Biotecnología para la Medicina de Precisión: Identificación de Variaciones Genómicas para el Diagnóstico de Cáncer de Mama

[ES] El manejo eficiente de datos es un problema esencial para garantizar un diagnóstico correcto en el ámbito de una Medicina de Precisión (MP) efectiva y adecuada. Ciencia de Datos y Biotecnología deben unir sus fuerzas para que ese objetivo sea alcanzable, obteniendo cada vez más datos y convirtiéndolos en información valiosa. En esa dirección, el objetivo de este trabajo es identificar las variaciones genómicas para el diagnóstico de cáncer de mama mediante la metodología SILE: Search-IdentificationLoad-Exploitation (Búsqueda-Identificación-Carga-Explotación). Basándose en dicha metodología, el primer paso es seleccionar un conjunto adecuado de fuentes de datos genómicas. Se iniciará el trabajo explorando cuatro bases de datos (ClinVar, Ensembl, GWAS catalog y SNPedia) y se buscarán en ellas todas las variaciones genómicas relacionadas con el cáncer de mama. Una vez obtenidas las variaciones candidatas, se filtrarán según un conjunto de criterios de calidad que guíen la selección definitiva: entre ellos, que al menos haya un artículo en PubMed (base de datos bibliográfica de referencia en la investigación bioinformática y biogenómica) que sustente la relación entre la variación y el fenotipo de estudio, que esta tenga un determinado significado clínico y que los resultados del estudio cumplan ciertos criterios estadísticos mínimos (lo cual se hace mediante una revisión bibliográfica manual). El objetivo final es identificar aquellas variaciones que tienen mayor riesgo de causar el fenotipo analizado, permitiendo por lo tanto que un diagnóstico fiable sea posible. El proceso de identificación debe de asegurar que las variaciones seleccionadas cumplen los criterios requeridos, y garantizar que los datos proporcionados por las bases de datos son correctos y se corresponden con lo que se busca en este estudio. Finalmente, las variaciones serán cargadas en una plataforma software para el diagnóstico genómico como evidencia experimental de que la MP de la que hablamos es efectivamente viable y puede ser explotada por el personal sanitario en el ámbito del diagnóstico clínico.[EN] The efficient management of data is an essential problem to ensure a correct diagnosis in the field of an effective and appropriate Precision Medicine (PM). Data Science and Biotechnology must join forces to make this goal reachable, obtaining more and more data and converting them into valuable information. In that direction, the objective of this work is identify genomic variations for the diagnosis of breast cancer using the SILE methodology: Search-Identification-Load-Exploitation. Based on this methodology, the first step is to select an appropriate set of genomic data sources. The work will begin by exploring four databases (ClinVar, Ensembl, GWAS catalog and SNPedia) and all the genomic variations related to breast cancer will be explored. Once the candidate variations have been obtained, they will be filtered according to a set of quality criteria that guide the final selection. Such criteria will include that at least one of the articles in which the variations (variation) is studied appears in PubMed (reference bibliographic database in bioinformatics and biogenomics research); that its mutation has a certain clinical significance; and that the article fulfils certain statistic data (which is checked using a literature review by hand). The final objective is to identify those variations that have a higher risk of causing the analysed phenotype, thus, allowing a reliable diagnosis to be possible. The identification process must ensure that the selected variations satisfy the required criteria, and that the data provided by the databases are correct and correspond to what is searched in this study. Finally, the variations will be loaded into a software platform for genomic diagnosis as experimental evidence that the PM we are talking about is indeed viable and can be exploited by health personnel to offer personalized genomic medicine to patients.Escalera Balsera, A. (2018). Biotecnología para la Medicina de Precisión: Identificación de Variaciones Genómicas para el Diagnóstico de Cáncer de Mama. http://hdl.handle.net/10251/107582TFG

Single-cell immune profiling of Meniere Disease patients

This work was supported by B-CTS-68-UGR20 Grant by FEDER Funds, PI17/1644 and PI20-1126 grants from ISCIII by FEDER Funds from the EU, CLINMON-2 from the Meniere's Society UK, and Impact Data Science (IMP0001) . MF is funded by F18/00228 grant from ISCIII by FEDER Funds from the EU. AEB is funded by the EU's Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261. LF is funded by CD20/0153 grant from ISCIII by FEDER Funds from the EU. Funding for open access charge: Universidad de Granada/CBUA.Background: Meniere Disease (MD) is an inner ear syndrome, characterized by episodes of vertigo, tinnitus and fluctuating sensorineural hearing loss. The pathological mechanism leading to sporadic MD is still poorly understood, however an allergic inflammatory response seems to be involved in some patients with MD. Objective: Decipher an immune signature associated with the syndrome. Methods: We performed mass cytometry immune profiling on peripheral blood from MD patients and controls. We analyzed differences in state and differences in abundance of the different cellular subsets. IgE levels were quantified through ELISA on supernatant of cultured whole blood. Results: We have identified two clusters of individuals according to the single cell cytokine profile. These clusters presented differences in IgE levels, immune cell population abundance, including a reduction of CD56dim NKcells, and changes in cytokine expression with a different response to bacterial and fungal antigens. Conclusion: Our results support a systemic inflammatory response in some MD patients that show a type 2 response with allergic phenotype, which could benefit from personalized IL-4 blockers.FEDER Funds B-CTS-68-UGR20, B-CTS-68-UGR20Instituto de Salud Carlos III Spanish Government PI17/1644, PI20-1126, CD20/0153, 848261EUMeniere's Society UKImpact Data Science F18/00228Horizon 2020 IMP0001Universidad de Granada/CBU

Phenotypic spectrum of tinnitus patients bearing rare ANK2 gene variants

Purpose To describe the clinical, audiological, and psychometric features observed in patients with chronic tinnitus and rare variants in the ANK2 gene. Methods We report a case series of 12 patients with chronic tinnitus and heterozygous variants in the ANK2 gene. Tinnitus phenotyping included audiological (standard and high-frequency audiometry, Auditory Brainstem Responses (ABR) and Auditory Middle Latency Responses (AMLR)), psychoacoustic and psychometric assessment by a Visual Analog Scale (VAS) for tinnitus annoyance, the Tinnitus Handicap Inventory (THI), the test on Hypersensitivity to Sound (THS-GÜF), the Patient Health Questionnaire (PHQ-9), the Hospital Anxiety and Depression Scale (HADS) and the Montreal Cognitive Assessment (MoCA). Results All patients reported a persistent, unilateral noise-type tinnitus, mainly described as white noise or narrowband noise. Seven patients (58%) were considered to have extreme phenotype (THI score > 76), and all patients reported some degree of hyperacusis (THS-GÜF score > 18 in 75% of patients). Seven patients scored MoCA < 26, regardless of the age reported, suggesting a mild cognitive disorder. ABR showed no significant differences in latencies and amplitudes between ears with or without tinnitus. Similarly, the latencies of Pa, Pb waves, and NaPa complex in the AMLR did not differ based on the presence of tinnitus. However, there were statistical differences in the amplitudes of Pa waves in AMLR, with significantly greater amplitudes observed in ears with tinnitus. Conclusion Patients with ANK2 variants and severe tinnitus exhibit an endophenotype featuring hyperacusis, persistent noise-like tinnitus, high-frequency hearing loss, and decreased amplitudes in AMLR. However, anxiety, depression, and cognitive symptoms vary among individuals.Open Access funding enabled and organized by CAUL and its Member InstitutionsInstituto de Salud Carlos III (PI22/01838, ISCIII; CD20/00153)Consejería de Salud y Familias, Junta de Andalucía (Grant RH-0150-2020)University of Sydney (K7013_B3413 Grant)HORIZON EUROPE Reforming and enhancing the European Research and Innovation system (Grant Agreement Number 848261)Asociacion Sindrome de Meniere España (ASMES), Meniere’s Society, U

Using coding and non-coding rare variants to target candidate genes in patients with severe tinnitus

Tinnitus is the phantom percept of an internal non-verbal set of noises and tones. It is reported by 15% of the population and it is usually associated with hearing and/or brain disorders. The role of structural variants (SVs) in coding and non-coding regions has not been investigated in patients with severe tinnitus. In this study, we performed whole-genome sequencing in 97 unrelated Swedish individuals with chronic tinnitus (TIGER cohort). Rare single nucleotide variants (SNV), large structural variants (LSV), and copy number variations (CNV) were retrieved to perform a gene enrichment analysis in TIGER and in a subgroup of patients with severe tinnitus (SEVTIN, n = 34), according to the tinnitus handicap inventory (THI) scores. An independent exome sequencing dataset of 147 Swedish tinnitus patients was used as a replication cohort (JAGUAR cohort) and population-specific datasets from Sweden (SweGen) and Non-Finish Europeans (NFE) from gnomAD were used as control groups. SEVTIN patients showed a higher prevalence of hyperacusis, hearing loss, and anxiety when they were compared to individuals in the TIGER cohort. We found an enrichment of rare missense variants in 6 and 8 high-constraint genes in SEVTIN and TIGER cohorts, respectively. Of note, an enrichment of missense variants was found in the CACNA1E gene in both SEVTIN and TIGER. We replicated the burden of missense variants in 9 high-constrained genes in the JAGUAR cohort, including the gene NAV2, when data were compared with NFE. Moreover, LSVs in constrained regions overlapping CACNA1E, NAV2, and TMEM132D genes were observed in TIGER and SEVTIN.La Caixa Foundation GNP-182 100010434 LCF/PR/DE18/52010002 H2020-SC1-2019-848261European Commission 848261 722046Svenska Lakaresalskapet SLS-779681 Hoerselforskningsfonden 503 Tysta Skolan and Forschung Fuer LebenAndalusian Goverment (CECEU) DOC_01677Sara Borrell postdoctoral Fellowship (ISCIII) CD20/00153Andalusian Health Government (CSyF 2020 POSTDOC) RH-0150-2020Swedish Research CouncilEuropean Commission 2018-0597

Using coding and non-coding rare variants to target candidate genes in patients with severe tinnitus

Tinnitus is the phantom percept of an internal non-verbal set of noises and tones. It is reported by 15% of the population and it is usually associated with hearing and/or brain disorders. The role of structural variants (SVs) in coding and non-coding regions has not been investigated in patients with severe tinnitus. In this study, we performed whole-genome sequencing in 97 unrelated Swedish individuals with chronic tinnitus (TIGER cohort). Rare single nucleotide variants (SNV), large structural variants (LSV), and copy number variations (CNV) were retrieved to perform a gene enrichment analysis in TIGER and in a subgroup of patients with severe tinnitus (SEVTIN, n = 34), according to the tinnitus handicap inventory (THI) scores. An independent exome sequencing dataset of 147 Swedish tinnitus patients was used as a replication cohort (JAGUAR cohort) and population-specific datasets from Sweden (SweGen) and Non-Finish Europeans (NFE) from gnomAD were used as control groups. SEVTIN patients showed a higher prevalence of hyperacusis, hearing loss, and anxiety when they were compared to individuals in the TIGER cohort. We found an enrichment of rare missense variants in 6 and 8 high-constraint genes in SEVTIN and TIGER cohorts, respectively. Of note, an enrichment of missense variants was found in the CACNA1E gene in both SEVTIN and TIGER. We replicated the burden of missense variants in 9 high-constrained genes in the JAGUAR cohort, including the gene NAV2, when data were compared with NFE. Moreover, LSVs in constrained regions overlapping CACNA1E, NAV2, and TMEM132D genes were observed in TIGER and SEVTIN.publishedVersio

Unification of Treatments and Interventions for Tinnitus Patients (UNITI): a study protocol for a multi-center randomized clinical trial

The UNITI project has received funding from the European Union's Horizon 2020 Research and Innovation Program (grant agreement number 848261).Background: Tinnitus represents a relatively common condition in the global population accompanied by various comorbidities and severe burden in many cases. Nevertheless, there is currently no general treatment or cure, presumable due to the heterogeneity of tinnitus with its wide variety of etiologies and tinnitus phenotypes. Hence, most treatment studies merely demonstrated improvement in a subgroup of tinnitus patients. The majority of studies are characterized by small sample sizes, unstandardized treatments and assessments, or applications of interventions targeting only a single organ level. Combinatory treatment approaches, potentially targeting multiple systems as well as treatment personalization, might provide remedy and enhance treatment responses. The aim of the present study is to systematically examine established tinnitus therapies both alone and in combination in a large sample of tinnitus patients. Further, it wants to provide the basis for personalized treatment approaches by evaluating a specific decision support system developed as part of an EU-funded collaborative project (Unification of treatments and interventions for tinnitus patients; UNITI project). Methods/study design: This is a multi-center parallel-arm randomized clinical trial conducted at five different clinical sites over the EU. The effect of four different tinnitus therapy approaches (sound therapy, structured counseling, hearing aids, cognitive behavioral therapy) applied over a time period of 12 weeks as a single or rather a combinatory treatment in a total number of 500 chronic tinnitus patients will be investigated. Assessments and interventions are harmonized over the involved clinical sites. The primary outcome measure focuses on the domain tinnitus distress assessed via the Tinnitus Handicap Inventory. Discussion: Results and conclusions from the current study might not only provide an essential contribution to combinatory and personalized treatment approaches in tinnitus but could also provide more profound insights in the heterogeneity of tinnitus, representing an important step towards a cure for tinnitus.European Union's Horizon 2020 Research and Innovation Program 84826

The statistical analysis plan for the unification of treatments and interventions for tinnitus patients randomized clinical trial (UNITI-RCT)

Background Tinnitus is a leading cause of disease burden globally. Several therapeutic strategies are recommended in guidelines for the reduction of tinnitus distress; however, little is known about the potentially increased effectiveness of a combination of treatments and personalized treatments for each tinnitus patient. Methods Within the Unification of Treatments and Interventions for Tinnitus Patients project, a multicenter, randomized clinical trial is conducted with the aim to compare the effectiveness of single treatments and combined treatments on tinnitus distress (UNITI-RCT). Five different tinnitus centers across Europe aim to treat chronic tinnitus patients with either cognitive behavioral therapy, sound therapy, structured counseling, or hearing aids alone, or with a combination of two of these treatments, resulting in four treatment arms with single treatment and six treatment arms with combinational treatment. This statistical analysis plan describes the statistical methods to be deployed in the UNITI-RCT. Discussion The UNITI-RCT trial will provide important evidence about whether a combination of treatments is superior to a single treatment alone in the management of chronic tinnitus patients. This pre-specified statistical analysis plan details the methodology for the analysis of the UNITI trial results. Trial registration ClinicalTrials.gov NCT04663828. The trial is ongoing. Date of registration: December 11, 2020. All patients that finished their treatment before 19 December 2022 are included in the main RCT analysis

Contribución de variantes raras en regiones codificantes al acúfeno severo

El acúfeno es la percepción subjetiva de un ruido tonal en ausencia de una fuente acústica externa. La prevalencia en la población mundial oscila entre el 10% y el 30%; no obstante, el acúfeno se considera un trastorno cuando se asocia con angustia emocional, disfunción cognitiva y/o activación autonómica. La Enfermedad de Meniere (EM) es un trastorno del oído interno caracterizado por vértigo episódico, hipoacusia neurosensorial, acúfeno y/o sensación de plenitud ótica. Aunque los ataques de vértigo son el síntoma principal en los primeros años de la enfermedad, muchos pacientes con EM describen el acúfeno como el síntoma más molesto. La hipótesis de esta tesis fue que la gravedad del acúfeno está relacionada con una carga de variantes raras en las regiones codificantes de los individuos; por lo tanto, el objetivo fue identificar los principales genes y procesos biológicos asociados con el acúfeno severo. Se realizó la secuenciación del exoma de los 310 pacientes con EM. En base al cuestionario Tinnitus Handicap Inventory (THI), se crearon cuatro subgrupos. Los individuos con un THI por encima del tercer cuartil conformaron el subgrupo de acúfeno severo (I4) y por debajo del primer cuartil el subgrupo sin molestias causadas por el acúfeno (I1). Se llevaron a cabo análisis de carga génica (Gene Burden Analyses, GBA) para identificar genes enriquecidos en variantes con alta probabilidad de ser de pérdida de función (loss-of-function, LoF) o variantes con baja probabilidad de ser LoF y con un impacto moderado en la proteína, siendo la mayoría de ellas de cambio de sentido (missense). Además, se estudiaron las variantes en el número de copias (copy number variants, CNVs) y las variantes estructurales (structural variants, SVs). Los resultados muestran que: (1) Once genes fueron asociados con la EM familiar a través de una revisión sistemática. (2) Veintiocho genes se identificaron como candidatos para el acúfeno severo, participando en la axogénesis, la respuesta auditiva del tronco cerebral, la transmisión sináptica y la organización de los estereocilios de las células ciliadas. (3) Dieciséis genes se identificaron como candidatos para individuos sin acúfeno, formando parte de la protección contra el daño de las células ciliadas cocleares y de la estructura de la membrana tectorial. (4) El acúfeno y la hiperacusia estaban fuertemente asociados en pacientes con la EM, compartiendo causas genéticas. Estos resultados respaldan que la molestia causada por los acúfenos que algunos pacientes con EM experimentan puede explicarse parcialmente por diferencias en las variantes genéticas raras que presentan.Tinnitus is the subjective perception of tonal broad-band noise without an external acoustic source. The prevalence in the world population is between 10% and 30%; nevertheless, tinnitus is considered a disorder when it is associated with emotional distress, cognitive dysfunction and autonomic arousal. Meniere Disease (MD) is an inner ear disorder characterised by episodic vertigo associated with sensorineural hearing loss, tinnitus and aural fullness. Although vertigo attacks are considered the main symptom in the first years of the disease, persistent tinnitus is described as the most troublesome symptom by many MD patients. This thesis hypothesis was that tinnitus severity is related to an overload of rare variants in the coding regions of the individuals; therefore, the aim was to identify the central genes and biological processes associated with severe tinnitus. Exome sequencing was performed for the 310 MD patients included in this study. According to the Tinnitus Handicap Inventory (THI) questionnaire, they were clustered into four subgroups. Individuals with a THI above the third quartile composed the severe tinnitus subgroup (I4) and those below the first quartile were the subgroup without disturbance caused by tinnitus (I1). Gene Burden Analyses (GBA) were carried out to target genes enriched in variants with high confidence of being loss-of-function (LoF) or variants with low confidence of being LoF and with a moderate impact in the protein, being missense most of them. Moreover, copy number variants (CNV) and structural variants (SV) were studied. The results show: (1) Eleven genes have been associated with familial MD through a systematic review. (2) Twenty-eight genes were identified as candidates for severe tinnitus, clustering in four groups according to their expression and involved axogenesis, auditory brainstem response, synaptic transmission and hair cell stereocilia organisation as main biological functions. (3) Sixteen genes were identified as candidates for individuals without tinnitus, clustering also in four subgroups by the expression and being part of the protection of cochlear hair cells against damage and the structure of the tectorial membrane. (4) Tinnitus and hyperacusis were strongly associated in MD patients, which shared genetic causes. These results support that MD patients who experience bothersome tinnitus can be partially explained by differences in the rare genetic variants that they carry.Tesis Univ. Granada.Unification of Treatments and Interventions for Tinnitus Patients (UNITI) Project, from the European Union's Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261.Proyecto IMPaCT-Data (Exp. IMP/00019), financiado por el Instituto de Salud Carlos III, co-financiado por el Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”).EMBO Scientific Exchange Grant Number 9783.Acciones de movilidad del CIBERER (Centro de Investigación Biomédica en Red de Enfermedades Raras)

Systematic Review of Sequencing Studies and Gene Expression Profiling in Familial Meniere Disease

Familial Meniere Disease (FMD) is a rare inner ear disorder characterized by episodic vertigo associated with sensorineural hearing loss, tinnitus and/or aural fullness. We conducted a systematic review to find sequencing studies segregating rare variants in FMD to obtain evidence to support candidate genes for MD. After evaluating the quality of the retrieved records, eight studies were selected to carry out a quantitative synthesis. These articles described 20 single nucleotide variants (SNVs) in 11 genes (FAM136A, DTNA, PRKCB, COCH, DPT, SEMA3D, STRC, HMX2, TMEM55B, OTOG and LSAMP), most of them in singular families—the exception being the OTOG gene. Furthermore, we analyzed the pathogenicity of each SNV and compared its allelic frequency with reference datasets to evaluate its role in the pathogenesis of FMD. By retrieving gene expression data in these genes from di erent databases, we could classify them according to their gene expression in neural or inner ear tissues. Finally, we evaluated the pattern of inheritance to conclude which genes show an autosomal dominant (AD) or autosomal recessive (AR) inheritance in FMD.Consejería de Salud y Familias PE-0356-201

DNA Methylation Signature in Mononuclear Cells and Proinflammatory Cytokines May Define Molecular Subtypes in Sporadic Meniere Disease

Meniere Disease (MD) is a multifactorial disorder of the inner ear characterized by vertigo attacks associated with sensorineural hearing loss and tinnitus with a significant heritability. Although MD has been associated with several genes, no epigenetic studies have been performed on MD. Here we performed whole-genome bisulfite sequencing in 14 MD patients and six healthy controls, with the aim of identifying an MD methylation signature and potential disease mechanisms. We observed a high number of differentially methylated CpGs (DMC) when comparing MD patients to controls (n= 9545), several of them in hearing loss genes, such as PCDH15, ADGRV1 and CDH23. Bioinformatic analyses of DMCs and cis-regulatory regions predicted phenotypes related to abnormal excitatory postsynaptic currents, abnormal NMDA-mediated receptor currents and abnormal glutamate-mediated receptor currents when comparing MD to controls. Moreover, we identified various DMCs in genes previously associated with cochleovestibular phenotypes in mice. We have also found 12 undermethylated regions (UMR) that were exclusive to MD, including two UMR in an inter CpG island in the PHB gene. We suggest that the DNA methylation signature allows distinguishing between MD patients and controls. The enrichment analysis confirms previous findings of a chronic inflammatory process underlying MD.PI17/1644 grant from ISCIII by FEDER Funds from the EUF18/00228 grant from ISCIII by FEDER Funds from the EUAEB is funded by the EU’s Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261