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  Director : Christine PETIT (cpetit@pasteur.fr)



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.



I. Kallmann syndrome

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

This syndrome associates an olfactory defect (anosmia) with the absence of spontaneous onset of puberty. Anosmia results from a developmental defect of the olfactory bulbs; hypogonadism is due to a failure in the embryonic migration of GnRH-synthesizing neurons (GnRH is 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-chromosome 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 the organogenesis period, 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. We have shown that anosmin-1 is involved in the formation of the collateral branches of the secondary olfactory axons that make the lateral olfactory tracts. In addition, we have discovered that loss-of-function mutations in the gene encoding fibroblast growth factor receptor 1 (FGFR1) underlie an autosomal dominant form of Kallmann syndrome.

II. The saga of the genes implicated in hereditary deafness

(Sébastien Chardenoux, Roney Coimbra, Sedigheh Delmaghani, Sophie Lainé, Michel Leibovici, Mirna Mustapha, Sylvie Nouaille, 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 35 genes (underlying 41 genetic forms of deafness) have been cloned. Twelve loci and the genes underlying 16 genetic forms of deafness have been identified in our laboratory.

We have 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 several novel loci responsible for isolated or syndromic deafness (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. During the last three years, we have discovered several deafness genes using this approach.

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

1. Connexin-26 and connexin-30 defects

(Martine Cohen-Salmon, Francisco DelCastillo, 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 stria vascularis 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. 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 of several types of cells. We also studied mutant mice with ubiquitous inactivation of the connexin-30 gene. Homozygous null mutants have a severe auditory defect, a complete lack of the endocochlear potential (i.e. the +80 mV transepithelial electric potential difference between the endolymphatic and perilymphatic spaces), and a progressive disorganisation of the sensory epithelium of the cochlea, due to cell death. We are currently trying to understand why these mice are not able to produce an endocochlear potential. In addition, we are in the process of generating a mouse model of DFNB1, i.e. mutant mice that lack connexin-26 in the two gap junction networks of the cochlea.

2. Usher Syndrome type I

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

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 proteins that bind to myosinVIIA. 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. 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. Now, we have extended our study to the proteins underlying the various genetic forms of USH1. We have shown that three of these proteins, namely myosin VIIA (USH1B), harmonin (USH1C) and cadherin-23 (USH1D), cooperate to shape the bundle of stereocilia in the developing hair cell (see photograph). In addition, we have recently identified the gene responsible for USH1G and shown that the encoded protein, SANS, interacts with harmonin; thus, SANS belongs to the aforementioned functional complex.

3. Otoferlin

(Mhamed Grati, Isabelle Roux, Saaid Safieddine)

Mutations in the OTOF gene underlie a recessive deafness form, DFNB9. The gene encodes otoferlin, a protein with C2 domains which, in the cochlea, is preferentially expressed in the inner hair cells. The protein is especially abundant in the synaptic region of these cells; hence we have hypothesized that it could be involved in the traffic of the synaptic vesicles. The presence of the C2 domains suggests that otoferlin could replace the calcium sensor synaptotagmin, which is not produced by inner hair cells. We are currently testing this hypothesis using different approaches. In addition, a search for proteins that bind to otoferlin is under way through the two-hybrid technique. This strategy should lead to a better understanding of the role of otoferlin in auditory signals' processing.

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

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  Office staff Researchers Scientific trainees Other personnel
  Gillet Dominique, Institut Pasteur Cohen-Salmon Martine, CNRS

El-Amraoui Aziz, Institut Pasteur

Ernest Sylvain, INSERM

Hardelin Jean-Pierre, INSERM

Leibovici Michel, CNRS

Levilliers Jacqueline, INSERM

Safieddine Saaid, CNRS

Soussi-Yanicostas Nadia, CNRS

Verpy Elisabeth , Institut Pasteur

Weil Dominique , INSERM

Adato Avital, Post-doc

DelCastillo Francisco , Post-doc

Delmaghani Sedigheh , PhD student

Étournay Raphaël, PhD student

Michel Vincent, Post-doc

Roux Isabelle, PhD student

Zwaenepoel Ingrid , PhD student

Blanchard Stéphane, Technician

Chardenoux Sébastien, Technician

Nouaille Sylvie, Technician

Perfettini Isabelle, Engineer

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