Biallelic nonsense mutations in the otogelin-like gene (OTOGL) in a child affected by mild to moderate hearing impairment.

Hearing impairment is characterized by great genetic heterogeneity. We report the identification, by whole exome sequencing, of two different nonsense mutations (c.1558C>T; p.Gln520 and c.2773C>T; p.Arg925) in the otogelin-like gene (OTOGL), in a child affected by mild to moderate isolated deafness. Parental genotypes allowed us to conclude that these mutations are present in the compound heterozygous state in the patient. In addition, our clinical data establish that the tectorial membrane and/or the outer hair cells are defective in this form of deafness.

Clinical genetics of Kallmann syndrome.

The Kallmann syndrome (KS) combines hypogonadotropic hypogonadism (HH) with anosmia. This is a clinically and genetically heterogeneous disease. KAL1, encoding the extracellular glycoprotein anosmin-1, is responsible for the X chromosome-linked recessive form of the disease (KAL1). Mutations in FGFR1 or FGF8, encoding fibroblast growth factor receptor-1 and fibroblast growth factor-8, respectively, underlie an autosomal dominant form with incomplete penetrance (KAL2). Mutations in PROKR2 and PROK2, encoding prokineticin receptor-2 and prokineticin-2, have been found in heterozygous, homozygous, and compound heterozygous states. These two genes are likely to be involved both in autosomal recessive monogenic (KAL3) and digenic/oligogenic KS transmission modes. Mutations in any of the above-mentioned KS genes have been found in less than 30% of the KS patients, which indicates that other genes involved in the disease remain to be discovered. Notably, KS may also be part of pleiotropic developmental diseases including CHARGE syndrome; this disease results in most cases from neomutations in CHD7 that encodes a chromodomain helicase DNA-binding protein.

The complex genetics of Kallmann syndrome: KAL1, FGFR1, FGF8, PROKR2, PROK2, et al.

Kallmann syndrome (KS) combines hypogonadotropic hypogonadism and anosmia. Anosmia is related to the absence or hypoplasia of the olfactory bulbs and tracts. Hypogonadism is due to gonadotropin-releasing hormone (GnRH) deficiency, which presumably results from a failure of the embryonic migration of neuroendocrine GnRH cells from the olfactory epithelium to the forebrain. This failure could be a consequence of the early degeneration of olfactory nerve and terminal nerve fibres, because the latter normally act as guiding cues for the migration of GnRH cells. Defects in GnRH cell fate specification, differentiation, axon elongation or axon targeting to the hypothalamus median eminence may, however, also contribute to GnRH deficiency, at least in some genetic forms of the disease. To date, five KS genes have been identified, namely, FGFR1, FGF8, PROKR2, PROK2, and KAL1. Mutations in these genes, however, account for barely 30% of all KS cases. Mutations in FGFR1, encoding fibroblast growth factor receptor 1, underlie an autosomal dominant form of the disease. Mutations in PROKR2 and PROK2, encoding prokineticin receptor-2 and prokineticin-2, have been found in heterozygous, homozygous or compound heterozygous states. These two genes are likely to be involved both in monogenic recessive and digenic or oligogenic KS transmission modes. Finally, KAL1, encoding the extracellular glycoprotein anosmin-1, is responsible for the X chromosome-linked form of the disease. It is believed that anosmin-1 acts as an enhancer of FGF signalling and perhaps of prokineticin signalling too.

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.

Kallmann syndrome: towards molecular pathogenesis.

Gonadotropin Releasing Hormone (GnRH) is a key regulator of reproduction and sexual behaviour. During the last decade, embryological studies have clarified the question of the early development of GnRH-synthesising neurones before the onset of neurosecretion. These studies have revealed the existence of a topographical link between GnRH-synthesising neurones and the embryonic olfactory system, thereby shedding new light on Kallmann syndrome, a developmental disease characterised by the association of hypogonadotropic hypogonadism and anosmia (or hyposmia). Although Kallmann syndrome was identified as an inherited disease in the forties, familial cases of the disease are infrequent. However, the identification, by positional cloning strategies, of the gene underlying the X-chromosome linked form of the disease (KAL-1) has opened the way to molecular pathophysiology. KAL-1 encodes an extracellular glycoprotein of compound modular structure. The protein, named anosmin-1, has been produced in a transfected mammalian cell line and purified. Polyclonal and monoclonal antibodies have been generated, which allowed us to study the distribution of the protein during the period of human organogenesis (4--10 embryonic weeks), by immunohistofluorescence. During this developmental period, anosmin-1 is a locally restricted component of various extracellular matrices (interstitial matrices and basement membranes). Later in embryonic life, KAL-1 expression apparently becomes restricted to definite neuronal populations. Based on the distribution of anosmin-1 in the early olfactory system, the pathogenesis of the olfactory loss and GnRH deficiency in X-linked Kallmann syndrome is discussed.

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.

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.

Pendred syndrome: phenotypic variability in two families carrying the same PDS missense mutation.

Pendred syndrome comprises congenital sensorineural hearing loss, thyroid goiter, and positive perchlorate discharge test. Recently, this autosomal recessive disorder was shown to be caused by mutations in the PDS gene, which encodes an anion transporter called pendrin. Molecular analysis of the PDS gene was performed in two consanguineous large families from Southern Tunisia comprising a total of 23 individuals affected with profound congenital deafness; the same missense mutation, L445W, was identified in all affected individuals. A widened vestibular aqueduct was found in all patients who underwent computed tomography (CT) scan exploration of the inner ear. In contrast, goiter was present in only 11 affected individuals, who interestingly had a normal result of the perchlorate discharge test whenever performed. The present results question the sensitivity of the perchlorate test for the diagnosis of Pendred syndrome and support the use of a molecular analysis of the PDS gene in the assessment of individuals with severe to profound congenital hearing loss associated with inner ear morphological anomaly even in the absence of a thyroid goiter.

[Genetic anomalies of the gonadotropic axis]. / Anomalies génétiques de l'axe gonadotrope.

Some forms of isolated hypogonadotropic hypogonadism are caused by mutations of the GnRH receptor gene. These mutations lead to inactivation of the receptor and are recessive. A unique disease that associates hypogonadotropic hypogonadism and congenital hyperplasia is caused by mutations in the DAX-1 gene, situated on chromosome X. The pathogenesis to these endocrine defects remains now elusive. Kallmann syndrome represents the association of hypogonadotropic hypogonadism due to GnRH deficiency, and anosmia. Additional developmental anomalies can be present. Three modes of inheritance have been described: X-linked, autosomal recessive and autosomal dominant. The X-linked KAL-1 gene has been cloned. It encodes an extracellular matrix protein, anosmin-1, the study of which should lead to a better understanding of this developmental disease.

Anosmin-1 is a regionally restricted component of basement membranes and interstitial matrices during organogenesis: implications for the developmental anomalies of X chromosome-linked Kallmann syndrome.

Kallmann syndrome is a developmental disease characterized by gonadotropin-releasing hormone (GnRH) deficiency and olfactory bulb hypoplasia. The gene underlying the X chromosome-linked form, KAL-1, has been identified for several years, yet the pathogenesis of the disease is not understood. By immunohistofluorescence and immunoelectron microscopy, we establish that the KAL-1 encoded protein, anosmin-1, is a transient and regionally restricted component of extracellular matrices during organogenesis in man. Anosmin-1 was detected in the basement membranes and/or interstitial matrices of various structures including bronchial tubes, mesonephric tubules and duct, branches of the ureteric bud, muscular walls of the digestive tract and larger blood vessels, precartilaginous models of skeletal pieces, muscle tendons, head mesenchymes, inner ear, and forebrain subregions. Our results suggest that this protein acts as a local, rather than a long-range, cue during organogenesis. In the olfactory system, anosmin-1 was detected from week 5 onward. The protein was restricted to the olfactory bulb presumptive region and later, to the primitive olfactory bulbs. We therefore suggest that the genetic defect underlying X-linked Kallmann syndrome disrupts the terminal navigation of the early olfactory axons or directly affects the initial steps of olfactory bulb differentiation. The mechanism of the GnRH deficiency is also discussed, relying on the evidence that anosmin-1 is present in the medial walls of the primitive cerebral hemispheres, along the rostro-caudal migratory pathway of the GnRH-synthesizing neurons, at 6 weeks. Finally, the present results strongly suggest that the renal aplasia observed in about one third of the affected individuals results from primary failure of the collecting duct system.

Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner.

Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.

Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system.

The KAL gene is responsible for the X-chromosome linked form of Kallmann's syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.

Migration of luteinizing hormone-releasing hormone (LHRH) neurons in early human embryos.

Luteinizing hormone-releasing hormone (LHRH) neurons originate in the epithelium of the medial olfactory pit and migrate from the nose into the forebrain along nerve fibers rich in neural cell adhesion molecule (N-CAM). The present study examined the ontogenesis of LHRH neurons in early human embryos and found a similar pattern of development of these cells. Luteinizing hormone-releasing hormone immunoreactivity was detected in the epithelium of the medial olfactory pit and in cells associated with the terminal-vomeronasal nerves at 42 (but not 28-32) days of gestation. The migration route of these cells was examined with antibodies to N-CAM and antibodies to polysialic acid (PSA-N-CAM), which is present on N-CAM at certain stages of development. Neural cell adhesion molecule immunoreactivity was present in a population of cells in the olfactory placode of the earliest embryos examined (28-32 days) and later (42 and 46 days) throughout the migration route. The PSA-N-CAM immunoreactivity was not detected until 42 days and was present in a more limited distribution in nerve fibers streaming from the olfactory placode and along the caudal part of the migration route below the forebrain. Previous studies have indicated that the highly sialated form of N-CAM is less adhesive. The PSA-N-CAM may therefore facilitate the migration of these cells by lessening the adhesion between the fascicles that make up the migration route, expediting the passage of cords of LHRH cells between the nerve fibers as these cells move toward the brain.

The human SOX11 gene: cloning, chromosomal assignment and tissue expression.

The mammalian testis determining gene SRY contains an HMG box-related DNA binding motif. By analogy a family of genes related to SRY in the HMG domain have been called SOX (SRY box-related genes). We have cloned and characterized the human SOX11 gene using the partial cloning of both human and mouse SOX11 genes and mapped it to chromosome 1p25. The SOX11 sequence is strongly conserved with the chicken homologue and is related to SOX4. It contains several putative transcriptional either activator or repressor domains. SOX11 expression pattern is consistent with the hypothesis that this gene is important in the developing nervous system.

A molecular approach to the pathophysiology of the X chromosome-linked Kallmann's syndrome.

The human KAL gene is responsible for the X chromosome-linked Kallmann's syndrome, which consists of an association between hypogonadotropic hypogonadism and anosmia (or hyposmia). Additional symptoms are occasionally observed. The olfactory defect is associated with hypoplasia of the olfactory bulbs and tracts. The hypogonadism may be due to a defect in the embryonic migratory process of GnRH-synthesizing neurones from the olfactory pits up to the brain. The human and chicken KAL genes have been isolated. From the amino acid sequences deduced, it has been postulated that the KAL protein is an extracellular matrix component, with putative antiprotease activity and adhesion function. Various point mutations and, in a few cases, deletions of KAL have been detected in patients. By in situ hybridization, KAL expression has been studied during embryonic development in the chick. From embryonic day 2 (ED2) to ED8, the KAL gene is expressed in various endodermal, mesodermal and ectodermal derivatives, whereas the expression from ED8 is almost entirely restricted to definite neuronal populations in the central nervous system, most of which still express the gene after hatching. According to such a spatiotemporal pattern of expression, we suggest that the KAL gene is involved both in morphogenetic events and in late neuronal differentiation and/or neuronal trophicity. With respect to the olfactory system, the KAL gene is expressed in the mitral cells of the olfactory bulbs from ED8 onwards. In contrast, no expression of the KAL gene is detected at any stage in either the embryonic olfactory epithelium or the surrounding nasal mesenchyme. Therefore, assuming that similar conditions are found in the human embryo, we suggest that the olfactory anomaly in X-linked Kallmann's syndrome results from a central target cell defect. Current hypotheses regarding the pathophysiology of the GnRH deficiency are also discussed. In situ hybridization experiments in the human embryo, as well as characterization of the KAL protein, are in progress.

Early expression of the KAL gene during embryonic development of the chick.

The human KAL gene is responsible for the X chromosome-linked Kallmann syndrome, which consists of the association of hypogonadotropic hypogonadism and anosmia. The human and chicken KAL genes have been isolated. Using in situ hybridization, we studied KAL gene expression during development of the chick. We have previously reported that, from embryonic day 8, the expression is almost restricted to definite neuronal populations in the central nervous system, most of which still express the gene after hatching. Here we report that the KAL gene is also expressed during early embryonic development (days 2-8) in various endodermal, mesodermal, and neurectodermal derivatives. In most endodermal and mesodermal derivatives, the expression is transient and precedes cell differentiation. In contrast, the expression in the nervous system concerns postmitotic central neuroblastic populations, most of which still express the gene after differentiation. In accordance with such a spatio-temporal pattern of expression, we suggest that the KAL gene is involved both in morphogenetic events and in neuronal late differentiation. In addition, the absence of detectable expression of the KAL gene either in the embryonic olfactory epithelium or in the surrounding nasal mesenchyme reinforces the hypothesis that Kallmann's syndrome results from a central olfactory target cell defect.

Analysis of a brain-specific isozyme. Expression and chromatin structure of the rat aldolase C gene and transgenes.

Aldolase C mRNA is detected by Northern blot in all fetal tissues in rat; it is very abundant in the adult brain and undetectable in the other adult tissues. However, reverse transcriptase polymerase chain reaction amplification indicates that this gene is not totally repressed in these tissues. A DNase-I hypersensitivity site located in a 115-base pair proximal promoter fragment is detectable in the brain as well as in other adult tissues. Two MspI/HpaII restriction sites located at -3800 and -450 base pairs are demethylated in the brain and totally or partially methylated in other tissues. In transgenic mice, a 12.5-kilobase genomic fragment is strongly and tissue specifically expressed in different lines, with conservation of a methylation pattern similar to that of the endogenous gene. A chloramphenicol acetyltransferase gene directed by either 800 or 115 base pairs of aldolase C 5'-flanking sequences is tissue specifically expressed in transgenic mice, but the level of expression is very low. This level is greatly increased when the transgene consists of a chloramphenicol acetyltransferase hybrid gene directed by 5.5 kilobases of aldolase C 5'-flanking sequences. We propose therefore that the chromatin structure around the aldolase C promoter is accessible in fetal tissues, then remains open in the adult brain, where the gene is very active, as well as in tissues in which it is practically inactive. The specificity of expression in the brain is conferred by a short 115-base pair proximal promoter fragment that needs more upstream sequences to be fully active.