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.

(Jean-Pierre Hardelin, Isabelle Perfettini, Nadia Soussi-Yanicostas)

This syndrome associates an olfactory defect (anosmia) with the absence of spontaneous onset of puberty. The hypogonadism is due to a GnRH deficiency, the hypothalamic hormone that controls puberty development in the gonads via the pituitary gland.

We have identified the gene KAL-1 as responsible for the X-linked form of the disease. The protein that it encodes, which we have called anosmin-1 on account of the olfactory defect that characterises the disease, is a 100 kDa extracellular glycoprotein. During organogenesis, anosmin-1 has a regional distribution within certain extracellular matrices. Later during development, the protein is produced by several neuronal populations, especially in the olfactory bulbs and the olfactory cortex. Two properties of this molecule have been discovered in 2001: a stimulating effect on axon growth and a guidance role, for which the physiological significance has been validated in organotypic cultures.

(Sébastien Chardenoux, Roney Coimbra, Sedigheh Delmaghani, Sophie Lainé, Michel Leibovici, Mirna Mustapha, Sylvie Nouaille, Joël Paronnaud, Elisabeth Verpy, Dominique Weil, Ingrid Zwaenepoel)

Deafness is the most common frequent sensory defect in children. It is sometimes associated with other anomalies, i.e. syndromic, but it is most often present as an isolated defect. Today, we know that 80% of the cases of profound congenital deafness are genetic in origin. Several dozen loci implicated in isolated deafness have been reported, and 26 genes have been cloned. Ten loci and 8 genes have been identified in our laboratory.

During 2001, we extended our collaboration network, which in addition to colleagues from Tunisia, Lebanon and Greece, now includes colleagues from Iran and Jordan. About one hundred large families affected with recessive isolated deafness have been collected. Genetic analysis of these families has already led to the identification of four novel loci responsible for isolated deafness and two loci for Usher syndrome type I (see § III-2). Our strategy for the isolation of the genes responsible for deafness is based on a candidate gene approach. Our working hypothesis is that genes whose expression is limited to the inner ear or which are preferentially expressed there must have a crucial role in hearing, and defects in these genes are likely to lead to deafness. In order to isolate these genes, we have generated subtractive cDNA libraries from inner ear sensory epithelia. In 2001, the gene responsible for recessive deafness DFNB16 was discovered using this approach. The production of antibodies directed against the encoded protein has revealed that it is exclusively present in the sensory cells of the ear; it was found to be associated with the stereocilia (see figure 1), i.e. the rigid apical villosities which form the receptor structures for the auditory stimulus, hence its name "stereocilin".

III. Physiology and molecular pathophysiology of the cochlea (the organ of audition)

1. Connexin-26 defect

(Martine Cohen-Salmon, Jean-Pierre Hardelin, Vincent Michel, Isabelle Perfettini)

We have shown that defects in the connexin-26 gene account for half of the cases of prelingual isolated deafness (i.e. with onset before the age of speech acquisition). The corresponding form of deafness, DFNB1, is therefore one of the most frequent monogenic diseases in western countries since it affects one child in 2 000 approximately.

In the inner ear, connexin-26 participates in the constitution of gap junctions that underlie the formation of two independent cellular networks. One of them, which is epithelial in nature, connects the supporting cells of the sensory epithelium to the adjacent epithelial cells. The other connects a group of fibrocytes, that are associated with basal and intermediate cells of the vascular stria in the cochlea (the epithelium responsible for the genesis of the endocochlear potential and for the secretion of potassium into the endolymph). Complete inactivation of the connexin-26 gene in the mouse is lethal in the embryo due to placental anomalies. The role of connexin-26 in the inner ear thus remains unknown. We have performed a conditional inactivation of the gene in the epithelial network of the inner ear by using a promoter from one of the genes isolated from the subtractive cDNA libraries mentioned above, and whose expression is strictly limited to the cells of this epithelial network. Homozygous mutant mice do not have any detectable anomalies of the vestibule. In contrast, they present an auditory defect accompanied by a progressive disorganisation of the sensory epithelium of the cochlea, due to death of several types of cells. This result demonstrates that, independently of the effect of the connexin-26 defect in the fibrocyte network of the cochlea, prevention of cell death in the sensory epithelium is a necessary condition for the restoration of auditory function in subjects affected with this form of deafness.

2. Usher Syndrome type I

(Batiste Boëda, Stéphane Blanchard, Aziz El-Amraoui, Sylvain Ernest)

This syndrome associates a profound congenital deafness and retinitis pigmentosa beginning around puberty, and evolving to blindness.

We have shown that a defect in the gene that encodes myosin VIIA is responsible most often for Usher syndrome type I, and more rarely causes isolated deafness. In order to understand the role of this unconventional myosin in the development and function of the cochlea, the yeast "double-hybrid" system was used to search for ligands of this protein. One of these is a novel ubiquitous protein found in intercellular adherens junctions, that we have called vezatin. Vezatin is a transmembrane protein characterised by numerous isoforms. In the inner ear, it is present not only at the adherens junctions between the hair cells and the surrounding supporting cells, but also at the base of the stereocilia (see figure 2). Thus, the vezatin-myosin VIIA complex most likely creates a tension between the actin filaments of the stereocilia and the transient basal links that hold the stereocilia together during development. This function can explain the disorganisation of stereocilia, which is observed in the shaker-1 mouse mutant, in which the myosin VIIA gene is mutated. We have also isolated another ligand of myosin VIIA, MyRIP (myosin VIIA Rab interacting protein), which is a novel effector of the rab27 GTPase. MyRIP and rab27 are colocalised at the surface of the melanosomes in cells of the pigmentary epithelium of the retina. Defects in the myosin VIIA-MyRIP-rab27 complex can explain the abnormal position of melanosomes in these cells in the shaker-1 mutant mice.

3. Otoferlin

(Mhamed Grati, Isabelle Roux, Saaid Safieddine)

The recessive isolated deafness DFNB9 is due to anomalies in otoferlin, a protein with C2 domains which, in the cochlea, is preferentially expressed in the inner hair cells. We have hypothesised that it could be involved in the traffic of the synaptic vesicles, and we are currently testing this hypothesis using different approaches.

Figure 1. Immunolabelling of stereocilin in the sensory macula of the mouse utricle, at postnatal day 15.

The immunoreactivity is located in the hair bundles, i.e. the mechanoreceptive structures of the sensory cells.

Figure 2. Immunolabelling of vezatin in a mouse cochlea, at 2 days postnatal.

The picture corresponds to a general view of the apical poles of hair cells, with their hair bundles. The 3 rows of outer hair cells (top) and single row of inner hair cells (bottom) are visible.

Vezatin is present at the base of hair bundles, at the place where transient basal links connecting the stereocilia are anchored.

Laganier Sylvie, Institut Pasteur

Weil Dominique, DR2 INSERM

Zwaenepoel Ingrid, Etudiante en thèse

Perfettini Isabelle, Ingénieur CNRS