic regions in genes associated with hearing loss, tinnitus, Meniere disease and expressed in the brain or the inner ear. Individual ID and sequencing depth on the MEI site will be added. Remaining MEs were manually analyzed. Each ME will be considered as ME candidate if at least two of tools can detect it in several individuals segregate the phenotype.

3 Audiovestibular phenotyping of an OTOG mouse model of Meniere disease
Summary of opportunity:

Meniere's disease (MD) is an inner ear disorder, characterized by episodes of vertigo associated with sensorineural hearing loss, tinnitus with a multifactorial origin including genetic mutations. Familial MD is associated with several genes, OTOG being the most commonly found with a compound recessive inheritance pattern. This research proposal plans to design and phenotype a mutant otogelin mouse model to validate the causal role of OTOG in Meniere disease. The PhD project goal is to perform a mouse phenotyping by audiological and vestibular testing during mouse development. The PhD work will also include histological studies including scanning electron microscopy and confocal microscopy.

Opportunity synopsis:

Background
Meniere's disease (MD) is an inflammatory inner ear disorder with a multifactorial origin, including a genetic contribution.Our research team has recently elucidated the relevance of two mutations in the OTOG that encodes otogelin, an extracellular protein that couples the hair cell stereocilia to the tectorial membrane (TM) in the organ of Corti, (OTOG^V269I and OTOG^A2037V) in multiple families with MD. The hypothesis is that these mutations will affect the interaction of otogelin with other TM proteins leading to morphological changes in the TM structure and eventually detachment of the TM, with hearing loss or vestibular loss. To better understand the pathophysiology of these mutations in the inner ear we plan to generate transgenic mice harboring these mutations and perform hearing testing and histopathological studies.

Aim of the study

To generate a transgenic mouse and to perform mouse phenotyping by audiological and vestibular testing during mouse development. The PhD work will also include histological studies including scanning electron microscopy and confocal microscopy.

Research Methods

1. Testing baseline hearing function of the OTOG^V269I and OTOG^A2037V mouse models

Otog knockout mice exhibit hearing deficits and have an impaired outer hair cell (OHC) function (12). OHC are the sensory cells in the cochlea responsible for amplifying sound signals. We hypothesis that OTOG knock in mice (OTOG^V269I and OTOG^A2037V) would have loss of function and would exhibit similar hearing deficits. OTOG knock in mice will be tested for hearing function using Auditory Brainstem response (ABR) using ‘click’ and tone-pip testing (8kHz32kHz). When comparing patients with familiar MD, hearing loss is typically seen at lower frequencies, but this needs to be established  in the mouse model. Having that function of OHC impaired in OTOG deficient mice, we hypothesis that OTOG^V269I and OTOG^A2037V heterozygous and/or double mutant will also exhibit this phenotype. ABR testing of auditory neurotransmission will be complemented with the use of cubic Distortion product otoacoustic emission (DPOAE) (8-24 kHz). Baseline hearing test will be performed at (6-8) weeks of age and again in 12 – 14 weeks to ascertain if the OTOG transgenic mice exhibit hearing impairment and if it progresses with age which would be tested at later time point (12-14) week  which can support if the mutation leads to progressive hearing loss. This assessment will be run against wildtype littermates by breeding heterozygous transgenic mice (C57Bl/6J background).

2. Testing susceptibility of OTOG^V269I and OTOG^A2037V mouse to noise.

We plan to test if noise leads to temporary hearing loss or permanent hearing loss in OTOG^V269I and OTOG^A2037V heterozygous and double mutants as compared to wild type littermates. To test this hypothesis, we would measure baseline hearing for ABR spanning (8-32kHz) and DPOAE across (8-24 kHz) for 12-14 week old mice followed by white noise exposure for 1 hour at 102 dB  (8-32KHZ). To test temporary hearing loss ABR and DPOAE measurement will be undertaken before and then immediately after noise exposure while the mice remain anaesthetized (isoflurane).

3. Testing vestibular function of the OTOG^V269I and OTOG^A2037V mouse.

Otog knockout mice exhibited vestibular dysfunction (12), hence we hypothesis that vestibular function will likely be degraded in OTOG^V269I and OTOG^A2037V transgenic knock-in mice. The heterozygous, double mutant and wild type littermates will be tested for its vestibular function and motor coordination at two time points (6-8) weeks and (12-14) weeks by gait test in open field and rotarod.

4. To study the structural morphology of the Organ of Corti and vestibular end organs (saccule and utricule) in OTOG^V269I and OTOG^A2037V mice.

Mice lacking Otog gene show defects in the morphology of the organ of Corti (12). The OHC hair bundles (stereocilia) lack horizontal connectors and are not anchored to the tectorial membrane. We hypothesis that a similar phenotype will be observed in the knock in mice. To study structural defects in OTOG knock in mice, cochlea from Postnatal P0-P20 of heterozygous and double mutants will be extracted after euthanizing the mice. Structural morphology of Inner and outer hair cells will be analyzed using a combination of techniques such as scanning electron microscopy, Transmission electron microscopy, whole mount Immunostaining and cryosection staining of the cochlear and vestibular tissues.

4 AI-driven multi-omic endotyping of Ménière disease using genomic, epigenomic and immune signatures

Aim of the study
The goal of the project is to identify biologically meaningful molecular endotypes, of Ménière disease by integrating whole-genome sequencing, DNA methylation, immune-related molecular data and clinical phenotypes. The project will use interpretable machine learning to discover genetic and epigenetic signatures that explain disease heterogeneity and support future precision diagnosis, prognosis and treatment stratification.

Objective 1: Identify rare and common genomic variants associated with Ménière disease, with a focus on non-coding genes (long non-coding RNA) involved in inner-ear structure, hair-cell function, gap junction signalling, immune regulation and oxidative stress.
Objective 2: Define DNA methylation and immune-related molecular signatures that distinguish subgroups of Ménière disease patients.
Objective 3: Develop interpretable machine learning models that integrate genomic, methylation, immune and clinical data to classify Ménière disease endotypes and predict clinically relevant outcomes.

5 Design and development of the Meniere disease Atlas of genes and cells (MDAtlas)
Summary of opportunity
Meniere Disease is a complex inner ear disorder characterised by episodic vertigo, fluctuating sensorineural hearing loss, tinnitus, and aural fullness [1]. Although research has identified genetic, immune, transcriptomic, and epigenomic factors that may contribute to the disease, these data are often difficult to connect, compare, and interpret across different datasets [2], [3], [4], [5], [6]. This project will contribute to the development of the Meniere Disease Atlas of Genes and Cells by improving how multi-omics data are integrated, visualised, and analysed within a web-based research platform. 
 
The research will focus on three connected areas. First, it will examine the current MDAtlas website, database structure, and available genomic, transcriptomic, and epigenomic datasets to identify requirements for further development [2]. Second, it will support the integration of novel datasets using a scalable and well-documented data model, with attention to metadata, data quality, filtering, and querying. Third, it will develop interactive visualisation tools and machine learning methods to help researchers explore genetic variants, methylation patterns, gene expression, and immune cell-related signatures in Meniere Disease. 
 
Aim of the study 
The goal of this project is the development of the Meniere Disease Atlas of Genes and Cells by improving how multi-omics data are integrated, visualised, and analysed. The project will focus on adding novel datasets, building clearer visualisation tools, and developing machine learning methods that can support researchers in exploring genetic and immune patterns related to Meniere Disease. 

Current Research Projects
Synopsis
 Meniere disease (MD) is the third most common cause of episodic vertigo after benign paroxysmal positional vertigo and vestibular migraine in neurotology clinics. It is considered a rare and debilitating disease within the general population.

MD is a multifactorial inner ear disorder characterized by episodes of spontaneous vertigo, associated with tinnitus, and fluctuating sensorineural hearing loss. It is a heterogeneous condition with a variable phenotype associated with other comorbidities such as migraine, several autoimmune and autoinflammatory disorders, including allergies.
According to epidemiological and genetic data, the condition has a significant genetic contribution. Familial MD is observed in 6-10% of cases and rare mutations in OTOG, MYO7A and TECTA genes have been found in several unrelated families. Although most patients are considered sporadic, hidden recessive inheritance andde novomutations are emerging as causative in MD, and genetic diagnosis is being implemented.

We have developed a humanized mouse to assess the functional role of OTOG-mediante compound recessive inheritance in familial MD.
Moreover, by keeping a persistent systemic inflammatory status, proinflammatory cytokines released by monocytes (IL-1? and TNF-?), and high levels of CCL2, IL-4, and IgE, support a disorder in the immune respose with several immunophenotypes. Some patients with an autoinflammatory phenotype mediated by IL-1?, while others show a autoimmune profile driven byTNF-? and usually associated with autoimmune thyroid disease or systemic rheumatoid disorders. Moreover, a relevant group of individuals have apersistent type 2 immune response with IL-4, IL-13, and IL-6 with high levels of IgE.
Therefore, genetic factors and innate/adaptive immune responses play a central role in the pathophysiology of the condition.

Tinnitus is the perception of a phantom sound that affects between 10 and 15% of the general population. Furthermore, 1-3% of the population can be diagnosed with a debilitating tinnitus disorder, a condition associated with sleep disturbance and psychological distress, with emotional and behavioral consequences with a significant impact on health-related quality of life.

Despite this high burden for the health system, treatments for tinnitus are presently lacking since the heterogeneity of the tinnitus patients represents a major barrier to the development of effective tinnitus therapies.Tinnitus is not only a symptom associated with hearing loss, but it is also considered the result of exacerbated plasticity of the central auditory system in response to the crosstalk with auditory nerve fibers. The development of tinnitus is related to different nuclei of the auditory pathway, particularly the cochlear nuclei and the primary auditory cortex. The perception of tinnitus involves different brain areas and neural networks in which other structures such as the hippocampus or prefrontal cortex. Likewise, it can involve unknown auditory and non-auditory networks and molecular pathways. This complex combination has hampered advancements in the field and the identification of a genetic contribution to tinnitus has been at the forefront of tinnitus research in the last five years.