RESUMO
BACKGROUND: There is a lack of information on prevalence, cause and consequences of slight/mild bilateral sensorineural hearing loss (SNHL) in children. We report the first systematic genetic analysis of the GJB2 gene in a population-derived sample of children with slight/mild bilateral SNHL. METHODS: Hearing tests were conducted in 6240 Australian elementary school children in Grades 1 and 5. 55 children (0.88%) were found to have a slight/mild sensorineural hearing loss. 48 children with slight/mild sensorineural hearing loss and a matched group of 90 children with normal hearing participated in a genetic study investigating mutations in the GJB2 gene, coding for connexin 26, and the presence of the del(GJB6-D13S1830) and del(GJB6-D13S1854) deletions in the GJB6 gene, coding for connexin 30. RESULTS: Four of 48 children with slight/mild sensorineural hearing loss were homozygous for the GJB2 V37I change. The four children with homozygous V37I mutations were all of Asian background and analysis of SNPs in or near the GJB2 gene suggests that the V37I mutation arose from a single mutational event in the Asian population. DISCUSSION: Based on the prevalence of carriers of this change we conclude that V37I can be a causative mutation that is often associated with slight/mild sensorineural hearing loss. No other children in the slight/mild hearing loss group had a hearing loss related to a GJB2 mutation. One child with normal hearing was homozygous for the R127H change and we conclude that this change does not cause hearing loss. Two children of Asian background were carriers of the V37I mutation. Our data indicate that slight/mild sensorineural hearing loss due to the GJB2 V37I mutation is common in people of Asian background.
Assuntos
Conexinas/genética , Perda Auditiva Bilateral/genética , Perda Auditiva Neurossensorial/genética , Mutação , Alelos , Austrália , Criança , Conexina 26 , Análise Mutacional de DNA , Feminino , Testes Genéticos , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Instituições AcadêmicasAssuntos
Conexinas/genética , Perda Auditiva/genética , Mutação , Deleção de Sequência , Austrália/epidemiologia , Sequência de Bases/genética , Bélgica/epidemiologia , Brasil/epidemiologia , Conexina 26 , Conexina 30 , Análise Mutacional de DNA , Efeito Fundador , França/epidemiologia , Frequência do Gene , Haplótipos , Perda Auditiva/epidemiologia , Humanos , Israel/epidemiologia , Itália/epidemiologia , Espanha/epidemiologia , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is a complex condition with high heritability. However, both biochemical investigations and association and linkage studies have failed to define fully the underlying genetic factors associated with ADHD. We have identified a family co-segregating an early onset behavioural/developmental condition, with features of ADHD and intellectual disability, with a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21). METHODS: We hypothesised that the inversion breakpoints affect a gene or genes that cause the observed phenotype. Large genomic clones (P1 derived/yeast/bacterial artificial chromosomes) were assembled into contigs across the two inversion breakpoints using molecular and bioinformatic technologies. Restriction fragments crossing the junctions were identified by Southern analysis and these fragments were amplified using inverse PCR. RESULTS: The amplification products were subsequently sequenced to reveal that the breakpoints lay within an intron of the dedicator of cytokinesis 3 (DOCK3) gene at the p arm breakpoint, and an intron of a novel member of the solute carrier family 9 (sodium/hydrogen exchanger) isoform 9 (SLC9A9) at the q arm. Both genes are expressed in the brain, but neither of the genes has previously been implicated in developmental or behavioural disorders. CONCLUSION: These two disrupted genes are candidates for involvement in the pathway leading to the neuropsychological condition in this family.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas de Transporte , Inversão Cromossômica , Cromossomos Humanos Par 3 , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina , Proteínas do Tecido Nervoso , Trocadores de Sódio-Hidrogênio/genética , Adulto , Animais , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Encéfalo/metabolismo , Linhagem Celular , Criança , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/metabolismo , Humanos , Camundongos , Fenótipo , Trocadores de Sódio-Hidrogênio/metabolismoAssuntos
Inversão Cromossômica , Cromossomos Humanos Par 3/genética , Deficiências do Desenvolvimento/genética , Transtornos Mentais/genética , Adulto , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Criança , Deficiências do Desenvolvimento/psicologia , Feminino , Humanos , Cariotipagem , Masculino , Transtornos Mentais/psicologiaRESUMO
OBJECTIVE: To determine (1) the prevalence and nature of connexin 26 mutations in a cohort of Australian children with non-syndromic hearing loss, and (2) the carrier frequency of the common connexin 26 mutation (35delG) in the general population. DESIGN: A cohort, case-finding study. Mutation analysis was performed on DNA extracted from white blood cells, buccal cells, or Guthrie blood spots. SETTING: A hearing loss investigation clinic and a deafness centre in two Australian capital cities, 1 January 1998 to 31 October 2000. PARTICIPANTS: (1) 243 children (age range, 4 weeks to 16 years; median, 4 years), attending hearing loss clinics in Sydney and Melbourne; (2) 1000 blood samples obtained from anonymous Guthrie card blood spots collected in 1984 [corrected] by the Victorian Clinical Genetics Service as part of the newborn screening program. MAIN OUTCOME MEASURES: (1) The prevalence and types of connexin 26 mutations in a cohort of children with prelingual deafness; (2) the carrier frequency of the common connexin 26 mutation, 35delG, in the general population. RESULTS: Connexin 26 mutations were identified and characterised in 52 (21%) of the 243 children; 14 different mutations, including four previously unreported mutations (135S, C53R, T123N and R127C), were identified. The common 35delG mutation was found in 56 of the 104 alleles (ie, 86 of the connexin 26 alleles in which a mutation was positively identified). The mutations V371 and M34T were also relatively common. The carrier frequency of connexin 26 mutations and of the common 35delG connexin 26 mutation in the Victorian population was estimated to be 1 in 54 and 1 in 100, respectively. CONCLUSIONS: Mutations in the connexin 26 gene (especially the 35delG mutation) are a common cause of prelingual hearing loss in Australia.
Assuntos
Conexinas/genética , Perda Auditiva Neurossensorial/genética , Mutação , Adolescente , Austrália/epidemiologia , Criança , Pré-Escolar , Conexina 26 , Genótipo , Perda Auditiva Neurossensorial/classificação , Perda Auditiva Neurossensorial/epidemiologia , Heterozigoto , Humanos , Lactente , Fenótipo , Prevalência , Índice de Gravidade de DoençaRESUMO
Most patients with mitochondrial disorders are diagnosed by finding a respiratory chain enzyme defect or a mutation in the mitochondrial DNA (mtDNA). The provision of accurate genetic counseling and reproductive options to these families is complicated by the unique genetic features of mtDNA that distinguish it from Mendelian genetics. These include maternal inheritance, heteroplasmy, the threshold effect, the mitochondrial bottleneck, tissue variation, and selection. Although we still have much to learn about mtDNA genetics, it is now possible to provide useful guidance to families with an mtDNA mutation or a respiratory chain enzyme defect. We describe a range of current reproductive options that may be considered for prevention of transmission of mtDNA mutations, including the use of donor oocytes, prenatal diagnosis (by chorionic villus sampling or amniocentesis), and preimplantation genetic diagnosis, plus possible future options such as nuclear transfer and cytoplasmic transfer. For common mtDNA mutations associated with mitochondrial cytopathies (such as NARP, Leigh Disease, MELAS, MERRF, Leber's Hereditary Optic Neuropathy, CPEO, Kearns-Sayre syndrome, and Pearson syndrome), we summarize the available data on recurrence risk and discuss the relative advantages and disadvantages of reproductive options.
Assuntos
DNA Mitocondrial/genética , Aconselhamento Genético , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Mutação , Diagnóstico Pré-Natal , Feminino , Humanos , Masculino , GravidezRESUMO
BACKGROUND: Cationic liposomes represent an important gene delivery system due to their low immunogenicity, but are relatively inefficient, with optimisation of DNA-liposome complexes (lipoplexes) for transfection necessary for each cell type of interest. There have been few studies examining optimisation in neuronal cell types or determining how the structure of lipoplexes affects transfection efficiency. METHODS: Four commercially available cationic liposome formulations were used to optimise transfection efficiency in neuronal cells. The DNA to liposome ratio and the amount of DNA used in transfections were varied. Transfection efficiency was determined by the percentage of cells positive for the micro-galactosidase reporter gene product. The structure of lipoplexes was studied using atomic force microscopy. Lipoplexes were characterised further using dynamic light scattering to determine size and fluorescence techniques to show DNA compaction. RESULTS: Optimal transfection conditions were found to differ between immortalised cell lines and primary cells. High transfection efficiencies in immortalised cell lines were achieved predominantly with multivalent cationic liposomes while primary neuronal cells showed optimal transfection efficiency with monovalent cationic liposomes. The structure of lipoplexes was observed with atomic force microscopy and showed globular complexes for multivalent cationic liposomes, while monovalent liposomes gave less compact structures. In support of this finding, high levels of DNA compaction with multivalent liposomes were observed using fluorescence quenching measurements for all DNA to liposome ratios tested. One monovalent liposome showed increasing levels of compaction with increasing liposome amount. Dynamic light scattering showed little change in complex size when the different lipoplexes were studied. CONCLUSIONS: Optimisation of transfection efficiency was different for cell lines and primary neurons. Immortalised cells showed optimal transfection with multivalent liposomes while primary neurons showed optimal transfection with monovalent liposomes. The charge ratio of the monovalent liposome was below one, suggesting a different mechanism of lipoplex binding and uptake in primary neurons. The structure of lipoplexes, as
Assuntos
DNA/genética , Neurônios/metabolismo , Transfecção , Animais , Células CHO , Cátions , Cricetinae , Luz , Lipossomos , Microscopia de Força Atômica , Espalhamento de RadiaçãoRESUMO
Mutations in the human gap junction beta-2 gene (GJB2) that encodes connexin-26 have been shown to cause non-syndromic sensorineural hearing loss (NSSNHL) at the DFNB1 locus on 13q11. Functional and genetic data regarding the disease causing potential of one particular GJB2 sequence variant, 101 T-->C (M34T), have proven contradictory. In this study, we found the prevalence of the M34T allele in a cohort of white sib pairs and sporadic cases with NSSNHL from the United Kingdom and Ireland to be 3.179% of chromosomes screened. Significantly, we identified the first M34T/M34T genotype cosegregating in a single family with mid to high frequency NSSNHL. Screening a control population of 630 subjects we identified 25 M34T heterozygotes; however, no M34T homozygotes were detected. Surprisingly, the majority of M34T alleles (88%) were in cis with a 10 bp deletion in the 5' non-coding sequence. This non-coding deletion was also homozygous in the homozygous M34T subjects. Microsatellite analysis of flanking loci in M34T heterozygotes and controls does not define an extensive ancestral haplotype but preliminary data suggest two common alleles in subjects with the M34T allele. In summary, we provide data that support M34T acting as a recessive GJB2 allele associated with mild-moderate prelingual hearing impairment.
Assuntos
Conexinas/genética , Perda Auditiva Neurossensorial/genética , Alelos , Substituição de Aminoácidos , Sequência de Bases , Segregação de Cromossomos , Conexina 26 , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Frequência do Gene , Testes Genéticos , Variação Genética , Genótipo , Perda Auditiva Neurossensorial/diagnóstico , Homozigoto , Humanos , Masculino , Mutação , Linhagem , Deleção de SequênciaRESUMO
Prenatal diagnosis of mitochondrial DNA (mtDNA) mutations is technically possible, but has only rarely been attempted. This is largely because of uncertainty about the effects of mtDNA heteroplasmy, the mtDNA bottleneck, random segregation or selection of mtDNA species, and difficulty in correlating a particular mtDNA mutant load with clinical outcome. We have investigated the feasibility of prenatal diagnosis for two common mtDNA mutations at nucleotide (nt)8993 by determining mtDNA mutant loads in human oocytes and by reviewing data on 56 pedigrees with these mutations, and by reviewing six studies on mtDNA mutations in human fetuses. Data from heteroplasmic human and mouse oocytes demonstrate that the bottleneck occurs in early oogenesis. Analysis of mutant loads of the nt8993 mutations in fetal and adult tissues confirms that there is no substantial tissue variation, implying that the mutant load in a prenatal sample will represent the mutant load in other fetal tissues. The two nucleotide 8993 mutations each show a strong correlation between mutant load and symptom severity and between maternal blood mutant load and risk of a severe outcome. We generated empirical data for calculating recurrence risk and predicting the clinical outcome of a given mutant load. These predictive data can be used (cautiously) for genetic counselling and prenatal diagnosis of nucleotide 8993 mutations.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Mutação Puntual , Diagnóstico Pré-Natal/métodos , Adulto , Criança , Análise Mutacional de DNA/métodos , DNA Mitocondrial/metabolismo , Feminino , Humanos , Miopatias Mitocondriais/genética , GravidezRESUMO
BACKGROUND: The most common form of nonsyndromic neurosensory autosomal recessive deafness, DFNB1, is caused by mutations in the connexin 26 gene (GJB2) on chromosome 13. One mutation, in which one guanosine (G) residue is deleted from a run of 6 Gs (35delG), is found in 40% to 70% of DFNB1 cases and has an expected population frequency of one in 40 to one in 100. METHODS AND RESULTS: Polymerase chain reaction (PCR)-based tests for the 35delG mutation were developed. They are based on mismatched PCR primers that produce novel EcoRII or DdeI restriction enzyme sites depending on the number of Gs at the 35delG locus. An EcoRII site is generated in the wild-type sequence (6 Gs), but not when the 35delG mutation is present. Alternatively, a DdeI site can be generated so that this enzyme cuts the PCR product when the 35delG mutation is present, but not the wild-type sequence. CONCLUSIONS: These tests enable a quick and reliable screen for the common 35delG mutation.
Assuntos
Conexinas/genética , Surdez/genética , Mutação , Reação em Cadeia da Polimerase/métodos , Criança , Pré-Escolar , Conexina 26 , Análise Mutacional de DNA , Testes Genéticos , HumanosRESUMO
Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.
Assuntos
Conexinas/genética , Genes Recessivos , Perda Auditiva de Alta Frequência/genética , Perda Auditiva Neurossensorial/congênito , Mutação , Audiometria , Austrália , Criança , Conexina 26 , Frequência do Gene , Genótipo , HumanosRESUMO
Mutations in the arylsulfatase E gene, located on the X chromosome, have been shown to cause chondrodysplasia punctata (CDP). A substitution of arginine with serine at amino acid 12 (R12S) was identified in a patient with typical features of mild symmetrical CDP including mild mental retardation. The proband was institutionalized and was found to have seven full and half siblings all of whom were microcephalic. Six siblings are alive and all are mentally retarded. The mother is borderline retarded. The mother and three daughters are carriers of the R12S change, but do not appear to have CDP. A son and three other daughters do not carry the R12S change. Further studies revealed that the mother had phenylketonuria (PKU) and the children maternal PKU. This suggests that the R12S change is not the primary cause of short stature, microcephaly, and mental retardation in this family. The relationship between CDP and PKU, both of which can cause short statue and mental retardation, is discussed.
Assuntos
Arilsulfatases/genética , Condrodisplasia Punctata/complicações , Fenilcetonúrias/diagnóstico , Substituição de Aminoácidos , Condrodisplasia Punctata/genética , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fenilcetonúrias/complicações , Fenilcetonúrias/genética , Reação em Cadeia da PolimeraseRESUMO
Two pathogenic mitochondrial DNA mutations, a T-to-G substitution (8993T > G) and a T-to-C substitution (8993T > C), at nucleotide 8993 have been reported. We describe 13 pedigrees with mitochondrial DNA mutations at nucleotide 8993; 10 pedigrees with the 8993T > G mutation and three with the 8993T > C mutation. Prenatal diagnosis of the nucleotide 8993 mutations is technically possible. However, there are three major concerns: (i) that there is variation in mutant loads among tissues; (ii) that the mutant load in a tissue may change over time; and (iii) that the genotype-phenotype correlation is not clearly understood. We have used the 13 pedigrees to determine specifically the extent of tissue- and age-related variation of the two mutations at nucleotide 8993 in the mitochondrial DNA. The tissue variation was investigated by analysing two or more different tissues from a total of 18 individuals. The age-related variation of the mutation was investigated by comparing the amount of both mutations in blood taken at birth and at a later age. No substantial tissue variation was found, nor was there any substantial change in the proportion of either mutation over periods of 8-23 years in the four individuals studied. In addition, we noted that two features were remarkably common in families with nucleotide 8993 mutations, namely (i) unexplained infant death (8 cases in 13 pedigrees); and (ii) de novo mutations (5 of the 10 8993T > G pedigrees).
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Mutação/fisiologia , Nucleotídeos/genética , Linhagem Celular , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Especificidade de Órgãos , Linhagem , Reação em Cadeia da PolimeraseRESUMO
Mitochondrial genetics is complicated by heteroplasmy, or mutant load, which may be from 1%-99%, and thus may produce a gene dosage-type effect. Limited data are available for genotype/phenotype correlations in disorders caused by mtDNA mutations; therefore, prenatal diagnosis for mtDNA mutations has been hindered by an inability to predict accurately the clinical severity expected from a mutant load measured in fetal tissue. After reviewing 44 published and 12 unpublished pedigrees, we considered the possibility of prenatal diagnosis for two common mtDNA mutations at nucleotide 8993. We related the severity of symptoms to the mutant load and predicted the clinical outcome of a given mutant load. We also used the available data to generate empirical recurrence risks for genetic counseling, which may be used in conjunction with prenatal diagnosis.
Assuntos
DNA Mitocondrial/genética , Dosagem de Genes , Aconselhamento Genético , Mutação , Diagnóstico Pré-Natal , Adenosina Trifosfatases/genética , Criança , Feminino , Genótipo , Humanos , Modelos Logísticos , Masculino , Dados de Sequência Molecular , Mães , Linhagem , Fenótipo , Prognóstico , Medição de RiscoRESUMO
Mutations in the connexin26 gene are the basis of much autosomal recessive sensorineural deafness. There is a high frequency of mutant alleles, largely accounted for by one common mutation, 35delG. We have studied a group of families, who had been brought together through marriages between Deaf persons, in which there are more than 30 Deaf people in four generations. We show that many of the several cases of deafness are the result of 35delG homozygosity or 35delG/Q57X compound heterozygosity at the connexin26 locus. A considerable range of audiographic phenotypes was observed. The combined effects of a high population frequency of mutant alleles, and of positive assortative marriage among the Deaf, led to an infrequently observed recessive pedigree pattern.
Assuntos
Conexinas/genética , Surdez/genética , Genes Recessivos , Alelos , Conexina 26 , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , LinhagemRESUMO
OBJECTIVE: To define the spectrum of clinical and biochemical features in 51 children with isolated complex I deficiency. BACKGROUND: Mitochondrial respiratory chain defects are one of the most commonly diagnosed inborn errors of metabolism. Until recently there have been technical problems with the diagnosis of respiratory chain complex I defects, and there is a lack of information about this underreported cause of respiratory chain dysfunction. METHODS: A retrospective review of clinical features and laboratory findings was undertaken in all diagnosed patients who had samples referred over a 22-year period. RESULTS: Presentations were heterogeneous, ranging from severe multisystem disease with neonatal death to isolated myopathy. Classic indicators of respiratory chain disease were not present in 16 of 42 patients in whom blood lactate levels were normal on at least one occasion, and in 23 of 37 patients in whom muscle morphology was normal or nonspecific. Ragged red fibers were present in only five patients. Tissue specificity was observed in 19 of 41 patients in whom multiple tissues were examined, thus the diagnosis may be missed if the affected tissue is not analyzed. Nine patients had only skin fibroblasts available, the diagnosis being based on enzyme assay and functional tests. Modes of inheritance include autosomal recessive (suggested in five consanguineous families), maternal (mitochondrial DNA point mutations in eight patients), and possibly X-linked (slight male predominance of 30:21). Recurrence risk was estimated as 20 to 25%. CONCLUSION: Heterogeneous clinical features, tissue specificity, and absence of lactic acidosis or abnormal mitochondrial morphology in many patients have resulted in underdiagnosis of respiratory chain complex I deficiency.
Assuntos
Transporte de Elétrons , Miopatias Mitocondriais/diagnóstico , NADH NADPH Oxirredutases/deficiência , Adolescente , Criança , Pré-Escolar , Feminino , Fibroblastos/patologia , Humanos , Lactente , Recém-Nascido , Fígado/patologia , Masculino , Mitocôndrias Musculares/patologia , Miopatias Mitocondriais/patologiaRESUMO
Sixteen males and two females with symmetrical (mild) type of chondrodysplasia punctata were tested for mutations in the X chromosome located arylsulphatase D and E genes. We identified one nonsense and two missense mutations in the arylsulphatase E gene in three males. No mutations were detected in the arylsulphatase D gene. Family studies showed segregation of the mutant genes establishing X linked inheritance for these families. Asymptomatic females and males were found in these studies. The clinical presentation varies not only between unrelated affected males, but also between affected males within the same family. We also conclude that clinical diagnosis of chondrodysplasia punctata in adults can be difficult. Finally, our results indicate that brachytelephalangy is not necessarily a feature of X linked symmetrical chondrodysplasia punctata.