RESUMO
X-linked Charcot-Marie-Tooth disease (CMT) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene. This gene has nerve specific P2 promoter that work synergistically with SOX10 and EGR2 genes to initiate transcription. Mutation in this region is known to cause Schwann cell dysfunction. A single large family of X linked peripheral neuropathy was identified in our practice. Next generation sequencing for targeted panel assay identified an upstream exon-splicing deletion identified extending from nucleotide c.-5413 to approximately - c.-49. This matches the sequence of 32 nucleotides at positions c.*218-*249 in the 3'UTR downstream of the GJB1 gene. The deleted fragment included the entire P2 promoter region. The deletion segregated with the disease. To our knowledge a deletion of the P2 promoter alone as a cause of CMT has not been reported previously.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Regiões Promotoras Genéticas , Deleção de Sequência , Adolescente , Adulto , Idoso , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Criança , Pré-Escolar , Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Nervo Sural/patologia , Nervo Sural/fisiopatologia , Adulto Jovem , Proteína beta-1 de Junções ComunicantesRESUMO
Mutations in the gene encoding the gap-junction protein connexin 26 (GJB2) on chromosome 13q11 (DFNB1 locus) have been shown as a major contributor to prelingual, non-syndromic, autosomal recessive deafness in Caucasian populations. One specific mutation, 35delG, has accounted for the majority of the mutations detected in the GJB2 gene and is one of the most frequent disease mutations identified to date. We have previously reported a carrier frequency of 35delG of 3.5% in the Greek population, and the 35delG mutation has been detected in one-third of the alleles in Greek patients with sensorineural, prelingual, non-syndromic deafness. The description of this common mutation has opened the way to prenatal diagnosis of prelingual deafness, and we here describe our experience with 29 couples requesting counseling, carrier testing and prenatal diagnosis of DFNB1 deafness.
Assuntos
Conexinas/genética , Surdez/diagnóstico , Surdez/genética , Triagem de Portadores Genéticos , Mutação , Diagnóstico Pré-Natal , Cromossomos Humanos Par 13 , Conexina 26 , Feminino , Frequência do Gene , Grécia , Humanos , GravidezRESUMO
To completely characterize the spectrum of mutations in the cystic fibrosis transmembrane conductance regulator gene in Greek cystic fibrosis (CF) patients, we screened 500 CF chromosomes by denaturing gradient gel electrophoresis followed by direct sequencing. We identified 48 mutations, accounting for 85.6% of CF chromosomes. They included eight novel mutations, three of which we have described before and five (E822X, Y247X, 2752-26A-->G, 3152delT, and 2751 + T-->A), which are described in this report. The detection of such a high proportion of Greek CF mutations is important for improving prenatal and genetic diagnosis of CF in Greece.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Mutação , Adulto , Alelos , Criança , Pré-Escolar , Fibrose Cística/fisiopatologia , Primers do DNA , Éxons , Feminino , Genótipo , Grécia , Haplótipos , Humanos , Íntrons , Masculino , Repetições de Microssatélites , Mutação PuntualRESUMO
The GJB2 (connexin 26) gene, one of the major genes responsible for autosomal recessive deafness, has been investigated previously by a variety of techniques, including PCR-SSCP and sequencing of the entire gene for screening of unknown mutations, and allele-specific PCR, ASO, and PCR-mediated site-directed mutagenesis for the detection of the common mutation 35delG. Here, we present the development of a DGGE method for the characterization of the full spectrum of mutations in the GJB2 gene. The GJB2 cDNA and flanking sequences were amplified in three overlapping segments. We screened 26 Greek patients with prelingual, sensorineural deafness, where syndromic forms and environmental causes of deafness had been excluded. The 35delG mutation was detected in 28 chromosomes (53.8%), while another three sequence variations accounted for 7.6% of the alleles. The sequence variation R127H, previously described in a few Spanish and Balkan patients, was detected in two patients as the sole mutation. A novel sequence variation, K224Q, was identified as the sole mutation in one patient. Use of this approach may contribute to the full description of mutations in this important deafness gene.
Assuntos
Conexinas/genética , Análise Mutacional de DNA , Perda Auditiva Neurossensorial/genética , Mutação , Alelos , Sequência de Bases , Conexina 26 , Primers do DNA/química , Eletroforese em Gel de Ágar , Feminino , Frequência do Gene , Genótipo , Grécia/epidemiologia , Perda Auditiva Neurossensorial/etnologia , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da PolimeraseRESUMO
The pathogenesis of venous thrombosis involves the interaction of genetic and environmental factors. In order to estimate the frequency of the factor V Leiden, the prothrombin G20210A, and the MTHFR C677T mutations in the Greek population, we analyzed 160 healthy Greek blood donors by PCR amplification and detected allele frequencies of 2.5%, 2.2%, and 35.3%, respectively. The allele frequencies were compared with reported frequencies of other populations of southern Europe. The identification of these common genetic risk factors for thrombosis should enable easy DNA diagnosis and carrier detection in a high proportion of cases and will contribute to a better understanding of the interaction of genetic and environmental risk factors.
Assuntos
Doadores de Sangue , Fator V/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Mutação Puntual , Protrombina/genética , Trombose Venosa/genética , Adulto , Alelos , Fator V/isolamento & purificação , Feminino , Frequência do Gene , Triagem de Portadores Genéticos , Grécia , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Reação em Cadeia da Polimerase , Prevalência , Protrombina/isolamento & purificação , Fatores de Risco , Trombose Venosa/etiologiaRESUMO
Congenital bilateral absence of the vas deferens (CBAVD) found in otherwise healthy infertile males, is associated with a high incidence of mutated cystic fibrosis transmembrane conductance regulator (CFTR) alleles, and is considered a genital form of cystic fibrosis (CF). The CF gene may also be involved in the aetiology of male infertility in cases other than CBAVD. The present study was undertaken to test the involvement of CFTR gene mutations in 14 CBAVD males and additionally in cases of male infertility caused by obstructive azoospermia (n = 10) and severe oligozoospermia (n = 3). The entire coding region of the CFTR gene was analysed using denaturing gradient gel electrophoresis (DGGE). The three allele (5T, 7T, 9T) polymorphic tract of thymidines in intron 8 (IVS8-polyT) of which the 5T allele acts as a mild mutation, causing reduced levels of normal CFTR mRNA due to deletion of exon 9, was also analysed. Of the 14 CBAVD cases, four (28.6%) were found to have mutations in both copies of the CFTR gene, six (42.8%) had one CFTR mutation, and in the remaining four (28.6%) no CFTR mutations were found. Of the 10 cases with obstructive azoospermia, three (30%) had one CFTR mutation and in the remaining seven (70%) no mutations were found. None of the three severe oligozoospermia cases carried a CFTR mutation. The frequency of the IVS8(5T) allele was 14.3% (4/28) for the CBAVD cases and 5% (1/20) for the obstructive azoospermia cases, none of the severe oligozoospermia males carried the IVS8-5(5T) allele. The data indicate that while there is a strong association between male infertility caused by CBAVD and mutations in the CFTR gene, cases of obstructive azoospermia without CBAVD also seem to be associated with CFTR gene mutations.
Assuntos
Fibrose Cística/genética , Testes Genéticos , Mutação , Oligospermia/genética , Ducto Deferente/anormalidades , Análise Mutacional de DNA , Humanos , Masculino , Oligospermia/patologiaRESUMO
The presence of maternal cells in fetal samples constitutes a serious potential source for prenatal misdiagnosis. Here we present our approach for detecting maternal cell contamination (MCC) at prenatal diagnosis for eight monogenic disorders (autosomal recessive: beta-thalassaemia, sickle-cell anaemia, cystic fibrosis, prelingual deafness; autosomal dominant: achondroplasia, Huntington disease, myotonic dystrophy, neurofibromatosis type I; X-linked: spinobulbar muscular atrophy). Our aim was to apply a simple and low-cost approach, which would easily and accurately provide information on the fetal tissue MCC status. MCC testing was applied to cases of recessive inheritance where the primary mutation screening of the fetus revealed the presence of the maternal mutation, to cases concerning dominant inheritance and to cases of multiple gestation. The potential presence of maternal cells was determined by the amplification of the 3'-HVR/APO B, D1S80, THO1 and VNTRI of vWf polymorphic loci, which have previously demonstrated high heterozygosity in Caucasians. Among 135 prenatal diagnoses, 44 finally needed to be tested for MCC (32.6%). MCC was detected in four cases, where DNA was isolated directly from chorionic villi samples (CVS), and in one case with DNA isolated directly from amniotic fluid (AF). In almost 90% of cases a simple test of one polymorphic locus provided sufficient information about MCC. The choice of the appropriate locus is therefore essential, while the simultaneous screening of both parents provides the means for distinguishing non-informative sites about MCC.
Assuntos
Artefatos , Amostra da Vilosidade Coriônica , Transtornos Cromossômicos/diagnóstico , Erros de Diagnóstico/prevenção & controle , Doenças Fetais/diagnóstico , Adulto , DNA/análise , Análise Mutacional de DNA , Feminino , Testes Genéticos/métodos , Humanos , Troca Materno-Fetal , Reação em Cadeia da Polimerase , Polimorfismo Genético , Gravidez , Manejo de EspécimesRESUMO
Two novel CFTR mutations were detected in Greek cystic fibrosis patients. One was a missense mutation, A46D, and the other a splice mutation, 296 + 1G-C. Neither was detected on normal chromosomes.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Adulto , Pré-Escolar , Éxons , Feminino , Grécia , Humanos , Masculino , Mutação , Splicing de RNARESUMO
To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (delta F 508, G542X, G551D, 621 + 1 G-->T, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the delta F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4A-->G) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.
Assuntos
Fibrose Cística/genética , Análise Mutacional de DNA , Proteínas de Membrana/genética , Alelos , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística , Eletroforese em Gel de Poliacrilamida , Éxons/genética , Feminino , Grécia , Humanos , Masculino , Dados de Sequência Molecular , Hibridização de Ácido NucleicoRESUMO
Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common.
Assuntos
Análise Mutacional de DNA/métodos , Eletroforese/métodos , Desenvolvimento Embrionário , Genótipo , Diagnóstico Pré-Implantação/métodos , Talassemia beta/genética , Blastômeros , Primers do DNA , Feminino , Humanos , Reação em Cadeia da Polimerase , GravidezRESUMO
Two rare de novo structural aberrations of the Y chromosome were detected during routine prenatal diagnosis: a satellited non-fluorescent Y chromosome (Yqs), the first de novo Yqs to be reported in a fetus, and a terminal deletion of the Y chromosome long arm del(Y)(q11). In both cases detailed cytogenetic and molecular analyses were undertaken. In the case of the Yqs it was demonstrated by fluorescence in situ hybridization (FISH) that the satellites were derived from chromosome 15. In the case of the del(Yq), it was shown with molecular analysis by polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS-PCR) that the deleted portion of the long arm of chromosome Y included the azoospermia factor loci, AZFb and AZFc. The clinical significance of these findings is discussed.
Assuntos
Diagnóstico Pré-Natal , Aberrações dos Cromossomos Sexuais/diagnóstico , Cromossomo Y , Adulto , Deleção Cromossômica , Diagnóstico Diferencial , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Reação em Cadeia da Polimerase , Gravidez , Primeiro Trimestre da Gravidez , Aberrações dos Cromossomos Sexuais/genéticaRESUMO
Mutations in the gene encoding the gap-junction protein connexin 26 (GJB2) on chromosome 13q11 have been shown as a major contributor to prelingual, sensorineural, nonsyndromic deafness. One specific mutation, 35delG, has accounted for the majority of the mutations detected in the GJB2 gene in Caucasian populations and is one of the most frequent disease mutations identified so far with highest carrier frequency of 3,5% in the Greek population. In a collaboration with the major referral centers for childhood deafness in Greece, patients were examined by an extensive questionnaire to exclude syndromic forms and environmental causes of deafness and by allele-specific PCR for the detection of the 35delG mutation. The 35delG mutation was found in 32.1% of the alleles in 173 unrelated cases of prelingual deafness: 50 homozygotes and 11 heterozygotes. Individuals heterozygous for the 35delG mutation were further analyzed by direct genomic sequencing of the coding region of the GJB2 gene, which revealed R184P and 486insT mutations in single alleles. We conclude that the 35delG GJB2 mutation is responsible for one third of prelingual, sensorineural deafness in Greece, which is higher than the usually quoted 20% for Caucasian populations.
Assuntos
Conexinas/genética , Perda Auditiva Neurossensorial/epidemiologia , Perda Auditiva Neurossensorial/genética , Mutação , Audiometria de Tons Puros , Conexina 26 , Análise Mutacional de DNA , Feminino , Testes Genéticos , Genótipo , Grécia/epidemiologia , Humanos , Masculino , Reação em Cadeia da Polimerase , Vigilância da População , Prevalência , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To evaluate hereditary and acquired hemostatic abnormalities associated with recurrent spontaneous early (first-trimester) abortions. METHOD: A group of 56 Greek women with two or more unexplained primary spontaneous abortions, and a reference group of 148 women without a history of recurrent abortions, were screened for hypercoagulability. A randomly selected population of first-trimester pregnant women was also chosen for factor V Leiden genetic screening. RESULTS: A total of 21% of the women with recurrent abortions, compared with 12% of the reference group, showed increased activated protein C resistance. Fourteen per cent had positive lupus anticoagulant, compared with 11.5% of the reference group. For the rest of the parameters, there was no difference between the two groups. Of 22 women studied for factor V Leiden, one was homozygous and one was heterozygous. Results were compared using Fisher's exact test and two-tailed Student's t tests. CONCLUSIONS: Increased activated protein C resistance appears to be an important factor in women with recurrent abortions. These data indicate the need for routine investigation of activated protein C resistance in women with recurrent abortions.