RESUMO
We demonstrate here the importance of interleukin signalling pathways in cognitive function and the normal physiology of the CNS. Thorough investigation of an MRX critical region in Xp22.1-21.3 enabled us to identify a new gene expressed in brain that is responsible for a non-specific form of X-linked mental retardation. This gene encodes a 696 amino acid protein that has homology to IL-1 receptor accessory proteins. Non-overlapping deletions and a nonsense mutation in this gene were identified in patients with cognitive impairment only. Its high level of expression in post-natal brain structures involved in the hippocampal memory system suggests a specialized role for this new gene in the physiological processes underlying memory and learning abilities.
Assuntos
Ligação Genética , Hipocampo/metabolismo , Deficiência Intelectual/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Cromossomo X , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , GTP Fosfo-Hidrolases/metabolismo , Deleção de Genes , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Bulbo Olfatório/metabolismo , Linhagem , Transdução de Sinais , Fatores de Tempo , Distribuição TecidualRESUMO
X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.
Assuntos
Encéfalo/metabolismo , Cromossomos Humanos Par 2 , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso/genética , Translocação Genética , Cromossomo X , Sequência de Aminoácidos , Sequência de Bases , Córtex Cerebral/metabolismo , Criança , Mapeamento Cromossômico , Éxons , Feminino , Hipocampo/metabolismo , Humanos , Cariotipagem , Masculino , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TetraspaninasRESUMO
INTRODUCTION: Tangier disease (TD) is a rare autosomal recessive disorder characterized by a deficiency or absence of high-density lipoprotein (HDL) caused by mutations in the adenotriphosphate-binding cassette transporter-1 gene (ABCA1). Mutations of ABCA1 lead to a defect in cellular cholesterol removal and to deposition of cholesterol esters throughout the body. OBSERVATION: We report here on the case of a 53-year-old woman with a severe phenotype of TD. The patient had a dizygous twin sister who had only asymptomatic corneal opacities and thrombopenia. CONCLUSION: This family demonstrates the wide intrafamilial phenotype diversity of TD.
Assuntos
Doença de Tangier/genética , Doença de Tangier/patologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Biópsia , Doenças Desmielinizantes/patologia , Eletrocardiografia , Eletroencefalografia , Eletromiografia , Feminino , Hormônios/sangue , Humanos , Lipídeos/sangue , Pessoa de Meia-Idade , Fibras Nervosas/patologia , Fenótipo , Células de Schwann/patologia , Células Receptoras Sensoriais/patologia , Doença de Tangier/líquido cefalorraquidiano , Gêmeos DizigóticosRESUMO
UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Plaquetas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Doença de Tangier/sangue , Transportador 1 de Cassete de Ligação de ATP/sangue , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Plaquetas/patologia , Tamanho Celular , Modelos Animais de Doenças , Retroalimentação Fisiológica , Feminino , Predisposição Genética para Doença , Transplante de Células-Tronco Hematopoéticas , Hemostasia , Humanos , Lipoproteínas HDL/sangue , Masculino , Camundongos Endogâmicos DBA , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Agregação Plaquetária , Doença de Tangier/genética , Doença de Tangier/patologia , Trombose/sangue , Trombose/genética , Tromboxano A2/metabolismo , Fatores de TempoRESUMO
Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in 70-85% of RTT cases. We report here five novel frameshift mutations (named 345delC, 895del202, 989ins18del8, 996insAG and 1124del53) in exon 3 and 4 of the MECP2 gene. To avoid the missing of few small deletions in RTT patients using classical mutation screening approaches, we suggest that screening of the mutations in the MECP2 gene in RTT girls should include at least a large PCR to amplify exon 4 entirely.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Éxons/genética , Proteínas Repressoras , Síndrome de Rett/genética , DNA/química , DNA/genética , Análise Mutacional de DNA , Mutação da Fase de Leitura , Humanos , Proteína 2 de Ligação a Metil-CpGRESUMO
Rett syndrome (RTT) is a neurodevelopmental disorder occurring almost exclusively in females as sporadic cases. Recently, DNA mutations in the MECP2 gene have been detected in approximately 70% of patients with RTT. To explain the sex-limited expression of RTT, it has been suggested that de novo X-linked mutations occur exclusively in male germ cells resulting therefore only in affected daughters. To test this hypothesis, we have analysed 19 families with RTT syndrome due to MECP2 molecular defects. In seven informative families we have found by DHPLC a nucleotide variant which could be used to differentiate between the maternal and the paternal allele. In each subject investigated from these families, we have amplified specifically each allele and sequenced allele-specific PCR products to identify the allele bearing the mutation as well as the parental origin of each X chromosome. This approach allowed us to determine the parental origin of de novo mutations in all informative families. In five cases, the de novo MECP2 mutations have a paternal origin and in the two other cases a maternal origin. In all transitions at CpG, the de novo mutation observed was of paternal origin. The high frequency of male germ-line transmission of the mutation (71% of RTT informative cases) is consistent with a predominant occurrence of the disease in females.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Impressão Genômica , Proteínas Repressoras , Síndrome de Rett/genética , Sequência de Bases , Primers do DNA , Humanos , Proteína 2 de Ligação a Metil-CpGRESUMO
The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metilação de DNA , DNA/genética , Impressão Genômica/genética , Animais , DNA/análise , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Genoma Humano , Humanos , Camundongos , Reação em Cadeia da Polimerase/métodos , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodosRESUMO
X-linked mental retardation is a very common condition that affects approximately 1 in 600 males. Despite recent progress, in most cases the molecular defects underlying this disorder remain unknown. Recently, a study using the candidate gene approach demonstrated the presence of mutations in PAK3 (p21-activating kinase) associated with nonspecific mental retardation. PAK3 is a member of the larger family of PAK genes. PAK proteins have been implicated as critical downstream effectors that link Rho-GTPases to the actin cytoskeleton and to MAP kinase cascades, including the c-Jun amino-terminal kinase (JNK) and p38. We screened 12 MRX pedigrees that map to a large region overlying Xq21-q24. Mutation screening of the whole coding region of the PAK3 gene was performed by using a combination of denaturing gradient gel electrophoresis and direct sequencing. We have identified a novel missense mutation in exon 2 of PAK3 gene (R67C) in MRX47. This confirms the involvement of PAK3 in MRX following the report of a nonsense mutation recently reported in MRX30. In the MRX47 family, all affected males show moderate to severe mental retardation. No seizures, statural growth deficiency, or minor facial or other abnormal physical features were observed. This mutation R67C is located in a conserved polybasic domain (AA 66-68) of the protein that is predicted to play a major role in the GTPases binding and stimulation of Pak activity.
Assuntos
Deficiência Intelectual/genética , Cromossomo X , Sequência de Aminoácidos , Análise Mutacional de DNA , Ligação Genética , Humanos , Masculino , Mutação , Linhagem , Proteínas Serina-Treonina Quinases/genética , Quinases Ativadas por p21RESUMO
We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX). The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library. Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1. OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation. Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C). The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.
Assuntos
Cromossomos Humanos Par 12 , Proteínas do Citoesqueleto , Proteínas Ativadoras de GTPase , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Polimorfismo Genético , Translocação Genética , Cromossomo X , Substituição de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Primers do DNA , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females, with an estimated prevalence of approximately one in 10 000-15 000 female births. Most cases are sporadic, but several reports about familial recurrence support X-linked dominant inheritance with male lethality. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in <25% of RTT cases in this first report. To characterize the spectrum of mutations in the MECP2 gene in RTT patients, we selected 46 typical RTT patients and performed mutation screening by denaturing gradient gel electrophoresis combined with direct sequencing. We identified 30 mutations, accounting for 65% of RTT patients. They include 12 novel mutations (11 located in exon 3 and one in exon 2). Mutations, such as R270X and frameshift deletions in a (CCACC) (n) rich region, have been found with multiple recurrences. Most of the mutations were de novo, except in one family where the non-affected transmitter mother exhibited a bias of X inactivation. Although this study showed that MECP2 mutations account for most cases of typical forms of RTT (65%) and mutations in non-coding regions cannot be excluded for the remaining cases, an alternative hypothesis that takes into account the homogeneous phenotype and exclusive involvement of females, could be the implication in RTT of a putative second X-linked gene.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Mutação , Proteínas Repressoras , Síndrome de Rett/genética , Sequência de Bases , Análise Mutacional de DNA , Eletroforese , Éxons , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Genótipo , Humanos , Proteína 2 de Ligação a Metil-CpG , Modelos Genéticos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Cromossomo XRESUMO
Following the recent discovery that the methyl-CpG binding protein 2 (MECP2) gene located on Xq28 is involved in Rett syndrome (RTT), a wild spectrum of phenotypes, including mental handicap, has been shown to be associated with mutations in MECP2. These findings, with the compelling genetic evidence suggesting the presence in Xq28 of additional genes besides RabGDI1 and FMR2 involved in non-specific X-linked mental retardation (MRX), prompted us to investigate MECP2 in MRX families. Two novel mutations, not found in RTT, were identified. The first mutation, an E137G, was identified in the MRX16 family, and the second, R167W, was identified in a new mental retardation (MR) family shown to be linked to Xq28. In view of these data, we screened MECP2 in a cohort of 185 patients found negative for the expansions across the FRAXA CGG repeat and reported the identification of mutations in four sporadic cases of MR. One of the mutations, A140V, which we found in two patients, has been described previously, whereas the two others, P399L and R453Q, are novel mutations. In addition to the results demonstrating the involvement of MECP2 in MRX, this study shows that the frequency of mutations in MECP2 in the mentally retarded population screened for the fragile X syndrome is comparable to the frequency of the CGG expansions in FMR1. Therefore, implementation of systematic screening of MECP2 in MR patients should result in significant progress in the field of molecular diagnosis and genetic counseling of mental handicap.
Assuntos
Proteínas Cromossômicas não Histona , Ilhas de CpG/genética , Proteínas de Ligação a DNA/genética , Ligação Genética , Deficiência Intelectual/genética , Mutação , Proteínas de Ligação a RNA , Cromossomo X/genética , Adulto , Sequência de Bases , Feminino , Proteína do X Frágil da Deficiência Intelectual , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Linhagem , Proteínas Repressoras/genética , Síndrome de Rett/genética , Fatores SexuaisRESUMO
Non-specific X-linked mental retardation (MRX) is a very common disorder which affects approximately 1 in 600 males. Despite this high frequency, little is known about the molecular defects underlying this disorder, mainly because of the clinical and genetic heterogeneity which is evident from linkage studies. Recently, a collaborative study using the candidate gene approach demonstrated the presence of mutations in GDIalpha, a Rab GDP-dissociation inhibitor encoded by a gene localized in Xq28, associated with non-specific mental retardation. GDIalpha is mainly a brain-specific protein that plays a critical role in the recycling of Rab GTPases involved in membrane vesicular transport. The study presented here was designed to assess the prevalence of mutations in the GDIalpha in mentally retarded patients and to discuss the clinical phenotypes observed in affected individuals. Mutation screening of the whole coding region of the GDIalpha gene, using a combination of denaturing gradient gel electrophoresis and direct sequencing, was carried out in 164 patients found negative for expansions across the FRAXA GCC repeat. In addition to the nonsense mutation recently reported in MRX48, we have identified a novel missense mutation in exon 11 of the GDIalpha gene in one familial form of non-specific mental retardation. In this family (family R), all affected males show moderate to severe mental retardation, and the X-linked semidominant inheritance is strongly suggested by the severe phenotypes in males with respect to mildly affected females or unaffected obligatory carriers. This study showed that the prevalence of GDIalpha mutations in non-specific mental retardation could be estimated to be 0.5-1%, and molecular diagnosis and genetic counselling in some cases of non-specific mental handicap can now be provided.