RESUMO
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is an early onset neurodegenerative disease with high prevalence (carrier frequency 1/22) in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec. We previously mapped the gene responsible for ARSACS to chromosome 13q11 and identified two ancestral haplotypes. Here we report the cloning of this gene, SACS, which encodes the protein sacsin. The ORF of SACS is 11,487 bp and is encoded by a single gigantic exon spanning 12,794 bp. This exon is the largest to be identified in any vertebrate organism. The ORF is conserved in human and mouse. The putative protein contains three large segments with sequence similarity to each other and to the predicted protein of an Arabidopsis thaliana ORF. The presence of heat-shock domains suggests a function for sacsin in chaperone-mediated protein folding. SACS is expressed in a variety of tissues, including the central nervous system. We identified two SACSmutations in ARSACS families that lead to protein truncation, consistent with haplotype analysis.
Assuntos
Ataxia/genética , Cromossomos Humanos Par 13 , Proteínas de Choque Térmico/genética , Mutação , Fases de Leitura Aberta , Degenerações Espinocerebelares/genética , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Sequência de Bases , Mapeamento Cromossômico , Éxons , Proteínas de Choque Térmico/química , Humanos , Desequilíbrio de Ligação , Camundongos , Dados de Sequência Molecular , Prevalência , Quebeque/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Epithelial and fibroblast cells were obtained from cultures of amniotic fluid cells. Epithelial cells demonstrated high activities of histidase. In contrast, histidase activity was not detected in fibroblasts derived from the same original culture. This observation indicates that cultures of amniotic fluid cells consist of cells with different biochemical properties as well as morphological characteristics.
Assuntos
Líquido Amniótico/citologia , Liases/análise , Amônia , Líquido Amniótico/enzimologia , Cromatografia em Papel , Técnicas de Cultura , Epitélio/enzimologia , Feminino , Fibroblastos/enzimologia , Histidina , Humanos , GravidezRESUMO
Sandhoff disease is caused by mutations affecting the beta subunit of lysosomal beta-hexosaminidase (EC 3.2.1.52) and displays a wide spectrum of clinical phenotypes. We report a 57-year-old patient with a very mild phenotype, although residual hexosaminidase A activity in his cultured fibroblasts was less than 3% of normal activity, a level observed in juvenile onset patients. Northern and Western blot analyses confirmed a similar low level of beta subunit-mRNA and mature beta-protein, respectively. Two mutations of the HEXB gene were identified in this patient, a partial 5' gene deletion (a null allele), and a C----T transition 8 nucleotides downstream from the intron 10/exon 11 junction affecting the splicing of the beta subunit-mRNA. In their homozygous forms, the 5' deletion has been previously shown to result in a severe infantile phenotype, and the C----T transition in a juvenile phenotype. The genotype and the low level of residual hexosaminidase A activity would be expected to produce a juvenile Sandhoff phenotype in this patient, as well as in four of his six clinically normal siblings. The biochemical basis of his mild phenotype is uncertain, but may result from genetic variations in the RNA splicing machinery.
Assuntos
Doença de Sandhoff/genética , beta-N-Acetil-Hexosaminidases/genética , Sequência de Bases , Genes , Glucuronidase/genética , Hexosaminidase A , Hexosaminidase B , Humanos , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Oligodesoxirribonucleotídeos/química , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Splicing de RNARESUMO
beta-N-Acetyl-hexosaminidase S (2-acetamido-2-deoxy-beta-hexoside acetamido-deoxyhexohydrolase, EC 3.2.1.52) was purified from liver and brain of a patient deceased of type O GM2 gangliosidosis (Sandhoff's disease). Brain beta-N-acetyl-hexosaminidase S was further purified by preparative polyacrylamide gel electrophoresis. The pH optimum of the purified liver and brain enzyme was 5.0 and Km values were 0.8--0.9 mM and 0.3--0.4 mM with 4-methylumbelliferyl-beta-D-N-acetylglucosamine and beta-D-N-acetylgalactosaminide derivatives, respectively. beta-N-Acetyl-hexosaminidase S was thermolabile losing most of its activity after 50 min at 50 degrees C. The apparent molecular weights of the purified liver and brain enzymes were 154 000 and 152 000, respectively. Hexosamines activated beta-N-acetyl-hexosaminidase S whereas the isoenzyme A and B were inhibited. The glycoprotein nature of beta-N-acetyl-hexosaminidase S was suggested by its affinity towards Concanavalin A-Sepharose.
Assuntos
Encéfalo/enzimologia , Hexosaminidases/isolamento & purificação , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Sítios de Ligação , Pré-Escolar , Feminino , Hexosaminidases/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Conformação Proteica , Doença de Sandhoff/enzimologiaRESUMO
A form of autosomal recessive spastic ataxia unique to the Charlevoix-Saguenay area was clinically identified 20 years ago in patients from that region. This region of Québec, Canada, was once considered a genetic isolate. First noted at gait initiation, signs of ataxia slowly progress along with spasticity of the four limbs, slurred speech, and followed by distal amyotrophy. Early diagnosis relies on the presence of prominent myelinated fibers embedding retinal blood vessels at funduscopy and marked saccadic alteration of ocular smooth pursuit. Imaging of the posterior fossa shows cerebellar vermis atrophy and nerve conduction studies reveal loss of sensory and reduced motor conduction velocities. The clinical features are consistent with a developmental defect in myelination of both retinal and peripheral nerve fibers. The cause of this defect and the progressive axonal degeneration in the corticospinal and spinocerebellar tracts, as well as in the peripheral nerves is still unknown. Results of recent molecular genetic linkage analysis have located the gene locus to chromosome 13q12. Further research is needed to define where this hereditary spastic ataxia stands in the classification of the early onset spinocerebellar degenerations.
Assuntos
Ataxia/genética , Genes Recessivos/genética , Ataxia/epidemiologia , Ataxia/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 13/genética , Eletrofisiologia , Feminino , Humanos , Masculino , Repetições de Microssatélites , Músculos/inervação , Músculos/patologia , Músculos/fisiopatologia , Quebeque/epidemiologiaRESUMO
Microvillar enzymes (disaccharidases, alkaline phosphatase, and gamma-glutamyltransferase) were assayed in amniotic fluid from pregnancies with normal and abnormal fetuses to determine their specificity and reliability for the prenatal detection of intestinal obstructions and cystic fibrosis. All fetuses with imperforate anus, duodenal atresia, jejuno-ileal atresia, multiple intestinal atresia, or other forms of intestinal obstructions, with or without associated ventral wall defect or aneuploidy syndrome, showed diminished microvillar enzyme activities below the normal range of control amniotic fluid samples. The exclusively intestinal hydrolases maltase, sucrase, palatinase, and alkaline phosphatase were the most reliable and sensitive markers to detect intestinal obstructions whereas more widely distributed trehalase and gamma-glutamyltransferase activities were less sensitive. The combination of intestinal disaccharidase maltase, sucrase or palatinase and ALP assays is more accurate for prenatal diagnosis of CF than a combination of intestinal ALP and GGTF assays.
Assuntos
Aneuploidia , Aberrações Cromossômicas/diagnóstico , Ensaios Enzimáticos Clínicos , Fibrose Cística/diagnóstico , Obstrução Intestinal/diagnóstico , Diagnóstico Pré-Natal , Fosfatase Alcalina/análise , Líquido Amniótico/enzimologia , Transtornos Cromossômicos , Dissacaridases/análise , Feminino , Feto/enzimologia , Humanos , Mucosa Intestinal/enzimologia , Microvilosidades/enzimologia , Gravidez , gama-Glutamiltransferase/análiseRESUMO
Infantile free sialic acid storage disease (ISSD) is a rare autosomal recessive metabolic disorder caused by a lysosomal membrane transport defect, resulting in accumulation of free sialic acid within lysosomes. Only a few cases have been described. We report on three new cases of ISSD with different modes of presentation: an infant with nephrotic syndrome, a case of fetal and neonatal ascites with heart failure, and a case of fetal ascites with esophageal atresia type III. From these patients and a review of the literature (27 cases total) we draw the following conclusions. 1) "Coarse facies," fair complexion, hepatosplenomegaly, and severe psychomotor retardation are constant findings in this disorder. 2) Nephrotic syndrome occurred in most cases (four in seven) in which renal evaluation was performed. Therefore, ISSD is an important cause of nephrosis in infants with a storage disorder phenotype. 3) Fetal/neonatal ascites or hydrops was the mode of presentation in 13 (60%) of 21 cases. Thus, ISSD enters in the differential diagnosis of hydrops fetalis with a storage disease phenotype. 4) Cardiomegaly was evident in nine cases. 5) Corneae were always clear, and albinoid fundi were reported in five cases. 6) Dysostosis multiplex was not prominent. 7) Bone marrow aspiration could be negative. 8) Death ensued in early infancy with a mean age of 13.1 months. All reported deaths were caused by respiratory infections.
Assuntos
Doenças Fetais/diagnóstico , Doenças por Armazenamento dos Lisossomos/diagnóstico , Ácido N-Acetilneuramínico/metabolismo , Ascite/congênito , Ascite/diagnóstico , Atresia Esofágica/diagnóstico , Feminino , Insuficiência Cardíaca/congênito , Insuficiência Cardíaca/diagnóstico , Humanos , Hidropisia Fetal/diagnóstico , Lactente , Recém-Nascido , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Masculino , Ácido N-Acetilneuramínico/urina , Síndrome Nefrótica/diagnósticoRESUMO
The significance of neuraminidase deficiency reported to be the primary defect in mucolipidosis II has been evaluated by determination of this enzyme activity in cultured fibroblasts, culture medium, and leucocytes from homozygote and heterozygous carriers of the disease. A new and sensitive fluorometric assay of neuraminidase was used with sodium (4-methylumbeliferyl-alpha-D-N-acetylneuraminate) as substrate. We report: 1) nearly total deficiency of neuraminidase in mucolipidosis fibroblasts, 2) partial deficiency of this enzyme in leucocytes of one patient, 3) this decreased activity ceases to exist following Triton X-100 treatment, and 4) intermediary mean neuraminidase activity in fibroblasts and leucocytes from obligate heterozygotes. Although these results would be consistent with the suggestion that neuraminidase deficiency is the primary defect in this disease, evidence from the work of other authors suggests that the enzyme deficiency results from a secondary effect of the mucolipidosis II mutation.
Assuntos
Fibroblastos/enzimologia , Leucócitos/enzimologia , Mucolipidoses/enzimologia , Neuraminidase/deficiência , Células Cultivadas , Pré-Escolar , Genes Recessivos , Genótipo , Glicosídeo Hidrolases/deficiência , Humanos , Lactente , Mucolipidoses/genética , Ácidos SiálicosRESUMO
The gene for Friedreich ataxia (FRDA), an autosomal-recessive neurodegenerative disease, remains elusive. The current candidate region of about 150 kb lies between loci FR2 and F8101 near the D9S15/D9S5 linkage group at 9q13-21.1. Linkage homogeneity between classical FRDA and a milder, slowly progressive Acadian variant (FRDA-Acad) has been demonstrated. An extended D9S15-D9S5 haplotype (C6) predominates in FRDA-Acad chromosomes from Louisiana. We studied 10 Acadian families from New Brunswick, Canada. In eight families, affected individuals conformed to the clinical description of FRDA-Acad; in one, 2 sibs presented with spastic ataxia (SPA-Acad). In the last family, 2 sibs had FRDA-Acad, and one had SPA-Acad. We found that SPA-Acad is linked to the FRDA gene region. The C6 haplotype and a second major haplotype (B7) were identified. The same ataxia-linked haplotypes segregated with both FRDA-Acad and SPA-Acad in two unrelated families. The parental origins of these haplotypes were different. Our observation of different phenotypes associated with the same combination of haplotypes may point to the influence of the parent of origin on gene expression, indicate the effect of modifier genes, or reflect the presence of different mutations on the same haplotypes. Our findings underline the need to investigate families with autosomal-recessive ataxias for linkage to the FRDA region, despite lack of key diagnostic manifestations such as cardiomyopathy or absent deep-tendon reflexes.
Assuntos
Ataxia de Friedreich/genética , Canadá , DNA/análise , Feminino , Ligação Genética/genética , Haplótipos , Humanos , Masculino , Linhagem , Polimorfismo GenéticoRESUMO
Lipoamide dehydrogenase was identified in cultured skin fibroblasts of normal individuals and patients with Friedreich's ataxia. The optimum conditions for its assay were defined. Data disclosed a normal range of 36--122 mumol/min/mg protein in control fibroblasts and 61--112 mumol/min/mg protein in patients fibroblasts. Numerous precautions should be taken in handling fibroblast cultures for lipoamide dehydrogenase determination.
Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Pele/enzimologia , Soluções Tampão , Células Cultivadas , Centrifugação , Coenzimas/metabolismo , Fibroblastos/enzimologia , Ataxia de Friedreich/enzimologia , Temperatura Alta , Humanos , Concentração Osmolar , Proteínas/metabolismo , Valores de Referência , Fatores de TempoRESUMO
The isoenzymes of N-acetyl-beta-hexosaminidase were quantitated in 30 amniotic fluid and 13 maternal serum samples collected between 11 and 40 weeks of gestation using DEAE-Sephadex A-25 chromatography. Isoenzymes A and B consitituted the major components of most amniotic fluids but seven samples characterized by high N-acetyl-beta-hexosaminidase activities contained high proportion of an isoenzyme apparently identical to isoenzyme P of maternal serum. This passage of maternal serum N-acetyl-beta-hexosaminidase into the amniotic cavity could lead to false negative diagnosis of type B and type O GM2 gangliosidoses if the diagnosis rely solely upon isoenzyme analysis in amniotic fluid. However, the release of maternal enzyme into amniotic fluid seems to be restricted to the third trimester of gestation and should not interfere with prenatal diagnosis of the GM2 gangliosidoses ususally performed at an earlier stage of gestation.
Assuntos
Líquido Amniótico/enzimologia , Gangliosidoses/diagnóstico , Hexosaminidases/metabolismo , Isoenzimas/metabolismo , Feminino , Gangliosidoses/enzimologia , Idade Gestacional , Hexosaminidases/sangue , Humanos , Isoenzimas/sangue , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Diagnóstico Pré-NatalRESUMO
Two neuraminidase (EC 3.2.1.18) comonents, A and B, were distinguished in cultured skin fibroblasts on the basis of thermolability at 37 degrees C. The more labile component (A) t1/2 = 4.7--5.3 min at 37 degrees C, comprises 66--90% of total neuraminidase activity when determined using sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) (MU-alpha-N) as substrate. Activity was assayed at 0 degrees C for 18 h instead of 37 degrees C to fully determine both thermolabile and thermostable components. Diminished activity was noted in cultured fibroblasts from mucolipidoses I, II and III (MLI, MLII, MLIII) and the cherry-red spot myoclonus syndrome (CRSM) patients when assayed at both 0 and 37 degrees C with either MU-alpha-N or each of a series alpha (2 leads to 3)- and alpha (2 leads to 6)-linked N-acetylneuraminyloligosaccharides. Increased sensitivity and rapidity of analyses were achieved using MJ-alpha-N as substrate in determining neuraminidase activity. Results from two obligate heterozygote MLI cell lines (14.5 and 8.0% of control activity) indicate that the MU-alpha-N substrate could be useful for heterozygote detection.
Assuntos
Mucolipidoses/enzimologia , Mioclonia/enzimologia , Neuraminidase/metabolismo , Pele/enzimologia , Células Cultivadas , Fibroblastos/enzimologia , Humanos , CinéticaRESUMO
Taurine and beta-alanine uptake kinetics were studied in cultured skin fibroblasts from 9 patients with Friedreich's Ataxia and 8 controls. No significant difference was observed. The data support the presence of normal beta-amino acid carrier protein in Friedreich's Ataxia cell membrane.
Assuntos
Alanina/metabolismo , Fibroblastos/metabolismo , Ataxia de Friedreich/metabolismo , Taurina/metabolismo , beta-Alanina/metabolismo , Células Cultivadas , Humanos , Técnicas In VitroRESUMO
Taurine and beta-alanine uptake in cultured skin fibroblasts proceeds through at least two distinct amino acid transport systems. The predominant beta amino acid uptake system which we refer to as the "Beta" system, incorporates taurine in a proportion of 95%. Beta-alanine in a proportion of 80% and does not incorporate beta-amino-isobutyric acid. A second transport system for beta-alanine seems to be operative cultured skin fibroblasts and this system shares the characteristics of system "L" for branched-chain and ring-side neutral amino acids. Results of ion depletion experiments, metabolic inhibition by drugs and blocking agents and previous kinetic studies of taurine and beta-alanine uptake in cultured skin fibroblasts failed to disclose any major difference in beta-amino acid transport between control individuals and patients with Friedreich's ataxia.
Assuntos
Alanina/metabolismo , Ataxia de Friedreich/metabolismo , Taurina/metabolismo , beta-Alanina/metabolismo , Aminoácidos/farmacologia , Cálcio/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Magnésio/farmacologia , Potássio/farmacologia , Pele/metabolismo , Sódio/farmacologiaRESUMO
The activity of lipoamide dehydrogenase and two closely related enzymes was studied simultaneously in early, mild, and late passage fibroblast cultures. Friedreich's ataxia fibroblasts tended to lose pyruvate dehydrogenase and citrate synthase activities, while lipoamide dehydrogenase activity remained constant with aging of the cells. Mean pyruvate dehydrogenase activity was lower over-all in fibroblasts from ataxics. Mean citrate synthase activity was higher in ataxic fibroblasts. Present tissue culture media do not represent the best conditions in which to reproduce cofactor binding defects such as those found in other genetic diseases with structural enzyme mutations.
Assuntos
Ataxia/enzimologia , Citrato (si)-Sintase/metabolismo , Di-Hidrolipoamida Desidrogenase/metabolismo , Fibroblastos/enzimologia , Ataxia de Friedreich/enzimologia , Oxo-Ácido-Liases/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Ataxia/genética , Células Cultivadas , Humanos , Espasticidade Muscular , SíndromeRESUMO
Serum lipoamide dehydrogenase activity and kinetics were studied in nine patients with Friedreich's ataxia before and three months after therapy with oral lecithin. Results disclosed a significant reduction in LAD inhibition by NADH in all patients after therapy. Three patients normalized their increased Km for lipoamide and one patient showed the opposite results after therapy. Two patients ceased lecithin after one month. All seven patients who remained in the trial group and one additional patient, showed subjective and objective signs of improvement in physical resistance. This study has offered some biochemical basis for the apparent clinical improvement in patients with Friedreich's ataxia who undergo lecithin therapy.
Assuntos
Di-Hidrolipoamida Desidrogenase/sangue , Ataxia de Friedreich/tratamento farmacológico , Fosfatidilcolinas/uso terapêutico , Adolescente , Adulto , Fatores Etários , Criança , Ataxia de Friedreich/enzimologia , Ataxia de Friedreich/genética , Humanos , CinéticaRESUMO
A clinical and biochemical evaluation of twenty-two patients with Friedreich's Ataxia and ten normal controls was undertaken in 1980 to assess the effect of lecithin and linoleic acid supplements on the course of the disease. The trial consisted of two consecutive six months periods on either supplements in a double-blind crossover fashion. Clinical appraisal was performed with regards to the following parameters: joints mobility, muscle strength, equilibrium, coordination, motor accuracy, speech and numerous day to day activities. Blood samples were obtained at the beginning and in the course of the trial for enzymatic determinations. This paper describes the methodology of the study.
Assuntos
Ataxia de Friedreich/tratamento farmacológico , Ácidos Linoleicos/uso terapêutico , Fosfatidilcolinas/uso terapêutico , Administração Oral , Adolescente , Adulto , Criança , Ensaios Clínicos como Assunto , Método Duplo-Cego , Feminino , Humanos , Ácido Linoleico , Locomoção/efeitos dos fármacos , Masculino , Destreza Motora/efeitos dos fármacos , Contração Muscular/efeitos dos fármacosRESUMO
We have measured the activity of malic enzyme NADP+ dependent in the nuclear, mitochondrial, lysosomal and cytosolic fractions of cultured skin fibroblasts from twelve patients with Friedreich's ataxia and nine control subjects. Hexosaminidase, cytochrome-C-oxidase, lactate dehydrogenase and malic enzyme NAD+ dependent were used as marker enzymes. The activity of malic enzyme NADP+ dependent was not significantly reduced in the mitochondrial fraction of patients with Friedreich's ataxia as compared with controls. When corrected for possible contamination between mitochondrial and cytosolic fractions, malic enzyme NADP+ dependent activity was still not significantly reduced in patients with Friedreich's ataxia. Unless critical methodological differences were overlooked in this or previously published studies, we conclude that mitochondrial malic enzyme deficiency is not the primary genetic defect underlying Friedreich's ataxia.
Assuntos
Ataxia de Friedreich/enzimologia , Malato Desidrogenase/metabolismo , Adolescente , Adulto , Núcleo Celular/enzimologia , Células Cultivadas , Criança , Citosol/enzimologia , Fibroblastos , Humanos , Lisossomos/enzimologia , Mitocôndrias/enzimologia , PeleRESUMO
Glutamic and aspartic acid uptake was measured in skin fibroblasts from patients with Friedreich's Ataxia, dicarboxylic aminoaciduria, and normal individuals. The results showed no difference in uptake kinetics of either dicarboxylic amino acids between Friedreich's Ataxia and normal cells, but reduced uptake velocities in dicarboxylic aminoaciduria fibroblasts. Friedreich's Ataxia fibroblasts were, however, less calcium-dependent and more magnesium and phosphate-dependent than controls in glucose-free incubation mixture. This difference might be related to some degree of glucose intolerance by Friedreich's Ataxia fibroblasts in culture.
Assuntos
Aminoácidos Dicarboxílicos/metabolismo , Fibroblastos/metabolismo , Ataxia de Friedreich/metabolismo , Aminoácidos/farmacologia , Aminoácidos Dicarboxílicos/urina , Ácido Aspártico/metabolismo , Células Cultivadas , Cloranfenicol/farmacologia , Glucose/farmacologia , Glutamatos/metabolismo , Humanos , Iodoacetatos/farmacologia , Potássio/farmacologia , Cianeto de Potássio/farmacologia , Sódio/farmacologia , Reagentes de Sulfidrila/farmacologia , Temperatura , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
Lecithin and safflower oil brought about the same changes in serum LAD activity and kinetics in patients with Friedreich's Ataxia as in controls when results of this double-blind crossover study were analyzed according to group assignation. According to functional stages, pretrial LAD activity decreased with advancing severity while Km for lipoamide increased. Lecithin and safflower oil supplements corrected the elevated Km for lipoamide but produced a further reduction in LAD activity. These changes may have been due to the increased intake of linoleic acid, a precursor of lipoic acid, which is present in high percentage in both lecithin and safflower oil. Results of the biochemical study thus agreed with the clinical data gathered during the course of the one-year trial in suggesting that linoleic acid may well have been the active factor through which biochemical and clinical improvement was previously observed in patients with Friedreich's Ataxia supplemented with lecithin.