Biomedical subjects

C Petit

Publications and source records attributed to C Petit.

At least 55 records · Page 3Linked to original sources

PubMed2001

Molecular genetics of hearing loss.

Hereditary isolated hearing loss is genetically highly heterogeneous. Over 100 genes are predicted to cause this disorder in humans. Sixty loci have been reported and 24 genes underlying 28 deafness forms have been identified. The present epistemic stage in the realm consists in a preliminary characterization of the encoded proteins and the associated defective biological processes. Since for several of the deafness forms we still only have fuzzy notions of their pathogenesis, we here adopt a presentation of the various deafness forms based on the site of the primary defect: hair cell defects, nonsensory cell defects, and tectorial membrane anomalies. The various deafness forms so far studied appear as monogenic disorders. They are all rare with the exception of one, caused by mutations in the gene encoding the gap junction protein connexin26, which accounts for between one third to one half of the cases of prelingual inherited deafness in Caucasian populations.

Animals↗

PubMed2001

Usher syndrome: from genetics to pathogenesis.

Usher syndrome (USH) is defined by the association of sensorineural deafness and visual impairment due to retinitis pigmentosa. The syndrome has three distinct clinical subtypes, referred to as USH1, USH2, and USH3. Each subtype is genetically heterogeneous, and 12 loci have been detected so far. Four genes have been identified, namely, USH1B, USH1C, USH1D, and USH2A. USH1B, USH1C, and USH1D encode an unconventional myosin (myosin VIIA), a PDZ domain-containing protein (harmonin), and a cadherin-like protein (cadherin-23), respectively. Mutations of these genes cause primary defects of the sensory cells in the inner ear, and probably also in the retina. In the inner ear, the USH1 genes, I propose, are involved in the same signaling pathway, which may control development and/or maintenance of the hair bundles of sensory cells via an adhesion force (a) at the junctions between these cells and supporting cells and (b) at the level of the lateral links that interconnect the stereocilia. In contrast, the molecular pathogenesis of USH2A, which is owing to a defect of a novel extracellular matrix protein, is likely to be different from that of USH1.

Animals↗

PubMed2001

Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness.

Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.

Animals↗

PubMed2001

In vitro maturation of bitch oocytes: effect of sperm penetration.

In contrast to most mammals, bitches ovulate immature oocytes at the germinal vesicle stage. Spermatozoa can be present in the oviduct of bitches at ovulation. The aim of this study was to investigate the influence of premature fertilization on the resumption of meiosis. In addition, the relative behaviour of male and female chromatin was observed. Canine cumulus-oocyte complexes cultured in medium 199 supplemented with 20% fetal calf serum for 24 h were incubated in vitro in the presence or absence of fresh dog semen for 24 h, and then rinsed and cultured in the same medium for a further 24 h. Chromatin was stained by propidium iodide and all oocytes were examined under laser scanning confocal microscopy. Results show that sperm penetration can occur in vitro in immature oocytes and that this induces a resumption of meiosis: at 72 h, the percentage of oocytes at the germinal vesicle stage decreased significantly and the percentage of oocytes beyond metaphase I increased. However, premature fertilization was ineffective for 40% of oocytes, which remained at the germinal vesicle stage. Measurement of chromatin areas in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage, indicating that male and female chromatins are sensitive to the same cell cycle regulators.

Animals↗

PubMed2000

Fdp, a new fibrocyte-derived protein related to MIA/CD-RAP, has an in vitro effect on the early differentiation of the inner ear mesenchyme.

During the course of a study aimed at isolating transcripts specifically or preferentially expressed in the inner ear, we identified a novel gene, encoding a fibrocyte-derived protein, that we named Fdp. Fdp is predicted to be a secreted 128-amino acid protein, which is highly homologous to the melanoma-inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP), a cartilage-specific protein also expressed in several tumors. Fdp and MIA/CD-RAP thus define a new family of proteins. Fdp is expressed from embryonic day 10.5 in the mesenchyme surrounding the otic epithelium. During development, these cells progressively aggregate, condense, and differentiate into cartilaginous cells forming the otic capsule, which no longer expresses Fdp, and into fibrocytes surrounding the epithelia, which strongly express Fdp. In order to address the function of Fdp, we developed an in vitro antisense oligonucleotide approach using microdissected periotic mesenchyme micromass cultures, and showed that Fdp antisense oligonucleotide treatment results in a significant reduction in chondrogenesis. Our results demonstrate that Fdp plays a role in the initiation of periotic mesenchyme chondrogenesis. Accordingly, Fdp and its human ortholog FDP, which map to chromosome 2 and band 20p11, respectively, could be candidate genes for forms of deafness associated with malformations of the otic capsule.

Amino Acid Sequence↗

PubMed2000

Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex.

Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two-hybrid screen. Vezatin is a ubiquitous protein of adherens cell-cell junctions, where it interacts with both myosin VIIA and the cadherin-catenins complex. Its recruitment to adherens junctions implicates the C-terminal region of alpha-catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin-catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell-cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane-membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA-defective mutants, inactivity of the vezatin-myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.

Alternative Splicing↗

PubMed2000

Unconventional myosin VIIA is a novel A-kinase-anchoring protein.

To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.

Animals↗

PubMed2000

Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action.

Selectively regulating gene expression in bacteria has provided an important tool for studying gene function. However, well-regulated gene control systems have been restricted primarily for use in laboratory non-pathogenic strains of bacteria (e.g. Escherichia coli, Bacillus subtilis). The development of analogous systems for use in bacterial pathogens such as Staphylococcus aureus would significantly enhance our ability to examine the contribution of any given gene product to pathogen growth and viability. In this report, we adapt, examine and compare three regulated gene expression systems in S. aureus, which had previously been used in B. subtilis. We demonstrate that all three systems function and exhibit titratable induction, together covering a dynamic range of gene expression of approximately 3000-fold. This dynamic range correlates well with the physiological expression levels of cellular proteins. Importantly, we show that one of these systems, the Spac system, is particularly useful for examining gene essentiality and creating specific conditional lethal phenotypes. Moreover, we find that titration of selective target gene products using this system allows direct demonstration of antibiotic mode of action.

Anti-Bacterial Agents↗

PubMed2000

Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness.

The zebrafish (Danio rerio) possesses two mechanosensory organs believed to be homologous to each other: the inner ear, which is responsible for the senses of audition and equilibrium, and the lateral line organ, which is involved in the detection of water movements. Eight zebrafish circler or auditory/vestibular mutants appear to have defects specific to sensory hair cell function. The circler genes may therefore encode components of the mechanotransduction apparatus and/or be the orthologous counterparts of the genes underlying human hereditary deafness. In this report, we show that the phenotype of the circler mutant, mariner, is due to mutations in the gene encoding Myosin VIIA, an unconventional myosin which is expressed in sensory hair cells and is responsible for various types of hearing disorder in humans, namely Usher 1B syndrome, DFNB2 and DFNA11. Our analysis of the fine structure of hair bundles in the mariner mutants suggests that a missense mutation within the C-terminal FERM domain of the tail of Myosin VIIA has the potential to dissociate the two different functions of the protein in hair bundle integrity and apical endocytosis. Notably, mariner sensory hair cells display morphological and functional defects that are similar to those present in mouse shaker-1 hair cells which are defective in Myosin VIIA. Thus, this study demonstrates the striking conservation of the function of Myosin VIIA throughout vertebrate evolution and establishes mariner as the first fish model for human hereditary deafness.

Alleles↗

PubMed2000

OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9.

We have recently reported that OTOF underlies an autosomal recessive form of prelingual sensorineural deafness, DFNB9. The isolated 5-kb cDNA predicted a 1,230 amino acid (aa) C-terminus membrane-anchored cytosolic protein with three C2 domains. This protein belongs to a family of mammalian proteins sharing homology with the Caenorhabditis elegans fer-1. The two other known members of this family, dysferlin and myoferlin, both have six predicted C2 domains. By northern blot analysis, a 7-kb otoferlin mRNA could be detected in the human brain. We isolated the corresponding cDNA, which is expected to encode a 1,977-aa-long form of otoferlin with six C2 domains. A 7-kb cDNA derived from the murine orthologous gene, Otof, was also identified in the inner ear and the brain. The determination of the exon-intron structure of the human and murine genes showed that they are composed of 48 coding exons and extend approximately 90 kb and approximately 80 kb, respectively. Alternatively spliced transcripts could be detected that predict several long isoforms (six C2 domains) in humans and mice and short isoforms (three C2 domains) only in humans. Primers were designed to explore the first 19 OTOF exons, henceforth permitting exploration of the complete coding sequence of the gene in DFNB9 patients. In a southwestern Indian family affected by DFNB9, a mutation in the acceptor splice site of intron 8 was detected, which demonstrates that the long otoferlin isoforms are required for inner ear function.

Alternative Splicing↗

PubMed2000

Physical map of the region surrounding the OTOFERLIN locus on chromosome 2p22-p23.

The autosomal recessive form of nonsyndromic deafness DFNB9 has been mapped to a 2-cM region on chromosome 2p22-p23, and the responsible gene, OTOF, has been recently identified by positional cloning combined with a candidate gene approach. In the course of this gene cloning, we established a contig of yeast artificial chromosomes, bacterial artificial chromosomes, and P1 phage artificial chromosomes delimited by polymorphic markers D2S2170 and D2S170, i.e. , extending over approximately 3500 kb. Sixty expressed sequence tags or genes and 14 sequence-tagged sites, 11 of which are polymorphic, were mapped to this contig and assigned to 21 chromosomal intervals.

Chromosomes, Human, Pair 2↗

PubMed2000

HIV-1 genome nuclear import is mediated by a central DNA flap.

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Amino Acid Sequence↗

PubMed2000

KCNQ4, a K+ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway.

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, an autosomal dominant form of progressive hearing loss in humans. In the mouse cochlea, the transcript has been found exclusively in the outer hair cells. By using specific antibodies, we now show that KCNQ4 is situated at the basal membrane of these sensory cells. In the vestibular organs, KCNQ4 is restricted to the type I hair cells and the afferent calyx-like nerve endings ensheathing these sensory cells. Several lines of evidence suggest that KCNQ4 underlies the I(K,n) and g(K,L) currents that have been described in the outer and type I hair cells, respectively, and that are already open at resting potentials. KCNQ4 is also expressed in neurons of many, but not all, nuclei of the central auditory pathway, and is absent from most other brain regions. It is present, e.g., in the cochlear nuclei, the nuclei of the lateral lemniscus, and the inferior colliculus. This is the first ion channel shown to be specifically expressed in a sensory pathway. Moreover, the expression pattern of KCNQ4 in the mouse auditory system raises the possibility of a central component in the DFNA2 hearing loss.

Amino Acid Sequence↗

PubMed2000

[Hereditary sensorineural deafness].

Deafness is the most common sensory defect. The investigation of the cause of deafness is critical for genetic counselling, and sometimes for appropriate management of associated pathologies. About two thirds of cases of congenital deafness are genetic forms, and the proportion is probably similar concerning the forms of deafness that appears during childhood. Some of the genetic forms are syndromic and the associated signs are sometimes inapparent or may appear during childhood. Consequently, a systematic search for the most frequent syndromes is necessary in each deaf individual. In the majority of genetic cases, deafness is the sole defect (non-syndromic deafness) and the major mode of transmission is autosomal recessive. The DFNB1 form of deafness, due to connexin 26 gene mutations, underlies half of the cases of non syndromic congenital deafness cases. The hearing loss has a prelingual onset, and it is most frequently severe or profound. There is no associated pathologies or radiological anomalies of the inner ear, and the vestibular tests are normal. The possibility of offering molecular diagnosis of connexin 26 gene defects is profoundly modifying daily medical practice in the investigation of the cause of deafness.

Connexin 26↗

PubMed2000

A simple and reliable method for the detection of the 30delG mutation of the CX26 gene.

Mutations in the CX26 gene (GJB2), encoding the gap-junction protein Connexin-26, have been shown to be the major cause of non-syndromic recessive deafness. Among these mutations, the deletion of a guanine within the stretch of six G between nucleotide positions +30 and +35 of the CX26 cDNA (30delG) accounts for the majority of this kind of deafness. Molecular detection of the 30delG mutation is usually performed by direct sequencing analysis of PCR products or by SSCP. To detect this mutation we developed an easy and reliable method, based on PCR, followed by a non-radioactive sandwich hybridization on microtiter plates. We tested 188 individuals recruited from the genetic counseling service for deaf people at the Pasteur Hospital and at the Armand-Trousseau Children's Hospital, Paris, France between April 1997 and September 1998. Our screening method is simple, uses stable and safe reagents, and employs inexpensive equipment. As such, it is suitable for widespread use in genetic diagnosis.

Connexin 26↗

PubMed2000

Characterization of the two zebrafish orthologues of the KAL-1 gene underlying X chromosome-linked Kallmann syndrome.

The gene underlying X chromosome-linked Kallmann syndrome, KAL-1, has been identified for several years, yet its role in development is still poorly understood. In order to take advantage of the zebrafish as a model in developmental genetics, we isolated the two KAL-1 orthologues, kal1.1 and kal1.2, in this species. Comparison of deduced protein sequences with the human one shows 75.5 and 66.5% overall homology, respectively. The most conserved domains are the whey acidic protein-like domain and the first of four fibronectin-like type III repeats. However, kal1.2 putative protein lacks the basic C-terminal domain (20 residues) found in kal1.1 and KAL-1. The expressions of kal1.1 and kal1.2 were studied in the embryo between 6 and 96 hours post fertilization using whole-mount in situ hybridization. Although a few structures express both genes, kal1.1 and kal1.2 expression patterns are largely non-overlapping. Taken together, these patterns match fairly well those previously reported for human KAL-1 and chicken kal1. As regards the olfactory system, kal1.1 is expressed, from 37 h.p.f. onward, in the presumptive olfactory bulbs, whereas kal1.2 transcript is only detected, from 48 h.p.f., in the epithelium of the nasal cavity. The relevance of the zebrafish as an animal model for studying both the function of KAL-1 in normal development and the developmental failure leading to the olfactory defect in Kallmann syndrome, is discussed.

Amino Acid Sequence↗

PubMed2000

Mutations of GJB2 in three geographic isolates from northern Tunisia: evidence for genetic heterogeneity within isolates.

Geographically isolated populations have been successfully used to localize genes for recessive inherited diseases, including non-syndromic sensorineural recessive deafness (NSRD). To date, 25 loci for NSRD have been localized on human chromosomes (DFNB loci), and six of the corresponding genes have been identified. Here, we report on the contribution of the DFNB1 locus (GJB2 gene) to NRSD in seven affected families living in three northern Tunisian geographic isolates, and we provide evidence for genetic heterogeneity within isolates. This finding challenges the classical view of a single 'founder' mutation segregating in such isolates.

Alleles↗

PubMed2000

Targeted disruption of otog results in deafness and severe imbalance.

Genes specifically expressed in the inner ear are candidates to underlie hereditary nonsyndromic deafness. The gene Otog has been isolated from a mouse subtractive cDNA cochlear library. It encodes otogelin, an N-glycosylated protein that is present in the acellular membranes covering the six sensory epithelial patches of the inner ear: in the cochlea (the auditory sensory organ), the tectorial membrane (TM) over the organ of Corti; and in the vestibule (the balance sensory organ), the otoconial membranes over the utricular and saccular maculae as well as the cupulae over the cristae ampullares of the three semi-circular canals. These membranes are involved in the mechanotransduction process. Their movement, which is induced by sound in the cochlea or acceleration in the vestibule, results in the deflection of the stereocilia bundle at the apex of the sensory hair cells, which in turn opens the mechanotransduction channels located at the tip of the stereo-cilia. We sought to elucidate the role of otogelin in the auditory and vestibular functions by generating mice with a targeted disruption of Otog. In Otog-/- mice, both the vestibular and the auditory functions were impaired. Histological analysis of these mutants demonstrated that in the vestibule, otogelin is required for the anchoring of the otoconial membranes and cupulae to the neuroepithelia. In the cochlea, ultrastructural analysis of the TM indicated that otogelin is involved in the organization of its fibrillar network. Otogelin is likely to have a role in the resistance of this membrane to sound stimulation. These results support OTOG as a possible candidate gene for a human nonsyndromic form of deafness.

Acoustic Stimulation↗