Academic literature on the topic 'DFNB16'

Autosomal dominant non-syndromic hearing loss (HL) typically occurs when only one dominant allele within the disease gene is sufficient to express the phenotype. Therefore, most patients diagnosed with autosomal dominant non-syndromic HL have a hearing-impaired parent, although de novo mutations should be considered in all cases of negative family history. To date, more than 50 genes and 80 loci have been identified for autosomal dominant non-syndromic HL. DFNA22 (MYO6 gene), DFNA8/12 (TECTA gene), DFNA20/26 (ACTG1 gene), DFNA6/14/38 (WFS1 gene), DFNA15 (POU4F3 gene), DFNA2A (KCNQ4 gene), and DFNA10 (EYA4 gene) are some of the most common forms of autosomal dominant non-syndromic HL. The characteristics of autosomal dominant non-syndromic HL are heterogenous. However, in most cases, HL tends to be bilateral, post-lingual in onset (childhood to early adulthood), high-frequency (sloping audiometric configuration), progressive, and variable in severity (mild to profound degree). DFNA1 (DIAPH1 gene) and DFNA6/14/38 (WFS1 gene) are the most common forms of autosomal dominant non-syndromic HL affecting low frequencies, while DFNA16 (unknown gene) is characterized by fluctuating HL. A long audiological follow-up is of paramount importance to identify hearing threshold deteriorations early and ensure prompt treatment with hearing aids or cochlear implants.

Non-syndromic hearing impairment (NSHI) is a very heterogeneous genetic condition, involving over 130 genes. Mutations in GJB2, encoding connexin-26, are a major cause of NSHI (the DFNB1 type), but few other genes have significant epidemiological contributions. Mutations in the STRC gene result in the DFNB16 type of autosomal recessive NSHI, a common cause of moderate hearing loss. STRC is located in a tandem duplicated region that includes the STRCP1 pseudogene, and so it is prone to rearrangements causing structural variations. Firstly, we screened a cohort of 122 Spanish familial cases of non-DFNB1 NSHI with at least two affected siblings and unaffected parents, and with different degrees of hearing loss (mild to profound). Secondly, we screened a cohort of 64 Spanish sporadic non-DFNB1 cases, and a cohort of 35 Argentinean non-DFNB1 cases, all of them with moderate hearing loss. Amplification of marker D15S784, massively parallel DNA sequencing, multiplex ligation-dependent probe amplification and long-range gene-specific PCR followed by Sanger sequencing were used to search and confirm single-nucleotide variants (SNVs) and deletions involving STRC. Causative variants were found in 13 Spanish familial cases (10.7%), 5 Spanish simplex cases (7.8%) and 2 Argentinean cases (5.7%). In all, 34 deleted alleles and 6 SNVs, 5 of which are novel. All affected subjects had moderate hearing impairment. Our results further support this strong genotype–phenotype correlation and highlight the significant contribution of STRC mutations to moderate NSHI in the Spanish population.

Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many mutant genes causing rare forms of deafness remain to be discovered. A consanguineous Pakistani family segregating nonsyndromic deafness in two sibships were studied using microarrays and exome sequencing. A 1.2 Mb locus (DFNB128) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. There are two previously reported Map3k1-kinase-deficient mouse models that are associated with recessively inherited syndromic deafness. MAP3K1 phosphorylates serine and threonine and functions in a signaling pathway where pathogenic variants of HGF, MET, and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97, and DFNB26, respectively. Our single-cell transcriptome data of mouse cochlea mRNA show expression of Map3k1 and its signaling partners in several inner ear cell types suggesting a requirement of wild-type MAP3K1 for normal hearing. In contrast to dominant variants of MAP3K1 associated with Disorders of Sex Development 46,XY sex-reversal, our computational modeling of the recessive substitution p.(Arg1487His) predicts a subtle structural alteration in MAP3K1, consistent with the limited phenotype of nonsyndromic deafness.

The function of outer hair cells (OHCs), the mechanical actuators of the cochlea, involves the anchoring of their tallest stereocilia in the tectorial membrane (TM), an acellular structure overlying the sensory epithelium. Otogelin and otogelin-like are TM proteins related to secreted epithelial mucins. Defects in either cause the DFNB18B and DFNB84B genetic forms of deafness, respectively, both characterized by congenital mild-to-moderate hearing impairment. We show here that mutant mice lacking otogelin or otogelin-like have a marked OHC dysfunction, with almost no acoustic distortion products despite the persistence of some mechanoelectrical transduction. In both mutants, these cells lack the horizontal top connectors, which are fibrous links joining adjacent stereocilia, and the TM-attachment crowns coupling the tallest stereocilia to the TM. These defects are consistent with the previously unrecognized presence of otogelin and otogelin-like in the OHC hair bundle. The defective hair bundle cohesiveness and the absence of stereociliary imprints in the TM observed in these mice have also been observed in mutant mice lacking stereocilin, a model of the DFNB16 genetic form of deafness, also characterized by congenital mild-to-moderate hearing impairment. We show that the localizations of stereocilin, otogelin, and otogelin-like in the hair bundle are interdependent, indicating that these proteins interact to form the horizontal top connectors and the TM-attachment crowns. We therefore suggest that these 2 OHC-specific structures have shared mechanical properties mediating reaction forces to sound-induced shearing motion and contributing to the coordinated displacement of stereocilia.

Mouse models for human deafness have not only proven instrumental in the identification of genes for hereditary hearing loss, but are excellent model systems in which to examine gene function as well as the resulting pathophysiology. One mouse model for human nonsyndromic deafness is the deafness (dn) mouse, a spontaneous mutation in the curly-tail (ct) stock. The dn gene is on mouse Chromosome 19 and it was recently shown to be a novel gene called Tmc1. A mutation in Tmc1 is also found in Beethoven (Bth), which is another deaf mouse mutant. In humans, one autosomal dominant form of nonsyndromic hearing loss (DFNA36) and two autosomal recessive forms (DFNB7 and DFNB11) are associated with mutations in TMC1, the human homologue of Tmc1. The transmembrane protein encoded by this gene is required for normal cochlear hair cell function and the mouse models will facilitate the elucidation of the molecular pathway that is disrupted when mutations are present.

Objectives: This study capitalizes on the unique molecular and developmental similarities between the auditory organs of Drosophila and mammals, to investigate genes implicated in human syndromic and nonsyndromic hearing loss in a genetically tractable experimental animal model, the fruit fly Drosophila. Methods: The Drosophila counterparts of 3 human deafness genes (DIAPH1/DFNA1, ESPN/DFNB36, and TMHS/DFNB67) were identified by sequence similarity. An electrophysiological assay was used to record sound-evoked potentials in response to an acoustic stimulus, the Drosophila courtship song. Results: Flies with mutations affecting the diaphanous, forked, and CG12026/TMHS genes displayed significant reductions in the amplitude of sound-evoked potentials compared to wild-type flies (p < 0.05 to p < 0.005). The mean responses were reduced from approximately 500 to 600 μV in wild-type flies to approximately 100 to 300 μV in most mutant flies. Conclusions: The identification of significant auditory dysfunction in Drosophila orthologs of human deafness genes will facilitate exploration of the molecular biochemistry of auditory mechanosensation. This may eventually allow for novel diagnostic and therapeutic approaches to human hereditary hearing loss.

La déficience auditive constitue un handicap fonctionnel majeur, affectant plus d'un demi-milliard de personnes dans le monde. Malgré sa prévalence élevée, aucun traitement curatif n'existe actuellement. Mon projet de thèse est translationnel et vise à établir la preuve de concept selon laquelle la thérapie génique virale peut restaurer l'audition dans le modèle préclinique de surdité DNFB16. La surdité DFNB16 est la deuxième cause de déficience auditive congénitale d'origine génétique. Elle est causée par des mutations du gène codant pour la stéréociline (STRC) et se caractérise par une surdité légère à modérée. La protéine STRC est principalement exprimée dans les cellules ciliées externes (CCE) de l'oreille interne, l'un des deux types de cellules sensorielles de la cochlée, responsables de l'amplification et la discrimination fréquentielle du signal sonore. La protéine STRC est cruciale pour le maintien de la morphologie des stéréocils des CCE. Les mutations du gène STRC sont responsables d'un dysfonctionnement des CCE conduisant à l'abolition de l'amplification cochléaire et donc à une augmentation des seuils auditifs. A ce jour, il n'existe aucun traitement curatif pour la surdité DFNB16.L'objectif principal de mon projet était de développer une thérapie génique basée sur les virus adéno-associés (AAV) pour remplacer le gène mutant par une copie fonctionnelle dans un modèle murin DFNB16. Compte tenu de la grande taille de la séquence codante du gène Strc, dépassant la capacité d'empaquetage de l'AAV, j'ai utilisé une stratégie hybride de double vecteur pour charger l'ADNc de la Strc dans les capsides de l'AAV. Sachant que les CCE sont intrinsèquement difficiles à transduire par les vecteurs AAV, j'ai tout d'abord effectué une analyse comparative du tropisme cellulaire de différents sérotypes d'AAV après administration dans l'oreille interne afin d'identifier le la capside la plus efficace pour cibler les CCE. Ensuite, j'ai utilisé le sérotype AAV le plus performant pour construire le vecteur thérapeutique qui a été administré dans les cochlées des souris DFNB16.Les résultats montrent que la thérapie génique a rétabli une expression robuste de la protéine STRC et ciblée dans les touffes ciliaires des CCE chez les souris traitées. Cette expression a entraîné la restauration de la morphostructure des touffes ciliaires et de l'amplification cochléaire, permettant une récupération stable et durable des seuils auditifs, similaires à ceux de souris sauvages. Par ailleurs, les mesures psychométriques de la perception des fréquences à l'aide d'une tâche de Go/NoGO ont montré que la discrimination fréquentielle du signal sonore chez les souris DFNB16 traitées étaient comparables à celles des souris sauvages. Ces résultats soulignent l'efficacité de la thérapie génique sur la restauration de la perception sonore dans un modèle préclinique de surdité DFNB16. Cette découverte jette les bases d'une thérapie génique translationnelle efficace pour les patients atteints de DFNB16<br>Hearing impairment stands as a significant contributor to disability, affecting over half a billion individuals throughout their lifespans. Despite its pervasive prevalence, no curative treatment currently exists. My Ph.D. project is translational, aiming to establish the proof of concept that viral gene therapy can restore hearing in a preclinical model for DFNB16 deafness. DFNB16, considered the second most common cause of hearing impairment, is caused by mutations in the stereocilin (STRC) gene and is characterized by mild-to-moderate deafness. The stereocilin (STRC) protein is predominantly expressed in outer hair cells (OHCs), one of the two types of cochlear sensory hair cells, responsible for sound amplification. STRC protein is crucial for the cohesion and maintenance of OHC bundles. Mutations in STRC result in defective OHCs, leading to abolished cochlear amplification and subsequent reduction in hearing sensitivity. As of now, there exists no cure for DFNB16.My main objective was to develop an adeno-associated virus (AAV)-based gene therapy to replace the mutant gene with its correct copy in a DFNB16 mouse model. Given the large size of the Strc coding sequence, exceeding AAV packaging capacity, I employed a hybrid dual-vector strategy to load Strc cDNA into AAV capsids. Since OHCs are inherently difficult to transduce with AAV vectors, we firstly conducted a comparative analysis of AAV cellular tropism within the inner ear to identify the most efficient AAV serotype for targeting OHCs. Secondly, I used the best performing AAV serotype to construct the therapeutic vector, which was administered into the cochleas of DFNB16 mice.Following the gene therapy, we found a robust restoration of STRC protein expression and its appropriate targeting at the tips of OHC stereocilia in treated mice. This process results in the restoration of the normal morphostructure of OHC bundles and cochlear amplification, ensuring stable and long-lasting restoration of hearing in the treated mice, similar to those of the wild-type mice. Notably, psychometric measurements of frequency perception using a Go/NoGo task demonstrated that frequency discrimination exhibited by the treated Strc-/- mice was comparable to those of wild-type mice, underscoring the efficacy of gene therapy in recovering essential features of natural sound perception associated with DFNB16. This finding lays the foundation for effective translational gene therapy for DFNB16 patients and facilitates the development of preclinical gene therapy studies for mouse models of human deafness

Par une analyse de liaison génétique dans des grandes familles iraniennes atteintes de surdité prélinguale de transmission autosomique récessive, j’ai pu définir deux nouveaux loci de surdité isolée: DFNB40, sur le chromosome 22 (22q11. 21-12. 1) et DFNB59, sur le chromosome 2 (2q31. 1-31. 3). J’ai ensuite identifié le gène DFNB59. Il code pour une nouvelle protéine de 352 acides aminés, que j’ai nommée pejvakine. Afin de valider l’implication de ce gène dans la surdité DFNB59, j’ai produit un modèle de souris knock-in pour l’une des deux mutations humaines identifiées. Ces souris ont une neuropathie auditive traduisant un dysfonctionnement des neurones de la voie auditive afférente. DFNB59 est le premier gène responsable de neuropathie auditive rétrocochléaire identifié. La production des souris knock-out pour Dfnb59 est en cours. Elle devrait nous aider à mieux comprendre le rôle de la pejvakine ainsi que le processus pathogénique associé à la forme génétique de surdité correspondante<br>By a genetic linkage analysis in the large consanguineous Iranian families with autosomal recessive, non-syndromic, prelingual sensorineural hearing impairment, I defined two novel non-syndromic deafness loci: DFNB40, on chromosome 22 (22q11. 21-12. 1) and DFNB59, on chromosome 2 (2q31. 1-31. 3). I then identified the gene DFNB59. It codes for a new protein of 352 amino acids, which was named pejvakin. In order to validate the implication of this gene in DFNB59 deafness, I generated a knock-in mouse model for one of the two mutations identified in human subjects. These mice present with auditory neuropathy, indicative of neuronal dysfunction along the afferent auditory pathway. DFNB59 is the first human gene implicated in nonsyndromic deafness due to a neuronal defect. Generation of Dfnb59 knockout mice is in progress. It should help us understand better the role of pejvakin, as well as the pathogenic process associated to this genetic form of deafness

Hearing loss is the most common form of sensory impairment, with approximately one infant/1000 born with profound congenital deafness. Nonsyndromic hearing loss and deafness (DFNB1) is an inherited condition with a mild to severe deafness phenotype caused by mutations in GJB2 (which encodes the protein connexin26) and GJB6 (which encodes connexin30). Gap junction channels formed primarily by these two connexin protein subunits couple non-sensory cells (supporting and epithelial cells) of the mammalian cochlea, forming vast functional syncytia. Previous work has shown that electrical and metabolic coupling mediated by gap junction channels is fundamental for the development and maintenance of hearing. However, precise estimates of the degree of coupling and its alterations under DFNB1 conditions are lacking, notwithstanding the vast body of studies conducted in recombinant expression systems. In this thesis work, we combined large scale optical recordings, single cell electrophysiology and computer simulations to elucidate the mechanisms that underlie intercellular communication in cochlear supporting cells from juvenile mice (first postnatal week). First, we developed a novel technique based on voltage imaging to map the extent and the degree of electrical coupling in non-sensory cell networks of the developing mouse cochlea. We also quantified precisely the reduction of electrical coupling in cochlear organotypic cultures from transgenic mice with hearing defects due to absence or mutation of connexin30 compared to wild type animals. By comparing our experimental results with numerical simulations, we estimated that cochlear supporting cells in the mouse are already well coupled in the first postnatal week by as many as ∼ 1500 channels per cell pair. In age-matched cultures from connexin30(T5M/T5M) and connexin30(−/−) mice, junctional conductance was reduced respectively by 14% and 91%, and these data account for the increased hearing thresholds exhibited by these animals in the adult stage. Besides electrical coupling, inner ear gap junction channels and hemichannels have been shown to participate in ATP- and IP3 - dependent intercellular Ca2+ signaling, and alterations of these signaling mechanisms in the postnatal cochlea have been linked to impairment of hearing acquisition. We thus performed Ca2+ imaging experiments aimed at elucidating the mechanisms underlying the generation and intercellular propagation of ATP-mediated Ca2+ signals in cochlear non-sensory cells. We determined that ATP- and IP3 - dependent Ca2+ oscillations in cochlear non-sensory cells can occur at constant intracellular IP3 concentration. We then combined the information gathered from the two types of experimental approaches in a mathematical model that (i) correctly reproduces the range and propagation speed of intercellular Ca2+ waves and (ii) indicates that inception and culmination of selfsustained Ca2+ oscillations are marked by supercritical Hopf bifurcations at ATP concentrations of ∼ 100 nM and ∼1 μM, respectively. Finally, we investigated the relationship between spontaneous Ca2+ transients in non-sensory cells and spontaneous Ca2+ action potentials in sensory inner hair cells. Our preliminary results suggest that Ca2+ signaling in non-sensory cells may have a modulating effect on spontaneous electrical activity, which is intrinsically generated in inner hair cells.<br>La perdita dell’udito è la forma più comune di disabilità sensoriale: circa un bambino su 1000, infatti, è affetto alla nascita da sordità congenita profonda. La sordità non sindromica (DFNB1) è una malattia ereditaria con un fenotipo di sordità che va da lieve a grave, causata da mutazioni nei geni GJB2 (che codifica la proteina connessina26) e GJB6 (che codifica la connessina30). Canali giunzionali formati prevalentemente da queste due proteine accoppiano le cellule non sensoriali (cellule di sostegno e cellule epiteliali) della coclea dei mammiferi, le quali formano vasti sincizi funzionali. Studi precedenti hanno dimostrato che l’accoppiamento elettrico e metabolico mediato da canali giunzionali è fondamentale per lo sviluppo e il mantenimento dell’udito. Tuttavia, nonostante il gran numero di studi condotti in sistemi di espressione, mancano stime precise del grado di accoppiamento e delle sue alterazioni in condizioni DFNB1. In questo lavoro di tesi, sono state combinate registrazioni ottiche su larga scala , registrazioni elettrofisiologiche su singola cellula e simulazioni al computer per chiarire i meccanismi che sono alla base della comunicazione intercellulare nelle cellule cocleari non sensoriali in topi giovani (prima settimana post-natale) . In primo luogo, abbiamo sviluppato una nuova tecnica basata sull’imaging del potenziale di membrana cellulare per mappare l’estensione e il grado di accoppiamento elettrico nelle reti cellulari non sensoriali della coclea in via di sviluppo . Abbiamo anche quantificato con precisione la riduzione dell’accoppiamento elettrico in colture organotipiche cocleari da topi transgenici con difetti uditivi causati dall’assenza o da mutazioni della connessina30 rispetto ad animali wild type. Confrontando i nostri risultati sperimentali con simulazioni numeriche , abbiamo stimato che le cellule non sensoriali della coclea nel topo sono già ben accoppiate nella prima settimana post-natale da ben ∼ 1500 canali per ogni coppia di cellule. Nelle colture di pari età provenienti da topi connexin30(T5M/T5M) e connexin30(−/−), la conduttanza giunzionale è ridotta rispettivamente del 14% e del 91% , e questi dati sono in accordo con l’aumento delle soglie uditive mostrato da questi animali nella fase adulta . Oltre a fornire l’accoppiamento elettrico , é stato dimostrato che i canali giunzionali dell’orecchio interno partecipano alla segnalazione Ca2+ intracellulare dipendente dall’ATP e dall’IP3, e l’alterazione di questi meccanismi di segnalazione nella coclea postnatale é stata collegata alla compromissione dell’acquisizione dell’udito. Abbiamo quindi eseguito esperimenti di imaging dello ione Ca2+ volti a chiarire i meccanismi alla base della generazione e propagazione intercellulare dei segnali Ca2+ mediati da ATP nelle cellule non sensoriali della coclea. Abbiamo determinato che le oscillazioni Ca2+ dipendenti dall’ATP e dall’IP3 nelle cellule non sensoriali cocleari possono verificarsi in presenza di una concentrazione intracellulare di IP3 costante. Abbiamo poi combinato le informazioni raccolte attraverso i due diversi approcci sperimentali in un modello matematico che (i) riproduce correttamente la velocità e il range di propagazione delle onde Ca2+ intercellulari e (ii) indica che l’inizio e il culmine delle oscillazioni Ca2+ sono contrassegnati da biforcazioni di Hopf supercritiche a concentrazioni di ATP di ∼ 100 nM e ∼ 1 μM, rispettivamente . Infine , abbiamo studiato la relazione tra transienti Ca2+ spontanei delle cellule non sensoriali e i potenziali d’azione spontanei delle cellule ciliate interne. I nostri risultati preliminari suggeriscono che i transienti Ca2+ delle cellule non sensoriali potrebbero avere un effetto modulatorio sull’attività elettrica spontanea , che è intrinsecamente generata nelle cellule ciliate interne.

L’acquedotto vestibolare è un canale osseo che si estende dalla parete mediale del vestibolo alla faccia cerebellare della piramide petrosa, contiene vasi e una componente del labirinto membranoso dell’orecchio interno, il dotto endolinfatico che si apre nel sacco endolinfatico. L’acquedotto vestibolare allargato (EVA) è la più comune malformazione congenita associata ad ipoacusia. Le mutazioni del gene SLC26A4 della pendrina sono considerate una delle più comuni cause di ipoacusia congenita ed EVA e sono coinvolte in circa il 10% di tutte le ipoacusie ereditate; due manifestazioni cliniche si associano alle mutazioni del gene della pendrina una forma sindromica (s. di Pendred: ipoacusia, EVA ed ipotiroidismo e due mutazioni patologiche del gene SLC26A4) e una forma non sindromica (DFNB4: ipoacusia ed EVA con una mutazione patologica del gene SLC26A4). Alcuni pazienti con ipoacusia ed EVA non segregano alcuna mutazione per il gene della pendrina (in questo caso si parla di ipoacusia associata ad EVA o S.EVA). L’intento di questo studio è di analizzare gli aspetti clinici, audiologici, radiologici e genetici in un gruppo di pazienti ipoacusici con acquedotto vestibolare allargato.

Plusieurs mutations du gèneOTOF ont été identifiées comme responsables de la surdité autosomique récessive DFNB9. Ce gène code pour l'otoferline, une protéine transmembranaire à 6 domaines C2, domaines d'interaction potentielle avec le calcium et les phospholipides. Dans la cochlée adulte, l'otoferline est spécifiquement exprimée dans les cellules ciliées internes qui sont les véritables cellules sensorielles de la cochlée puisqu'elles transmettent le message auditif au système nerveux central. Dans ces cellules, l'otoferline est associée aux vésicules synaptiques entourant le ruban et à la membrane plasmique présynaptique. Des tests de liaison in vitro indiquent que l'otoferline interagit de façon directe et dépendante du calcium avec la syntaxinel et la SNAP25, deux protéines du complexe SNARE. Afin d'étudier la fonction de l'otoferline in vivo et la physiopathologie de DFNB9, j'ai généré des mutants nuls du gène Otof par recombinaison homologue. Les souris Otof''' reproduisent le phénotype des patients DFNB9. Des mesures de variation de la capacitance membranaire ont montré l'abolition quasi-complète de l'exocytose des cellules ciliées internes chez ces mutants, malgré la présence de courants calciques normaux, et une morphogenèse normale des cellules ciliées internes et de leurs synapses à ruban. L'otoferline pourrait donc jouer un rôle de détecteur calcique, similaire à celui joué par la synaptotagmine I dans l'exocytose des synapses du système nerveux central, en contrôlant la dernière étape de la fusion vésiculaire au niveau des synapses à ruban des cellules ciliées internes<br>Mutations in the OTOF gene have been identified as responsible for the DFNB9 autosomal recessive form of human deafness. This gene encodes otoferlin, a transmembrane C2 domain protein; C2 domains are known to bind calcium and to interact with phopholipids. In the adult cochlea, otoferlin is specifically expressed in the inner hair cells, which are the genuine sensory cells as they transmit sound information to the central nervous System. In these cells, otoferlin is associated with synaptic vesicles surrounding the ribbon and with the presynaptic plasma membrane. In vitro binding assays indicate that otoferlin binds, in a calcium dependent manner, syntaxinl and SNAP25, two proteins of the SNARE complex. In order to study otoferlin function in vivo and to characterize DFNB9 pathophysiology, I have generated knock-out mice for the Otof gene using homologous recombination. Otof7' mice reproduce DFNB9 patients phenotype. Besides, exocytosis of Otof7' inner hair cells, as monitored by membrane capacitance measurements, was nearly completely abolished, despite normal ribbon synapse morphogenesis and normal calcium current. Furthermore, these cells lacked the fast secretory component of the exocytic burst in calcium-uncaging experiments. Therefore, otoferlin is essential for a late step of synaptic vesicle exocytosis, probably by acting as the major calcium sensor triggering fusion at the auditory hair cell ribbon synapse

L'audition joue un role majeur dans la communication entre individus dans la societe puisqu'elle permet la perception de la parole, vehicule du langage. Par une approche de clonage positionnel et de gene candidat, nous avons identifie le gene otof dont les mutations sont responsables de la surdite isolee autosomique recessive humaine dfnb9. Il coderait pour deux proteines, l'une de 140 et l'autre de 257 kda contenant respectivement 3 et 6 domaines c2, et un domaine transmembranaire cooh-terminal. Le gene otof et son orthologue murin otof comprennent 48 exons. Ils codent pour plusieurs transcrits generes par epissage alternatif. Nous avons decele deux mutations dans otof : (i) une mutation non sens, presumee deleter les deux formes de la proteine de leur domaine c2 cooh-terminal et de leur domaine transmembranaire ; (ii) une mutation au niveau d'un site d'epissage qui n'affecterait que la forme longue de la proteine. Cette derniere mutation demontre l'implication de la forme longue de l'otoferline dans la fonction auditive. Afin d'etudier la localisation de l'otoferline nous avons genere un anticorps contre la proteine. Les etudes d'immunohistochimie ont revele la presence de l'otoferline dans l'appareil de golgi et dans la membrane plasmique basale et baso-laterale de la cellule ciliee (cc) sensorielle interne (i) de la cochlee. Son expression au cours du developpement est parallele a la maturation des synapses des cellules sensorielles. L'expression de l'otoferline dans les pc12 a montre qu'elle serait transportee vers les terminaisons neurite-like grace a la presence de motifs d'adressage. Le profil d'expression de l'otoferline et ces domaines c2 homologues a ceux de la synaptotagminei, absente dans les cci, nous ont

TMPRSS3 est une sérine protéase mutée dans les surdités humaines DFNB8/10. Pour déterminer le rôle de cette protéine dans la physiologie cochléaire, nous avons généré puis caractérisé le phénotype auditif d'un modèle murin mimant la pathologie humaine. Les souris mutantes homozygotes sont profondément sourdes, due à une perte rapide et complète des cellules sensorielles cochléaires au moment de leur entrée en fonction. Afin de caractériser les mécanismes moléculaires responsables de la perte de ces cellules, nous avons comparé le protéome de souris sauvages et mutantes par des gels en 2-dimensions. Nous avons ensuite analysé les spots variants par spectrométrie de masse, ce qui nous a permis de reconstruire les réseaux dans lesquels les protéines variantes interviennent. Parmi les réseaux identifiés, nous avons focalisé notre analyse sur celui du canal potassique BK, puisque sa mise en place est concomitante de la dégénérescence des cellules sensorielles. Par des expériences d'immunohistochimie et de patch-clamp, nous avons pu montrer dans les cellules sensorielles de la cochlée, une réduction de l'expression membranaire et de l'activité de ce canal en l'absence de Tmprss3 fonctionnelle. Le résultat original de notre travail est qu'une sérine protéase est capable de réguler le canal potassique BK<br>TMPRSS3 is a type II serine protease mutated in human DFNB8/10 deafness. In order to determine the role of this protein in the cochlear physiology, we generated a mutant mice and phenotyped it. We found that homozygous mutant mice are profoundly deaf, due to a rapid and drastic degeneration of cochlear sensory cells at the onset of hearing. In order to decipher the molecular mechanism leading to sensory cells degeneration, we compared the cochlear proteome of wild type and homozygous mice using 2-dimensions gels. Then, we analyzed variant spots using mass spectrometry. Using bioinformatics, we clustered the protein in signaling pathways. We focused on the network centered on BK potassium channel because this channel appears at the onset of hearing, the time when sensory cells degenerate. Using immunohistochemistry and patch-clamp techniques, we were able to show that in the absence of a functional Tmprss3, membranous expression and activity of BK channel are altered in cochlear sensory cells. The original finding of our work is that a serine protease is able to modulate BK potassium channel

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2017<br>Introdução: O ouvido é um órgão sensorial que tem como função a transmissão e a tradução de sons para o cérebro assegurando assim a audição essencial a uma comunicação oral eficaz. A surdez é considerada a deficiência sensorial mais comum na população humana, comprometendo a integração social do indivíduo afetado e envolve a perda total ou parcial da capacidade de um indivíduo detetar sons. Aproximadamente 1/1000 recém-nascidos apresentam surdez, bem como cerca de 1/3 dos indivíduos com idade superior a 65 anos. A surdez está descrita como sendo a terceira doença sensorial crónica do mundo, prevendo-se um aumento de 25% dos casos até ao ano de 2020. A surdez associada à idade ou presbiacusia é uma doença multifatorial, representando a sequela final de diversos fatores intrínsecos e extrínsecos, que atuam no ouvido interno ao longo da vida. Esta forma de surdez caracteriza-se por uma perda auditiva progressiva, que começa nas altas frequências e está descrita como afetando mais homens do que mulheres. Esta forma de perda auditiva é também referida como surdez social por estar na origem do isolamento social e mesmo depressão, observados em alguns idosos onde a perda auditiva é maior. As causas de surdez podem ser genéticas ou ambientais como a associada a situações de anoxia, a doenças infeciosas ou infeções crónicas no ouvido, ao uso de medicamentos ototóxicos, exposição ao ruído e envelhecimento, como já referido. O locus DFNB1 foi o primeiro a ser identificado na surdez autossómica recessiva, contendo dois genes vizinhos no cromossoma 13, GJB2 e GJB6, que pertencem por isso ao mesmo cluster e que codificam individualmente duas proteínas transmembranares, a conexina 26 e a conexina 30, respetivamente. As conexinas são as subunidades dos conexões, estruturas que constituem as “gap-junctions” que funcionam como canais intercelulares. Ambas as conexinas, 26 e 30, são expressas na cóclea, entre as células ciliadas, pelo que possuem um papel fundamental no processo auditivo. Atualmente encontram-se descritas mais de 100 mutações e polimorfismos no gene GJB2. O espectro destas mutações varia entre populações, existindo mutações típicas das populações caucasianas, das asiáticas, etc, pelo que a identificação de mutações neste gene são muito relevantes em cada população. Duas grandes deleções no gene GJB6 são também responsáveis por casos de surdez. Dada a relevância dos genes GJB2 e GJB6 na etiologia da surdez em várias populações, o diagnóstico molecular de casos de surdez neurosensorial começa pelo seu estudo. Assim, faz sentido que na presbiacusia se estudem também estes genes procurando conhecer o seu efeito na causa deste tipo de surdez. Existem ainda mutações no gene GJB2 cuja patogenicidade é controversa, pelo que a realização de estudos funcionais que ajudem a clarificar o efeito destas mutações, identificadas de novo ou já conhecidas, é uma forma de prever a sua patogenicidade dessas mutações e assim estudar a sua associação com a surdez. A investigação com base em estudos genéticos e moleculares tem permitido grandes avanços na área da surdez, sugerindo que esta condição pode ser evitável e também pode ser tratada mais precocemente. Objetivo: O presente estudo teve como objetivo geral aumentar o conhecimento da surdez associada à idade em idosos da população portuguesa. Como objetivos específicos podem definir-se: 1) o papel dos genes GJB2/GJB6 na surdez associada à idade; 2) o estudo de novas mutações identificadas de novo na população Portuguesa com vista a clarificar a sua patogenicidade. Materiais e métodos: Foram analisadas 200 amostras de DNA, obtidas a partir de sangue colhido em idosos da população portuguesa, provenientes de diferentes regiões de Portugal. Todos os indivíduos assinaram um consentimento informado, responderam a um inquérito sobre o seu estado geral de saúde e antecedentes familiares, realizaram um audiograma com vista a identificar a presença de presbiacusia e aceitaram voluntariamente participar neste estudo pelo que também forneceram uma amostra de sangue colhida em cartão FTA. A pesquisa de mutações no gene GJB2 realizou-se em 80 amostras de DNA e em 120 amostras de DNA para o gene GJB6. Para isso foi amplificado por PCR e sequenciado em ambas as direções a região codificante (exão 2) do gene GJB2. As grandes deleções descritas no gene GJB6, foram estudadas por PCR multiplex, onde os primers usados permitem distinguir pelo padrão de amplificação a presença e a ausência das deleções. Foram estudadas três mutações p.Leu213X, p.Gly160Ser, p.Gly160Cys previamente identificadas de novo na população Portuguesa. Assim, realizaram-se culturas “in vitro” de células HeLa para efetuar estudos de expressão e de imunolocalização. Usaram-se também programas de modelação tridimensional de proteínas (CHIMERA, PYMOL e PDB) para tentar esboçar a conformação da proteína mutada comparativamente com a conexina selvagem (wild type) ou não mutada. Esta última abordagem foi também aplicada no estudo da mutação p.Ala40Gly identificada na amostra em estudo. Resultados e Discussão: Os 200 indivíduos considerados no presente estudo incluem 68.5% de mulheres (n=137) e 31.5% de homens (n=63), com idades compreendidas entre os 50 e os 90 anos de idade no geral. O estudo dos audiogramas mostrou que 4.5% (n=9) dos indivíduos apresenta uma audição normal (até 20dB de perda auditiva no melhor ouvido) e 16.5% (n=32) não possuem informação, pelo que os restantes 79% (n=159) apresentam presbiacusia. Destes, 1% dos indivíduos (n=2) apresentam surdez profunda (acima dos >81dB) sendo um homem com 70 anos de idade e uma mulher de 90 anos de idade. A pesquisa de mutações no gene GJB2 encontrou 5% dos indivíduos (n=4/80) com mutações em heterozigotia. Assim, nenhum deles apresentava surdez associada a GJB2 e todos estes indivíduos foram ouvintes durante toda a sua vida enquanto jovens e adultos. Observaram-se as mutações p.Arg143Gln (n=1) p.Met93Ile (n=1) e p.Ala40Gly (n=2) identificadas pela primeira vez na população Portuguesa. No gene GJB6 não foi observada nenhuma das grandes deleções já descritas. Os estudos realizados “in vitro”, em células HeLa que passaram a expressar a proteína conexina 26 (Cx26) selvagem (wt) e mutada com p.Leu213X, p.Gly160Ser ou com p.Gly160Cys, permitiam pela técnica Western Blot, verificar a expressão apenas da conexina 26 wt não tendo sido possível quantificar os níveis de expressão da conexina mutada com nenhuma das três mutações dado que não se observaram as bandas correspondentes. A repetição destes resultados sugere a não expressão das Cx26. Os resultados de imunofluorescência na presença da mutação p.Leu213X, evidenciam uma marcação perinuclear, enquanto que tanto com a mutação p.Gly160Ser como com a mutação p.Gly160Cys se observa uma marcação mais forte no núcleo, estes dados de alguma forma apoiam os dados obtidos com o Western Blot, mas, nos controlos não foi possível observar a imunomarcação da Cx26. Da análise comparativa da hipotética sequência tridimensional da Cx26 wt com cada uma das sequências referentes às quatro mutações em estudo (p.Leu213X, p.Gly160Ser, p.Gly160Cys e p.Ala40Gly), observaram-se diferenças entre os modelos obtidos para as 4 mutações. Estes resultados parecem sugerir a patogenicidade das mutações estudadas, já que as diferenças observadas poderão levar a alterações na função da proteína expressa o que justificaria os resultados obtidos nos estudos funcionais. Conclusões: Em termos epidemiológicos, as principais conclusões deste estudo permitem indicar que na sua maioria: 1) os idosos portugueses apresentam presbiacusia (79% dos casos da amostra), Quanto aos resultados genéticos resultantes do estudo do locus DFNB1 permitem concluir que: 1) apenas 5% dos indivíduos possuem mutações em GJB2 e nenhum em GJB6, pelo que o locus DFNB1 não parece estar associado à origem da presbiacusia ainda que esta amostra apresente uma incidência de portadores maior do que a população em geral; 2) As mutações p.Leu213X, p.Gly160Ser e p.Gly160Cys parecem ser patogénicas dado que não parecem expressar-se ao contrário da proteína wt, o que é suportado por não se ter conseguido as proteínas nas células HeLa e também porque se observam diferenças nas conformações da proteína normal e das proteínas mutadas nos modelos preliminares desenvolvidos; 3) a proteína p.Ala40Gly identificada em dois indivíduos desta amostra e de patogenicidade controversa segundo a bibliografia, poderá ser patogénica considerando as diferenças observadas nas hipotéticas conformações, mas não se realizaram estudos funcionais que apoiem este dado.<br>Introduction: The ear is a sensory organ which function is the transmission and translation of sounds to the brain. Hearing loss is a condition where a person loses part or all of their ability to hear sound. Age-related hearing loss or presbycusis is a multifactorial illness resulting from years of intrinsic and extrinsic factors affecting the inner ear during a life time. Locus DFNB1 was the first to be identified in autosomal recessive hearing loss and contains two neighbouring genes in chromosome 13, GJB2 and GJB6, which belong to the same cluster and codify two transmembrane proteins, connexin 26 and connexin 30, respectively. Research based on genetic and molecular studies has allowed us to make huge advances in understanding hearing loss, suggesting that this condition could be avoided and treated early on. Objectives: Our specific objectives are to: 1) understand the role played by genes GJB2/GJB6 in age-related hearing loss; 2) study the new mutations that have been identified in the Portuguese population, to better understand their pathogenicity. Material and methods: Analysis of 200 DNA samples taken from the blood of elderly Portuguese volunteers. Research into the mutations was carried out on 80 samples of gene GJB2 and 120 samples of gene GJB6, amplified by PCR. Four mutations were studied: p.Leu213X, p.Gly160Ser p.Gly160Cys and p.Ala40Gly, identified in the Portuguese population. In vitro cultures of HeLa cells were performed for expression and immunolocalization studies and functional studies using 3-D protein modelling programmes. This latter approach was also applied in the study of the identified p.Ala40Gly mutation in the study sample. Results and discussion: The group of 200 people has 68.5% women (n=137) and 31.5% men (n=63) between 50 and 90 years of age. The research into the mutations in gene GJB2 found that 5% of people had a heterozygote mutation. Three mutations, p.Arg143Gln, p.Ala40Gly and p.Met93Ile, were identified for the first time in Portuguese people. Using Western Blot it was not possible to quantify the levels of expression in the mutated connexin with the three mutations. Using the immunofluorescence technique, the location of connexin 26 on the cellular membrane was not observed. In the comparative analysis of the structure of the wild Cx26 and the protein containing one of the four mutations under study, differences were seen in all four cases. The results suggest these mutations are pathogenic as the differences observed may explain alterations in the function of the protein expressed and so they may affect hearing loss. Conclusions: The main conclusions of this study, epidemiologically speaking show: 1) Portuguese elderly present presbycusis (79% dos cases).The genetic results of the studies of the DFNB1 locus allow to conclude that: 1) only 5% of individuals have GJB2 mutations and none present GJB6 mutations, so the locus DFNB1 does not seem to be an important factor in presbycusis, although a high level was found in a prevalent number of carriers of the mutations in GJB2, above that found in the population at large; 2) Mutations p.Leu213X, p.Gly160Ser and p.Gly160Cys seem to be pathogenic as they do not appear to be expressed in the cellular membranes of the cells nor is it possible to quantify their low level of expression in those cells. The differences observed in the conformations created point to this conclusion, when compared to a normal protein; 3) The Ala40Gly protein was identified in two individuals from a controversial pathogenicity sample.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2019<br>A surdez é a deficiência sensorial mais comum e afecta cerca de 466 milhões de pessoas em todo o mundo, das quais 34 milhões são crianças. Apesar de a surdez hereditária poder estar associada a causas adquiridas, estima-se que, de todos os casos de surdez, mais de 50% tenha uma causa genética, tendo já sido identificados mais de 100 genes em humanos associados a esta patologia. A surdez não sindrómica, corresponde a cerca de 70% dos casos congénitos e destes, cerca de 80% está associado a um padrão de hereditariedade autossómico recessivo. Neste contexto, o locus DFNB1, que inclui os genes GJB2 e GJB6, que codificam as conexinas 26 e 30, associa-se a mais de 50% dos casos. De forma a diagnosticar o mais precocemente possível estes doentes, está implementado, na maioria dos países desenvolvidos, um rastreio neonatal auditivo. Adicionalmente, recorre-se cada vez mais ao diagnóstico molecular que, para além de outras vantagens, permite identificar a etiologia nos casos de surdez genética. Actualmente, os implantes cocleares constituem uma opção segura e eficaz de reabilitação dos doentes com surdez severa a profunda neuro-sensorial. No entanto, uma maior compreensão dos mecanismos genéticos moleculares subjacentes a esta patologia possibilita o desenvolvimento de novas terapias, nomeadamente a génica coclear.<br>Deafness is the most common sensory impairment and affects about 466 million people worldwide, of which 34 million are children. Although hereditary deafness may be associated with acquired causes, it is estimated that, of all cases of deafness, more than 50% have a genetic cause, having already been identified more than 100 genes in humans associated with this pathology. Non-syndromic deafness accounts for about 70% of congenital cases and of which about 80% are associated with an autosomal recessive inheritance pattern. In this context, the DFNB1 locus, which includes the GJB2 and GJB6 genes, that encode the 26 and 30 connexins, is associated with more than 50% of the cases. In order to diagnose these patients as early as possible, a neonatal auditory screening test is implemented in most developed countries. In addition, molecular diagnosis is increasingly used, which, in addition to other advantages, makes it possible to identify the etiology in cases of genetic deafness. Currently, cochlear implants are a safe and effective option for the rehabilitation of patients ranging from severe to profound sensorineural deafness. However, a better understanding of the molecular genetic mechanisms underlying this pathology is enabling the development of new therapies such as cochlear genetics.

Pendrin is an anion transporter that is expressed in several organs. In the thyroid gland, pendrin is localized at the apical pole of thyrocytes and it is responsible for the iodide efflux from thyrocytes into the colloid in the follicular lumen where iodide is organificated. The extrathyroidal expression was shown in the inner ear, kidney, placenta and mammary gland. Carriers of mutations in the pendrin gene (PDS, SLC26A4) display variable phenotypical features following the autosomal recessive manner of the inheritance: combined thyroid and hearing affection (Pendred syndrome - OMIM274600), nonsyndromic autosomal recessive neurosensory deafness (DFNB4 - OMIM600791) or isolated enlarged vestibular aqueduct (EVA - OMIM603545). The thyroid affection is usually manifested as euthyroid or hypothyroid goitre in the second decade of life. In a minority of patients, dyshormonogenesis is present at birth, and the disease is diagnosed in the frame of the nation-wide neonatal screening for congenital hypothyroidism.