Unit: Sensory Deficit Genetics - INSERM (U587)

Director: Christine PETIT

Research in the laboratory of Génétique des Déficits Sensoriels aims at elucidating the molecular bases of hereditary sensory defects in humans, mainly auditory defects. Expected outcomes of this research include medical applications (molecular diagnosis and development of novel therapies) as well as an understanding of the development and function of sensory organs in molecular terms.

Understanding the mechanisms which underlie the function of sensory systems is an objective to which the study of hereditary dysfunctions of these systems can contribute, following the development of tools for genetic and genomic analysis. We first became interested in the study of the Kallmann syndrome, which includes a defect in olfaction, and identified the first gene implicated, KAL1, which encodes a component of the extracellular matrix, anosmin-1. Recently, we have identified a second gene implicated in this syndrome, KAL2, which codes for the FGFR1 receptor. We believe that anosmin-1 modulates the FGF signalling implicated in the formation of the olfactory bulb.

At the beginning of the 1990s, we chose to study hereditary forms of human deafness for two reasons: (1) at the time they constituted an unexplored domain of hereditary sensory pathology, and (2) they should lead to an approach to the molecular basis of the development and functioning of the cochlea (the hearing receptor organ), which was then totally unknown.

Since 1995, we have identified the genes responsible for two forms of type I Usher syndrome (USH1B and USH1C), in which there is an association of profound sensorineural deafness and progressive retinitis pigmentosa leading to blindness, as well as a gene which is defective in branchio-oto-renal syndrome. We have also identified the genes affected in five forms of isolated recessive deafness (DFNB2, DFNB9, DFNB16, DFNB18 and DFNB21), and two forms of isolated dominant dearness (DFNA2 and DFNA3). In 2002-2003 we have added DFNB22 and DFNB31 to this list of genes implicated in isolated deafness, and identified USH1G as the causative gene in another form of type I Usher syndrome. These genes code for otoancorin, whirlin and the SANS protein respectively.

Using complementary experimental approaches, we have obtained an ensemble of results concerning the function of the proteins encoded by these genes. We have observed that, for the most part, these genes participate in the following processes: (1) the structure of the tectorial membrane, an acellular membrane which covers the auditory sensory epithelium and which participates in the transmission of the energy of the sound wave to the hair bundle of the sensory cells, (2) the development of the hair bundle, the receptor structure to sound, which is composed of a group of rigid microvilli, the stereocilia, and which contains the machinery for mechanotransduction, (3) the function of the synapse of the sensory cells ("ribbon" synapse), and (4) the communication between cells via gap junctions.

Development of the hair bundle

Four components of the hair bundle have been identified: myosin VIIa, whirlin, harmonin and stereocilin. Mutations of the genes coding for myosin VIIa and harmonin are responsible for type I Usher syndrome (USH1B and USH1C forms), and more rarely for an isolated form of deafness. Harmonin and whirlin are two molecules with PDZ domains, submembrane proteins which organise protein complexes. The tails of unconventional myosins, such as myosin VIIa, are linked to the proteins on which the motor force of myosin is directed. The structures with which these proteins are associated are thus placed under tension, and may even move under certain circumstances. With the aim of understanding the role of myosin VIIa, a study of its ligands has been undertaken. This has allowed us to identify a new ubiquitous transmembrane molecule present in intercellular adhesion junctions, vezatin, which belongs to the same complex as E-cadherin, and thus we could propose a role for myosin VIIa in cell adhesion. We then demonstrated that myosin VIIa is an anchor protein for protein kinase A. A third ligand for this myosin binds to rab27, which is present at the surface of the melanosomes, thus explaining the abnormal position of melanosomes in the cells of the pigmented epithelium of the retina in patients with USH1B syndrome. Finally, we have shown that the b isoforms of harmonin, which are present in the developing hair bundle both bind to actin filaments and induce their clustering into bundles, and also interact with cadherin-23, another protein implicated in Usher syndrome type I (USH1D), which forms the transitory interstereociliar links. Thus, three proteins which are defective in three forms of type I Usher syndrome work together in a network of molecular interactions which contributes to the cohesion of the developing hair bundle and stabilises the stereocilia by anchoring the links which connect them to the actin filaments which form the cytoskeleton of each stereocilium. Furthermore, we have shown that the protein encoded by the SANS gene also interacts with harmonin.

Synapses of the sensory cells

In a collaborative study, we have demonstrated that the DFNA2 form of deafness is due to a defect in the KCNQ4 potassium channel, which is expressed principally by the outer hair cells (for which the essential function is the amplification of the sound stimulus), and by the neurons of the central auditory pathway. Especially interesting, the identification of the gene responsible for another form of recessive deafness, DFNB9, has led to the discovery of otoferlin, a protein from the dysferlin family, which is implicated in the synaptic function of inner hair cells (the authentic auditory sensory cells).

Gap junctions

Connexin26 is defective in a form of recessive deafness, DFNB1, which, we have shown, accounts for about one-third of the cases of deafness in children. The two cochlear cellular networks formed by the gap junctions, the epithelial network and the fibrocyte network, express connexin26 and connexin30, for which a deficit also leads to deafness. Because of the high frequency for the DFNB1 form of deafness, we have undertaken a study of its pathogenicity. Ubiquitous inactivation of the gene which codes for connexin26 (Cx26) is lethal in the mouse. We therefore performed a conditional inactivation of Cx26 in the epithelial network, and analysed the phenotype of the mutant mice, as well as that of mice in which the gene which encodes connexin30 (Cx30) was inactivated ubiquitously. In both cases, death by apoptosis was observed in cells of the sensory auditory epithelium beginning in the 3rd week postnatally, i.e. a little after wild-type mice begin to hear. Furthermore, the endocochlear potential, a transepithelial potential difference between endolymphatic and perilymphatic compartments, which normally appears in the 2nd week of life, is not detected in Cx30-/- mice. This finding led us to conclude that connexin30 plays an essential role in the stria vascularis, the site of production of this difference of potential.

These results have been obtained because of the synergy in the activities of the various members of our laboratory. The subtracted cDNA libraries which were generated by some have also helped others in isolating deafness genes; they have been the source of promoters utilised by several scientists to generate conditional inactivations of genes in the ear. Because of the complex structure of the inner ear, the knowledge of histology and embryology of some has been essential to permit the transition from the isolation of genes responsible for deafness to the study of the pathogenicity of the corresponding forms. A real transfer of knowledge between specialists in molecular biology, embryologists and ENT physicians has taken place very rapidly. A dialog between those who are looking for new genes implicated in deafness and those who are trying to

define the networks of molecular interactions in which the products of already-identified genes participate has been a source of ideas and discoveries for both. A continuous articulation with the clinic has made it possible to establish the frequency of defects in the connexin26 gene, to provide the first clinical description of a genetic form of isolated deafness, and above all to evaluate the medical implications of our studies. Genetic counselling for families with deaf individuals has been considerably improved.


(A,B) Localisation of myosin VIIa (A) and of one of its transmembrane protein ligands, in the hair bundle of inner (IHC) and outer (OHC, V shape) hair cells. (C) A Schematic representation illustrating how myosin VIIa may exert a tension force between the plasma membrane, through its transmembrane (TM) protein ligand, and the underlying actin cytoskeleton, via its motor head domain. (A. El-Amraoui).

Keywords: Human genetics, sensorineural defects, deafness, cell biology , sensory physiology

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