RESUMO
Gene targeting in somatic cells represents a potentially powerful method for gene therapy, yet with the exception of pluripotent mouse embryonic stem (ES) cells, homologous recombination has not been reported for a well characterized, non-transformed mammalian cell. Applying a highly efficient strategy for targeting an integral membrane protein--the interferon gamma receptor--in ES cells, we have used homologous recombination to target a non-transformed somatic cell, the mouse myoblast, and to compare targeting efficiencies in these two cell types. Gene-targeted myoblasts display the properties of normal cells including normal morphology, ability to differentiate in vitro, stable diploid karyotype, inability to form colonies in soft agar and lack of tumorigenicity in nude mice.
Assuntos
Terapia Genética/métodos , Músculos/imunologia , Receptores de Interferon/genética , Animais , Sequência de Bases , Técnicas Genéticas , Vetores Genéticos , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Músculos/citologia , Receptores de Interferon/antagonistas & inibidores , Recombinação Genética , Células-Tronco/citologia , Células-Tronco/imunologia , Receptor de Interferon gamaRESUMO
Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild-type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L-selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF-alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P-selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E-selectin in leukocyte rolling in mice.
Assuntos
Moléculas de Adesão Celular/fisiologia , Leucócitos/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Anticorpos Monoclonais/farmacologia , Adesão Celular , Linhagem Celular , Quimiotaxia de Leucócito , Humanos , Selectina L , Selectina-P , Fator de Necrose Tumoral alfa/farmacologia , Vênulas/citologia , Vênulas/efeitos dos fármacosRESUMO
The dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) gene encodes a protein kinase known to play a critical role in neurodevelopment. Mice with one functional copy of Dyrk1A (Dyrk1A(+/-)) display a marked hypoactivity and altered gait dynamics in basal conditions and in novel environments. Dopamine (DA) is a key neurotransmitter in motor behavior and genetic deletion of certain genes directly related to the dopaminergic system has a strong impact on motor activity. We have studied the effects of reduced Dyrk1A expression on the function of the nigrostriatal dopaminergic system. To characterize the dopaminergic system in DYRK1A(+/-) mice, we have used behavioral, pharmacological, histological, neurochemical and neuroimaging (microPET) techniques in a multidisciplinary approach. Dyrk1A(+/-) mice exhibited decreased striatal DA levels, reduced number of DA neurons in the substantia nigra pars compacta, as well as altered behavioral responses to dopaminergic agents. Moreover, microdialysis experiments revealed attenuated striatal DA release and positron emission tomography scan display reduced forebrain activation when challenged with amphetamine, in Dyrk1A(+/-) compared with wild-type mice. These data indicate that Dyrk1A is essential for a proper function of nigrostriatal dopaminergic neurons and suggest that Dyrk1A(+/-) mice can be used to study the pathogenesis of motor disorders involving dopaminergic dysfunction.
Assuntos
Dopamina/metabolismo , Atividade Motora/fisiologia , Neostriado/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Substância Negra/enzimologia , Animais , Feminino , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Vias Neurais/metabolismo , Tomografia por Emissão de Pósitrons , Prosencéfalo/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Quinases DyrkRESUMO
Pendred syndrome is an autosomal-recessive disorder characterized by congenital sensorineural hearing loss combined with goiter. This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS/SLC26A4) has been mapped to chromosome 7q22-q31 and encodes a chloride-iodide transport protein. Mutations in this gene are also a cause of non-syndromic autosomal recessive hearing impairment (DFNB4). We have analyzed the PDS/SLC26A4 gene in Spanish and Italian families and we have detected five new mutations (X871M, T132I, IVS1-2A>G, Y556H and 406del5).
Assuntos
Proteínas de Transporte/genética , Transtornos da Audição/genética , Proteínas de Membrana Transportadoras , Alelos , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Genótipo , Bócio/complicações , Bócio/genética , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/genética , Humanos , Itália , Mutação , Fenótipo , Espanha , Transportadores de Sulfato , SíndromeRESUMO
Pendred syndrome is an autosomal-recessive disorder characterized by congenital sensorineural hearing loss combined with goiter. This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS/SLC26A4) has been mapped to chromosome 7q22-q31 and encodes a chloride-iodide transport protein. Mutations in this gene are also a cause of non-syndromic autosomal recessive hearing impairment (DFNB4). We have analyzed the PDS/SLC26A4 gene in Spanish and Italian families and we have detected five novel mutations (X781W, T132I, IVS2-2A>G, Y556H and 406del5).
Assuntos
Proteínas de Transporte/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana Transportadoras , Alelos , Processamento Alternativo/genética , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Genótipo , Bócio/complicações , Bócio/genética , Perda Auditiva Neurossensorial/complicações , Humanos , Itália , Região do Mediterrâneo , Mutação , Mutação de Sentido Incorreto , Fenótipo , Deleção de Sequência , Espanha , Transportadores de Sulfato , SíndromeRESUMO
Connexin31 (GJB3) has been associated with hearing impairment and erythrokeratodermia variabilis. We have analyzed this gene in samples from patients with peripheral neuropathies, deafness and controls and have found several single nucleotide polymorphisms (SNPs). In the noncoding exon 1 of GJB3 two small deletions, 581del2 and 632del4 (GenBank accession number AF052692), were found at frequencies of 30% and 14%, respectively. In exon 2 we found two amino acid changes, R32W (1227C-T) and V200I (1731G-A), and three nucleotide variants not affecting the amino acid sequence, 1610G-A, 1700C-T and 1931C-T. Most of these changes were found at similar frequencies in patients with deafness, patients with peripheral neuropathies and control subjects. V200I, 1700C-T and 1610G-A were found associated in three unrelated patients with deafness and in a fourth patient with peripheral neuropathy, but were not detected in control subjects.
Assuntos
Substituição de Aminoácidos/genética , Conexinas/genética , Polimorfismo de Nucleotídeo Único/genética , Éxons/genética , Humanos , Regiões não Traduzidas/genéticaRESUMO
By Alu-splice PCR we have trapped two exons and subsequently identified the full length cDNA of a human gene, Intersectin (ITSN), which maps to chromosome 21q22.1 between markers D21S320 and D21S325. The gene has the potential to code for at least two different protein isoforms by alternative splicing (ITSN-L and ITSN-S). Intersectin exists with a high degree of similarity in flies, frogs and mammals, suggesting a conserved role in higher eukaryotes. Analysis of the expression pattern of human and mouse Intersectin detected mRNAs in all adult and foetal tissues tested, with the longer isoform present in brain. In situ hybridisation studies in the developing mouse brain showed ITSN expression in both proliferating and differentiating neurons. The genomic structure of ITSN was determined using the chromosome 21 sequences deposited in the public databases. The protein contains several known motifs which implicate ITSN in clathrin mediated endocytosis and synaptic vesicle recycling. The expression pattern of Intersectin in mouse brain, its presumed function and its overexpression in brains from Down syndrome patients, suggest that Intersectin may contribute in a gene dosage-dependent manner to some of the abnormalities of Down syndrome.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Elementos Alu/genética , Proteínas de Transporte/genética , Síndrome de Down/genética , Síndrome de Down/metabolismo , Neurônios/metabolismo , Aneuploidia , Animais , Northern Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular , Cromossomos Humanos Par 21 , Clonagem Molecular , DNA Complementar/análise , Éxons , Etiquetas de Sequências Expressas , Dosagem de Genes , Humanos , Hibridização In Situ , Íntrons , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
The availability of the DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS). Trisomy 21, full or partial, is a major cause of mental retardation and other phenotypic abnormalities, collectively known as Down syndrome (DS), a disorder affecting 1 in 700 births. The identification of genes on HSA21 and the elucidation of the function of the proteins encoded by these genes have been a major challenge for the human genome project and for research in DS. Over 100 of the estimated 300-500 genes of HSA21 have been identified, but the function of most remains largely unknown. It is believed that the overexpression of an unknown number of HSA21 genes is directly or indirectly responsible for the mental retardation and the other clinical features of DS. For this reason, HSA21 genes that are expressed in tissues affected in DS patients are of special interest.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Síndrome de Down/genética , Genoma Humano , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Endopeptidases , Genômica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Musculares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Quinases DyrkRESUMO
In the brain, programmed cell death (PCD) serves to adjust the numbers of the different types of neurons during development, and its pathological reactivation in the adult leads to neurodegeneration. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in neural proliferation and cell death, and its role during brain growth is evolutionarily conserved. Human DYRK1A lies in the Down syndrome critical region on chromosome 21, and heterozygous mutations in the gene cause microcephaly and neurological dysfunction. The mouse model for DYRK1A haploinsufficiency (the Dyrk1a(+/-) mouse) presents neuronal deficits in specific regions of the adult brain, including the substantia nigra (SN), although the mechanisms underlying these pathogenic effects remain unclear. Here we study the effect of DYRK1A copy number variation on dopaminergic cell homeostasis. We show that mesencephalic DA (mDA) neurons are generated in the embryo at normal rates in the Dyrk1a haploinsufficient model and in a model (the mBACtgDyrk1a mouse) that carries three copies of Dyrk1a. We also show that the number of mDA cells diminishes in postnatal Dyrk1a(+/-) mice and increases in mBACtgDyrk1a mice due to an abnormal activity of the mitochondrial caspase9 (Casp9)-dependent apoptotic pathway during the main wave of PCD that affects these neurons. In addition, we show that the cell death induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that activates Casp9-dependent apoptosis in mDA neurons, is attenuated in adult mBACtgDyrk1a mice, leading to an increased survival of SN DA neurons 21 days after MPTP intoxication. Finally, we present data indicating that Dyrk1a phosphorylation of Casp9 at the Thr125 residue is the mechanism by which this kinase hinders both physiological and pathological PCD in mDA neurons. These data provide new insight into the mechanisms that control cell death in brain DA neurons and they show that deregulation of developmental apoptosis may contribute to the phenotype of patients with imbalanced DYRK1A gene dosage.
Assuntos
Apoptose , Neurônios Dopaminérgicos/metabolismo , Intoxicação por MPTP/metabolismo , Mesencéfalo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Caspase 9/genética , Caspase 9/metabolismo , Sobrevivência Celular/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Humanos , Intoxicação por MPTP/genética , Intoxicação por MPTP/patologia , Mesencéfalo/patologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Quinases DyrkRESUMO
To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-ß42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/genética , Biomarcadores/sangue , Marcadores Genéticos/genética , Proteínas Serina-Treonina Quinases/sangue , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/sangue , Proteínas Tirosina Quinases/genética , Idoso , Doença de Alzheimer/diagnóstico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Estudos de Associação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Valor Preditivo dos Testes , Quinases DyrkRESUMO
The gene encoding the dual-specificity tyrosine-regulated kinase DYRK1A maps to the chromosomal segment HSA21q22.2, which lies within the Down syndrome critical region. The reduction in brain size and behavioral defects observed in mice lacking one copy of the murine homologue Dyrk1A (Dyrk1A+/-) support the idea that this kinase may be involved in monosomy 21 associated mental retardation. However, the structural basis of these behavioral defects remains unclear. In the present work, we have analyzed the microstructure of cortical circuitry in the Dyrk1A+/- mouse and control littermates by intracellular injection of Lucifer Yellow in fixed cortical tissue. We found that labeled pyramidal cells were considerably smaller, less branched and less spinous in the cortex of Dyrk1A+/- mice than in control littermates. These results suggest that Dyrk1A influences the size and complexity of pyramidal cells, and thus their capability to integrate information.
Assuntos
Neocórtex/anormalidades , Neocórtex/patologia , Malformações do Sistema Nervoso/genética , Proteínas Serina-Treonina Quinases/genética , Células Piramidais/patologia , Animais , Diferenciação Celular/genética , Forma Celular/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/patologia , Síndrome de Down/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/metabolismo , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Fenótipo , Proteínas Tirosina Quinases , Células Piramidais/metabolismo , Quinases DyrkRESUMO
We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.
Assuntos
Astrócitos/enzimologia , Cerebelo/enzimologia , Isoenzimas/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Western Blotting , Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Imuno-Histoquímica , Isoenzimas/análise , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase/análise , Ratos , Ratos Sprague-DawleyRESUMO
L-selectin, a cell adhesion molecule expressed by leukocytes, mediates the attachment of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes and mediates the earliest interactions between leukocytes and activated vascular endothelium. Mice possessing a mutant L-selectin gene that results in the complete loss of cell surface receptor expression were generated by gene targeting. Lymphocytes from these mice did not bind to peripheral lymph node HEV and these mice had a severe reduction in the number of lymphocytes localized to peripheral lymph nodes. Short-term homing experiments demonstrated that L-selectin was also involved in lymphocyte migration to mucosal lymph nodes, Peyer's patches, and spleen. Furthermore, significant defects in leukocyte rolling and neutrophil migration into the peritoneum in response to an inflammatory stimulus were observed. Thus, L-selectin plays an essential role in leukocyte homing to lymphoid tissues and sites of inflammation.
Assuntos
Moléculas de Adesão Celular/fisiologia , Leucócitos/fisiologia , Linfócitos/fisiologia , Receptores de Retorno de Linfócitos/fisiologia , Animais , Moléculas de Adesão Celular/genética , Movimento Celular/fisiologia , Marcação de Genes , Técnicas In Vitro , Selectina L , Leucócitos/patologia , Linfonodos/patologia , Linfócitos/patologia , Camundongos , Camundongos Mutantes , Mutação , Neutrófilos/fisiologia , Receptores de Retorno de Linfócitos/genética , Mapeamento por RestriçãoRESUMO
Mutations in the GJB2 gene have been identified in many patients with childhood deafness, 35delG being the most common mutation in Caucasoid populations. We have analyzed a total of 576 families/unrelated patients with recessive or sporadic deafness from Italy and Spain, 193 of them being referred as autosomal recessive, and the other 383 as apparently sporadic cases (singletons). Of the 1,152 unrelated GJB2 chromosomes analyzed from these patients, 37% had GJB2 mutations. Twenty-three different mutations were detected (1 in-frame deletion, 4 nonsense, 5 frameshift, and 13 missense mutations). Mutation 35delG was the most common, accounting for 82% of all GJB2 deafness alleles. The relative frequency of 35delG in Italy and Spain was different, representing 88% of the alleles in Italian patients and only 55% in the Spanish cases. Eight non-35delG mutations were detected more than once (V37I, E47X, 167delT, L90P, 312de114, 334delAA, R143W, and R184P), with relative frequencies ranging between 0.5 and 1.6% of the GJB2 deafness alleles. The information based on conservation of amino acid residues, coexistence with a second GJB2 mutation or absence of the mutation in non-deaf control subjects, suggests that most of these missense changes should be responsible for the deafness phenotype.
Assuntos
Conexinas/genética , Perda Auditiva Neurossensorial/genética , Mutação , Alelos , Criança , Conexina 26 , Mutação da Fase de Leitura , Deleção de Genes , Genes Recessivos , Perda Auditiva Neurossensorial/congênito , Humanos , Mutação de Sentido IncorretoRESUMO
Pendred syndrome is a recessive inherited disorder that consists of developmental abnormalities of the cochlea, sensorineural hearing loss, and diffuse thyroid enlargement (goiter). This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS) has been mapped to chromosome 7q22-q31, and encodes a chloride-iodide transport protein. We performed mutation analysis of individual exons of the PDS gene in one Spanish family that shows intrafamilial variability of the deafness phenotype (two patients with profound and one with moderate-severe deafness). We identified a new splice-site mutation affecting intron 4 of the PDS gene, at nucleotide position 639+7. RNA analysis from lymphocytes of the affected patients showed that mutation 639+7A-->G generates a new donor splice site, leading to an mRNA with an insertion of six nucleotides from intron 4 of PDS. Since the newly created donor splice site is likely to compete with the normal one, variations of the levels of normal and aberrant transcripts of the PDS gene in the cochlea may explain the variability in the deafness presentation.
Assuntos
Proteínas de Transporte/genética , Bócio/genética , Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana Transportadoras , Mutação , Sequência de Aminoácidos , Audiometria , Sequência de Bases , Cóclea/anormalidades , DNA/genética , Feminino , Genes Recessivos , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Masculino , Linhagem , Fenótipo , Splicing de RNA/genética , Transportadores de Sulfato , SíndromeRESUMO
Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snell's waltzer (sv) mouse. The corresponding human gene, MYO6, is located on chromosome 6q13. We describe the mapping of a new deafness locus, DFNA22, on chromosome 6q13 in a family affected by a nonsyndromic dominant form of deafness (NSAD), and the subsequent identification of a missense mutation in the MYO6 gene in all members of the family with hearing loss.
Assuntos
Cromossomos Humanos Par 6 , Surdez/genética , Cadeias Pesadas de Miosina/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Cadeias Pesadas de Miosina/química , Linhagem , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Mutations in the connexin 31 (GJB3) gene have been found in subjects with dominant and recessive deafness and in patients with erythrokeratodermia variabilis. We report here a dominant mutation in the GJB3 gene (D66del) in a family affected with peripheral neuropathy and sensorineural hearing impairment. A wide range of disease severity for peripheral neuropathy, from asymptomatic cases to subjects with chronic skin ulcers in their feet and osteomyelitis leading to amputations, was detected in D66del patients. Mild, often asymmetrical, hearing impairment was found in all but one patient with mutation D66del of this family and the same mutation was present in an independent family ascertained because of hearing impairment. We have found mouse connexin 31 (Gjb3) gene expression in the cochlea and in the auditory and sciatic nerves, showing a pattern similar to that of Gjb1 (connexin 32), of which the human ortholog (GJB1) is involved in X-linked peripheral neuropathy. This expression pattern, together with auditory-evoked brainstem anomalous response in D66del patients, indicates that hearing impairment due to GJB3 mutations involves alterations in both the cochlea and the auditory nerve. Peripheral neuropathy is the third phenotypic alteration linked to GJB3 mutations, which enlarges the list of genes that cause this group of heterogeneous disorders.