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1.
J Comp Neurol ; 391(4): 444-55, 1998 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-9486824

RESUMO

We have identified a novel receptor-like protein tyrosine phosphatase (RPTPrho) transcript whose expression in the cerebellar cortex is restricted to the granule cell layer of lobules 1-6. Acidic fibroblast growth factor (FGF-1) mRNA follows a similar cerebellar expression pattern. Together, the two markers define a sharp boundary in lobule 6, slightly caudal to the primary fissure. Anterior and posterior compartments became discernible only during postnatal weeks two and six, for RPTPrho and FGF-1, respectively. A rostrocaudal boundary in lobule 6 of the murine cerebellar cortex has also been identified morphologically by the effects of the meander tail mutation. The position of the RPTPrho and FGF-1 boundary on the rostrocaudal axis of the cerebellar cortex was close to, but not coincident with, the caudal extent of the disorganized anterior lobe of meander tail and the rostral extent of Otx-2 expression. The restricted pattern of FGF-1 and RPTPrho implies that these molecules may have specific signaling roles in the tyrosine phosphorylation/dephosphorylation pathway in the anterior compartment of the adult cerebellar cortex.


Assuntos
Córtex Cerebelar/química , Fator 1 de Crescimento de Fibroblastos/genética , Mesencéfalo/química , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/análise , Receptores de Superfície Celular/genética , Rombencéfalo/química , Animais , Biomarcadores/química , Córtex Cerebelar/citologia , Córtex Cerebelar/embriologia , Desenvolvimento Embrionário e Fetal/fisiologia , Genes Homeobox , Mesencéfalo/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Rombencéfalo/embriologia , Transdução de Sinais/fisiologia
2.
Brain Res Mol Brain Res ; 56(1-2): 9-21, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9602027

RESUMO

We describe the cloning, chromosomal localization and characterization of RPTPrho, a new member of the RPTPmu/kappa phosphatase subfamily. Receptor tyrosine phosphatases in this subfamily are comprised of a MAM domain near the N-terminal, an immunoglobulin-like domain, four fibronectin type III repeats, a single transmembrane domain, and a large juxtamembrane segment followed by two intracellular phosphatase domains. An alternatively spliced mini-exon was identified in the extracellular segment of RPTPrho, between the fourth fibronectin type III repeat and the transmembrane domain. The RPTPrho gene was mapped to human chromosome 20 and mouse chromosome 2. Northern blot analysis demonstrated that RPTPrho expression was restricted to the central nervous system, and in situ hybridization studies showed that the RPTPrho transcript was distributed throughout the murine brain and spinal cord. Exceptionally high levels of the transcript were present in the cortex and olfactory bulbs during perinatal development, but were down-regulated during postnatal week two. The motifs found in the extracellular segment of type II receptor protein tyrosine phosphatases are commonly found in neural cell adhesion molecules, suggesting that RPTPrho may be involved in both signal transduction and cellular adhesion in the central nervous system.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Receptores de Superfície Celular/metabolismo , Medula Espinal/metabolismo , Adulto , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores
3.
Thromb Res ; 83(2): 195-8, 1996 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8837319

RESUMO

Increased levels of homocysteine have been linked to both arterial and venous thromboembolic problems (1,2). Homocystinuria is a relatively rare disorder caused by a deficiency of cystathione synthase and is characterized by markedly increased levels of homocysteine and premature vascular disease (3-5). Epidemiological studies have suggested that mild elevations of homocysteine are also associated with vascular disease (2). Recent evidence suggests that a polymorphism of the gene encoding for 5,10-methylene tetrahydrofolate reductase (MTHFR) gives rise to a thermolabile form of the enzyme that is associated with increased levels of homocysteine when inherited as a homozygous trait (6). This polymorphism is due to a C --> T substitution at nucleotide 677 which converts an alanine to valine in a conserved portion of the molecule (6). The allele frequency for the thermolabile form of the enzyme was quite high (0.38) in a population of French Canadians. This polymorphism thus appears to be a common risk factor for increased plasma levels of homocysteine and vascular diseases. As the incidence of such genetic polymorphisms often varies among ethnic populations, we were interested in comparing the incidence of this polymorphism in Caucasians and African Americans.


Assuntos
Negro ou Afro-Americano , Tetra-Hidrofolato Desidrogenase/genética , Alelos , Frequência do Gene , Humanos , Polimorfismo Genético
4.
Arch Pathol Lab Med ; 122(7): 633-7, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9674544

RESUMO

BACKGROUND: Hereditary hemochromatosis, a common autosomal recessive trait caused by mutations in the HLA-H gene, is often diagnosed by the pathologist at the time of histologic examination. Unfortunately, histologic parameters alone do not differentiate between hereditary hemochromatosis and other causes of iron overload. We performed a retrospective study to determine the frequency of familial hemochromatosis in patients diagnosed with he mochromatosis by abnormal liver histology. METHODS AND RESULTS: DNA was isolated from paraffin-embedded tissue sections from 15 patients and used in a polymerase chain reaction-based assay in which we tested for the C282Y and H63D mutations. We found that in this group of patients, 5 (33%) were homozygous for the common C282Y genetic mutation, 3 (20%) were heterozygous, and 7 (47%) were normal. CONCLUSIONS: Our study shows that the molecular assay is the gold standard for the diagnosis of hereditary hemochromatosis. The case study also illustrates that a definitive diagnosis of familial hemochromatosis has significant counseling implications allowing for accurate family studies.


Assuntos
Biópsia , Análise Mutacional de DNA , Hemocromatose/diagnóstico , Hemocromatose/genética , Hepatopatias/genética , Adulto , Idoso , Diagnóstico Diferencial , Eletroforese em Gel de Poliacrilamida , Feminino , Hemocromatose/patologia , Heterozigoto , Homozigoto , Humanos , Hepatopatias/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Reação em Cadeia da Polimerase
5.
Am J Physiol ; 251(1 Pt 1): C32-40, 1986 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2425629

RESUMO

As part of a detailed study of cell volume regulation in high-potassium mammalian erythrocytes, we have characterized ouabain-insensitive sodium transport in normal and osmotically shrunken rabbit red cells. In cells of normal volume and physiological pH, there is no amiloride-inhibited component of the sodium efflux (into either sodium-containing or sodium-free media). Osmotic shrinkage activates an amiloride-sensitive (50% inhibitory concentration = 10(-5) M) sodium transport system that can catalyze net sodium movement in either direction. This system appears to be distinct from the sodium-sodium (sodium-lithium) counter-transporter that operates in cells of normal volume. Replacement of chloride with acetate does not inhibit the sodium flux, but replacement with either nitrate or thiocyanate is inhibitory. An inward sodium gradient in shrunken cells induces a net uphill efflux of acid equivalents, indicating that the sodium transport is a sodium-hydrogen exchange. However, a sevenfold inward gradient of hydrogen ions (pHo = 6.4; pHi = 7.2) does not stimulate net sodium efflux in shrunken cells. This suggests that the extracellular affinity of the transport site for hydrogen ions is high, and that there is an extracellular noncompetitive inhibitory site for proton binding. Bilateral pH reduction stimulates an amiloride-inhibitable sodium flux in cells of normal volume; this indicates that, as has been found in kidney, brain, and lymphocytes, there is an intracellular protonation site that can activate the transport. Shrinkage of the cells shifts the pH dependence of the transport, suggesting that part of the signal for the osmotic activation of the transport is a shift in the pKa of this modifier site.


Assuntos
Amilorida/farmacologia , Eritrócitos/metabolismo , Canais Iônicos/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico Ativo , Permeabilidade da Membrana Celular , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Concentração de Íons de Hidrogênio , Canais Iônicos/efeitos dos fármacos , Fragilidade Osmótica , Ouabaína/farmacologia , Coelhos
6.
Hum Mol Genet ; 5(11): 1727-32, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8922999

RESUMO

The gene for autosomal recessive spinal muscular atrophy (SMA) has been mapped to 5q12 in a region that contains repeated markers and genes. Three cDNAs that detect deletions in SMA patients have been reported. One of these, the survival motor neuron (SMN) cDNA, is encoded by two genes (SMNT and SMNC) which are distinguished by base changes in exons 7 and 8. Exon 7 of the SMNT gene is not detectable in approximately 95% of SMA cases, due either to deletion or sequence conversion. There is limited information on the mutations in SMA patients that have detectable SMNT, these are critical for confirmation of SMNT as the SMA gene. Using SSCP analysis of the SMN exons we screened our SMA patients that possess at least one intact SMNT allele for mutations in SMNT. We identified one type I SMA patient with an 11 bp duplication in exon 6 which causes a frameshift and premature termination of the deduced SMNT protein. Dosage and SSCP analysis of SMNT in this family indicated that the father contributed a SMNT-deleted allele to the affected child whereas the mother passed on the 11 bp exon 6 duplication SMNT allele. Analysis of RNA by RT-PCR conclusively demonstrated that the 11 bp duplication is associated with the SMNT locus and not SMNC. This mutation provides strong support for SMN as the SMA-determining gene and indicates that disruption of SMNT on its own is sufficient to produce a severe type I SMA phenotype.


Assuntos
Éxons/genética , Mutação da Fase de Leitura/genética , Proteínas do Tecido Nervoso/genética , Atrofias Musculares Espinais da Infância/genética , Alelos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Feminino , Dosagem de Genes , Humanos , Lactente , Masculino , Fenótipo , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor
7.
J Med Genet ; 35(8): 674-6, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9719377

RESUMO

We report a child with clinical findings consistent with Werdnig-Hoffmann disease (spinal muscular atrophy type I) who was found not to have the homozygous absence of the survival motor neurone (SMN(T)) gene observed in approximately 95% of spinal muscular atrophy patients. A quantitative PCR based dosage assay for SMN(T) copy number showed that this patient possessed a single copy of the SMN(T) gene. Heteroduplex and sequence analysis of the remaining copy of SMN(T) showed a 2 base pair deletion within exon 4 which produces a frameshift and premature termination of the deduced SMN(T) protein. This protocol of initial SMN(T) gene dosage analysis followed by mutation detection allows identification of SMA compound heterozygotes (patients lacking one copy of SMN(T) and having another mutation in their other copy), thereby increasing the sensitivity of SMA molecular diagnosis.


Assuntos
Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase , Atrofias Musculares Espinais da Infância/diagnóstico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Análise Mutacional de DNA , Deleção de Genes , Testes Genéticos , Humanos , Lactente , Masculino , Ácidos Nucleicos Heteroduplexes , Proteínas de Ligação a RNA , Proteínas do Complexo SMN , Atrofias Musculares Espinais da Infância/genética
8.
Am J Hum Genet ; 63(6): 1712-23, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9837824

RESUMO

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.


Assuntos
Efeito Fundador , Dosagem de Genes , Atrofia Muscular Espinal/genética , Mutação , Proteínas do Tecido Nervoso/genética , Alelos , Centrômero/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Análise Mutacional de DNA , Éxons/genética , Mutação da Fase de Leitura/genética , Proteínas Fúngicas/metabolismo , Análise Heteroduplex , Heterozigoto , Humanos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Polimorfismo Genético , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Complexo SMN , Deleção de Sequência , Telômero/genética
9.
Am J Hum Genet ; 60(6): 1411-22, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9199562

RESUMO

The survival motor neuron (SMN) transcript is encoded by two genes, SMNT and SMNC. The autosomal recessive proximal spinal muscular atrophy that maps to 5q12 is caused by mutations in the SMNT gene. The SMNT gene can be distinguished from the SMNC gene by base-pair changes in exons 7 and 8. SMNT exon 7 is not detected in approximately 95% of SMA cases due to either deletion or sequence-conversion events. Small mutations in SMNT now have been identified in some of the remaining nondeletion patients. However, there is no reliable quantitative assay for SMNT, to distinguish SMA compound heterozygotes from non-5q SMA-like cases (phenocopies) and to accurately determine carrier status. We have developed a quantitative PCR assay for the determination of SMNT and SMNC gene-copy number. This report demonstrates how risk estimates for the diagnosis and detection of SMA carriers can be modified by the accurate determination of SMNT copy number.


Assuntos
Cromossomos Humanos Par 5 , Dosagem de Genes , Triagem de Portadores Genéticos , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA/sangue , DNA/genética , Primers do DNA , Éxons , Feminino , Humanos , Masculino , Neurônios Motores/patologia , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/epidemiologia , Ácidos Nucleicos Heteroduplexes/genética , Linhagem , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA , Medição de Risco , Proteínas do Complexo SMN , Medula Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Transcrição Gênica
10.
Neurogenetics ; 1(2): 141-7, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10732817

RESUMO

The telomeric survival motor neuron (SMN(T)) gene is a valuable molecular diagnostic tool for childhood-onset spinal muscular atrophy (SMA) as homozygous deletions of SMN(T) exon 7 (delta7SMN(T)) are present in approximately 94% of patients. In this report, we provide the first comprehensive study of 32 unrelated non-deletion SMA patients. Quantitative polymerase chain reaction (PCR) studies established that 90% had two intact copies of SMN(T) exon 7 suggesting that these patients do not have 5q SMA. Once 5q SMA is confirmed, the SMN(T) gene can be screened for subtle mutations. Using single strand conformation analysis, we identified two missense mutations (P245L and Y272C) in exon 6 of the SMN(T) gene of two SMA patients shown to have a single copy of SMN(T) exon 7. Y272 is most likely critical for SMN(T) function as it is a target for recurring mutations and is associated with type I SMA. These results emphasize the need for dosage analysis in the differential diagnosis of 5q SMA in nondeletion patients, consistent with extensive clinical heterogeneity and some genetic heterogeneity in this disease. Homozygosity or heterozygosity for a delta7SMN(T) allele confirms the diagnosis of 5q SMA with greater precision than clinical examination alone.


Assuntos
Éxons/genética , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Substituição de Aminoácidos , Sequência de Bases , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Masculino , Atrofia Muscular Espinal/diagnóstico , Mutação , Linhagem , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Proteínas de Ligação a RNA , Proteínas do Complexo SMN
11.
N Engl J Med ; 333(13): 832-8, 1995 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-7651473

RESUMO

BACKGROUND: Myoblast transfer has been proposed as a technique to replace dystrophin, the skeletal-muscle protein that is deficient in Duchenne's muscular dystrophy. Donor myoblasts injected into muscles of affected patients can fuse with host muscle fibers, thus contributing their nuclei, which are potentially capable of replacing deficient gene products. Previous controlled trials involving a single transfer of myoblasts have been unsuccessful. METHODS: We injected donor muscle cells once a month for six months to the biceps brachii muscles of one arm of each of 12 boys with Duchenne's muscular dystrophy. The opposite arms served as sham-injected controls. In each procedure 110 million cells donated by fathers or brothers were transferred. The patients were randomly assigned to receive either cyclosporine or placebo. Strength was measured by quantitative isometric muscle testing. Six months after the final myoblast transfer, the presence of dystrophin was assessed with the use of peptide antibodies specific to the deleted exons of the dystrophin gene. RESULTS: There was no significant difference in muscle strength between arms injected with myoblasts and sham-injected arms. In one patient, 10.3 percent of muscle fibers expressed donor-derived dystrophin after myoblast transfer. Three other patients also had a low level of donor dystrophin (< 1 percent); eight had none. CONCLUSIONS: Myoblasts transferred once a month for six months failed to improve strength in patients with Duchenne's muscular dystrophy. The value of exon-specific peptide antibodies in the interpretation of myoblast-transfer results was demonstrated in a patient with Duchenne's muscular dystrophy who had a high percentage of donor-derived dystrophin. Specific variables affecting the efficiency of myoblast transfer need to be identified in order to improve upon this technique.


Assuntos
Transplante de Células , Distrofina/genética , Músculo Esquelético/citologia , Distrofias Musculares/terapia , Anticorpos/análise , Braço/fisiopatologia , Fenômenos Biomecânicos , Criança , Pré-Escolar , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Método Duplo-Cego , Distrofina/análise , Éxons , Terapia Genética , Humanos , Injeções Intramusculares , Contração Isométrica/efeitos dos fármacos , Masculino , Fibras Musculares Esqueléticas/química , Distrofias Musculares/genética , Distrofias Musculares/fisiopatologia , Peptídeos/imunologia
12.
Hum Mol Genet ; 6(8): 1205-14, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9259265

RESUMO

The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMN(T)) and centromeric SMN (SMN(C)). Mutations in SMN(T), but not SMN(C), cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMN(T) and SMN(C) genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non-SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMN(T) in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.


Assuntos
Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Western Blotting , Células Cultivadas , Centrômero , Galinhas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA , Proteínas do Complexo SMN , Medula Espinal/metabolismo , Telômero
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