RESUMO
Neurologic deterioration began in a girl before age 2 years. By 4 she was spastic and decerebrate. GM1 gangliosidosis was diagnosed by absence of beta-galactosidase activity in leukocytes and fibroblasts. She died at 17 years. Her small brain contained only 2.61 mumole glycolipid N-acetylneuraminic acid per gram, and was filled with autofluorescent material. GM1 gangliosidosis was confirmed by the presence of membranous cytoplasmic bodies, by the absence of beta-galactosidase, and by failure of complementation when the patient's fibroblasts were fused with cells from other forms of GM1 gangliosidosis. The autofluorescent material probably accumulated because of the long survival rather than the primary enzyme defect.
Assuntos
Gangliosidoses/diagnóstico , Lipofuscina/metabolismo , Transtornos da Pigmentação/diagnóstico , Pigmentos Biológicos/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Pré-Escolar , Feminino , Gangliosidoses/enzimologia , Gangliosidoses/patologia , Humanos , Ácidos Siálicos/metabolismo , beta-Galactosidase/metabolismoRESUMO
In order to determine the status of Tay-Sachs disease carrier identification in Toronto, Canada, since a change was made in 1978 from testing in the context of large-scale community clinics (up to 1,200 individuals tested in 1 day) to a case-finding approach to screening, a sample of area Jews was surveyed by questionnaire. The results indicated that a trend has developed for individuals at risk to delay testing until pregnancy when carrier detection is technically more difficult and the time available for retesting and organizing prenatal diagnosis is limited. If the trend continues, the full potential of chorionic villus sampling (CVS) for the prenatal diagnosis of the disease will be difficult to realize.
Assuntos
Triagem de Portadores Genéticos , Doença de Tay-Sachs/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Judeus , Masculino , Pessoa de Meia-Idade , Ontário , Inquéritos e QuestionáriosRESUMO
In a child with enzymatically and histopathologically proven metachromatic leukodystrophy (MLD), the disease pursued a course typical of juvenile MLD characterized by neurological degeneration beginning at age 9 years and ending in death at age 18. A younger brother of the patient was found to have profound deficiency of arylsulfatase A in leukocytes and to excrete five- to 20-fold greater-than-normal amounts of sulfatide in the urine. He was completely free of symptoms attributable to MLD until age 16 when he developed acute cholecystitis caused by sulfatide accumulation in the gallbladder. Results of detailed neurological examination at age 21 years were normal; formal psychometric assessment showed a full-scale IQ of 105 (Wechsler). Studies on cultured skin fibroblasts from the brother showed defects in arylsulfatase A activity, measured with the use of synthetic and natural substrates, and in radiolabeled sulfatide turnover. Cellulose acetate gel electrophoresis of fibroblast extracts from the patient showed no detectable arylsulfatase A isozyme under conditions that clearly distinguished pseudo-arylsulfatase A deficiency from classical MLD. Biochemically, the patient was indistinguishable from patients with classical MLD; on the other hand, his clinical course is dramatically more benign than that of his sister who was affected with severe MLD.
Assuntos
Leucodistrofia Metacromática/genética , Adolescente , Cerebrosídeo Sulfatase/deficiência , Criança , Eletroforese em Acetato de Celulose , Feminino , Fibroblastos/enzimologia , Humanos , Leucócitos/enzimologia , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/enzimologia , Masculino , Mutação , Exame Neurológico , Linhagem , Sulfoglicoesfingolipídeos/urinaRESUMO
Galactosialidosis (GS) is an autosomal recessive condition caused by combined deficiency of the lysosomal enzymes beta-galactosidase and alpha-neuraminidase. The combined deficiency has been found to result from a defect in protective protein/cathepsin A (PPCA), an intralysosomal protein which protects these enzymes from premature proteolytic processing. The most severe form of GS, the early-infantile form, results in early onset of edema, ascites, visceromegaly, and skeletal dysplasia. We report a case of early-infantile GS in a male infant who presented with nonimmune fetal hydrops (NIH), "coarse" facial appearance, massive fluid-filled inguinal hernias, multiple telangiectasia, and diffuse hypopigmentation; he subsequently developed visceromegaly. The diagnosis of GS was confirmed biochemically and the defect in PPCA characterized at the protein level. Examination of fetal peripheral blood smears sampled at 30 weeks gestation demonstrated vacuolation of lymphocytes, suggesting blood film examination may be a useful screening tool for cases of NIH where a metabolic disorder is suspected. Skeletal radiography at birth demonstrated punctate epiphyses of the femora, calcanei, and sacrum. We present a discussion of and differential diagnosis for this radiographic finding. To the best of our knowledge, this is the first case of early-infantile GS presenting with stippled epiphyses.
Assuntos
Doenças por Armazenamento dos Lisossomos/patologia , Western Blotting , Osso e Ossos/anormalidades , Osso e Ossos/diagnóstico por imagem , Anormalidades do Olho/patologia , Fibroblastos/enzimologia , Humanos , Recém-Nascido , Doenças por Armazenamento dos Lisossomos/diagnóstico , Doenças por Armazenamento dos Lisossomos/enzimologia , Masculino , Neuraminidase/metabolismo , Diagnóstico Pré-Natal , Radiografia , beta-Galactosidase/metabolismoRESUMO
Metachromatic leukodystrophy (MLD) presents as six separate variant forms, four allelic and two non-allelic. It is diagnosed in the laboratory by a decrease in the fibroblast or leukocyte arylsulfatase A activity, generally against an artificial substrate. Since residual enzyme activity is not always an indicator of presence or absence of disease, it may be helpful to supplement this information with that of the presence or absence of sulfatide storage in the body. We have improved the HPLC analysis of sulfatide by the use of a sulfated internal standard, sulfatoxymonoalkylmonoacylgalactosylglycerol. Normal urines contain approximately 0 to 0.2 nmol sulfatide/mg creatinine, whereas MLD urines may contain 5 to 7.5 nmol/mg. There is no increase in plasma sulfatide compared to controls in the age group of MLD patients which we studied (up to 4 years).
Assuntos
Leucodistrofia Metacromática/urina , Sulfoglicoesfingolipídeos/urina , Cerebrosídeo Sulfatase/sangue , Cromatografia Líquida de Alta Pressão/métodos , Glicolipídeos/análise , Glicoesfingolipídeos/urina , Humanos , Leucócitos/enzimologia , Leucodistrofia Metacromática/diagnósticoRESUMO
Uncultured and cultured embryonic trophoblastic tissue obtained by chorionic villus sampling (CVS) displays enzyme activity towards 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranosyl-6-sulfate (MUGS), a specific substrate for Hexosaminidase A (Hex A), the enzyme deficient in Tay-Sachs disease (TSD). Specific activity is comparable to that found in cultured amniocytes and fibroblasts. The enzyme activity has a pH optimum of 4.1 and an apparent Km of 6 x 10(-4) mol/L. Thirteen pregnancies in eight families at risk for TSD were monitored by CVS using MUGS as the Hex A substrate. Four fetuses were proven affected by enzyme analysis of fetal tissues and cultured fetal fibroblasts obtained at the time of termination of the pregnancies. Nine fetuses were judged to be unaffected. Eight babies were clinically normal while the other pregnancy is continuing. The use of MUGS as substrate for Hex A makes prenatal diagnosis by CVS of families at risk for TSD simple, direct and accurate.
Assuntos
Himecromona/análogos & derivados , Diagnóstico Pré-Natal/métodos , Doença de Tay-Sachs/diagnóstico , beta-N-Acetil-Hexosaminidases/análise , Células Cultivadas , Vilosidades Coriônicas/enzimologia , Feminino , Hexosaminidase A , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Fatores de Risco , Especificidade por SubstratoRESUMO
Concentrations of GL-la (glucocerebroside) (8.36 nmol/ml), GL-2a (lactosylceramide) (4.03 nmol/ml), GL-3a (globotriosylceramide) (2.25 nmol/ml) and GL-4a (globotetraosylceramide) (2.87 nmol/ml) have been determined in normal plasma and compared to concentrations in the plasma from patients with Gaucher, Krabbe, Fabry, Sandhoff and Tay-Sachs diseases as well as with hypercholesterolemia. HPLC analysis of perbenzoylated glycolipid derivatives (isolated and purified by modification of an existing procedure) was performed on samples equivalent to 50 to 100 microliter of plasma. The sensitivity could be readily increased ten-fold. We have employed a novel internal standard-monogalactosyl diglyceride, a plant glycolipid, commercially available in pure form. Analysis was performed on a 5 micron ultrasphere silica column, using a gradient of isopropanol in hexane rather than the more usual dioxane in hexane. Our gradient exhibited an essentially flat baseline precluding the necessity of a reference cell. Recoveries of glycolipids added to plasma (95%), experimental yields (60%) and standard curves are presented and discussed. A method is also presented for the separation of GL-la and monogalactosyl diglyceride derivatives for rapid (8 minute) isocratic analysis of multiple samples from Gaucher patients. The benefits of such a simple, reproducible HPLC technique are discussed.
Assuntos
Glicolipídeos/sangue , Erros Inatos do Metabolismo/sangue , Cromatografia Líquida de Alta Pressão/métodos , Doença de Gaucher/sangue , Humanos , Hipercolesterolemia/sangueRESUMO
We report an unusual case of multiple sulfatase deficiency in which neurodegeneration was accompanied by early, severe visual impairment associated with prominent pigmentary retinopathy, suggesting a diagnosis of neuronal ceroid-lipofuscinosis. The levels of arylsulfatases A, B, and C, heparan N-sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, and iduronate-2-sulfate sulfatase were all markedly decreased in cultured skin fibroblasts. Screening tests for mucopolysacchariduria were consistently negative; however, thin-layer chromatographic analysis of isolated urinary glycosaminoglycans showed increased amounts of heparan sulfate.
Assuntos
Degeneração Retiniana/etiologia , Sulfatases/deficiência , Criança , Humanos , MasculinoAssuntos
Estudos de Avaliação como Assunto , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Triagem de Portadores Genéticos , Leucodistrofia de Células Globoides/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Leucócitos , Leucodistrofia de Células Globoides/enzimologia , Masculino , Linhagem , Contagem de CintilaçãoRESUMO
Glycogen storage disease, type II (GSDII; Pompe disease; acid maltase deficiency) is an autosomal recessive disease caused by mutations of the GAA gene that lead to deficient acid alpha-glucosidase enzyme activity and accumulation of lysosomal glycogen. Although measurement of acid alpha-glucosidase enzyme activity in fibroblasts remains the gold standard for the diagnosis of GSDII, analysis of the GAA gene allows confirmation of clinical or biochemical diagnoses and permits predictive and prenatal testing of individuals at risk of developing GSDII. We have developed a clinical molecular test for the detection of GAA mutations based on cycle sequencing of the complete coding region. GAA exons 2-20 are amplified in six independent PCR using intronic primers. The resulting products were purified and sequenced. Preliminary studies using this protocol were conducted with DNA from 21 GSDII-affected individuals from five centers across Canada. In total, 41 of 42 mutations were detected (96.7% detection rate). Mutations spanned intron 1 through exon 19 and included nine novel mutations. Haplotype analysis of recurrent mutations further suggested that three of these mutations are likely to have occurred independently at least twice. Additionally, we report the identification of the c.-32-13T>G GAA mutation in an individual with infantile variant GSDII, despite reports of this mutation being associated almost exclusively with late-onset forms of the disease. The development of a clinical molecular test provides an important tool for the management and counseling of families and individuals with GSDII, and has provided useful information about the GAA mutation spectrum in Canada.
Assuntos
Predisposição Genética para Doença/genética , Doença de Depósito de Glicogênio Tipo II/genética , alfa-Glucosidases/genética , Alelos , Análise Mutacional de DNA , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Mutação , alfa-Glucosidases/deficiênciaRESUMO
We describe a method for isolating milligram quantities of the four neutral glycosphingolipids, glucocerebroside, lactosylceramide, traiosylceramide, and globoside, from human placental tissue. This procedure is carried out on a silicic acid column eluted with a continuous chloroform-methanol gradient (19:1 to 4:1); the four glycosphingolipids elute as separate fractions with no need for further separation. The method is simple, rapid, and yields sufficient material to use as analytical standards for several hundred runs. The lipids have been identified by NMR spectroscopy. Placental tissue is freely available in most centers and is an excellent untapped source for these compounds. Given that lactosylceramide is not commercially available and that triaosylceramide (ceramide trihexoside) cannot be obtained in a reliable state, this technique represents an effective solution to this dilemma.
Assuntos
Glicoesfingolipídeos/análise , Placenta/análise , Humanos , Espectroscopia de Ressonância Magnética , MétodosRESUMO
Heterozygotes for Tay-Sachs disease can be distinguished by measuring the serum hexosaminidase activity and calculating the percentage of the heat-labile (A) form. We tested 7565 Ashkenazi Jews in Metropolitan Toronto and found a carrier frequency of 0.071. This figure was similar to the predicted frequency on the basis of caseload over a five-year period. We also found that 15% of women taking oral contraceptives were false-positive carriers. As with pregnant women, these false-positive carriers could be distinguished from true carriers by assaying leukocyte hexosaminidases.
Assuntos
Lipidoses/prevenção & controle , Programas de Rastreamento , Adulto , Anticoncepcionais Orais , Custos e Análise de Custo , Arquitetura de Instituições de Saúde , Reações Falso-Positivas , Feminino , Educação em Saúde , Heterozigoto , Hexosaminidases/sangue , Temperatura Alta , Humanos , Judeus , Leucócitos/análise , Masculino , Métodos , Ontário , Gravidez , VoluntáriosRESUMO
A female child of healthy, unrelated parents presented at 12 months of age with a history of moderately severe developmental delay, macrocephaly, dysmorphic facies, hypotonia, hepatosplenomegaly, mild generalized dysostosis multiplex, mucopolysacchariduria (dermatan and heparan sulfates), and Alder-Reilly bodies in peripheral blood leukocytes. Iduronate sulfatase activity in plasma was markedly depressed: 0.11 units/ml/h (normal, 1.75 +/- 0.56, N = 6). Analyses of arylsulfatases A, B, and C, heparan N-sulfatase, alpha-mannosidase, beta-mannosidase, beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, and alpha-fucosidase activities in plasma, leukocytes, and/or cultured skin fibroblasts were all normal. Urinary sulfatide excretion was also within normal limits. Karyotypes of peripheral blood leukocytes and cultured skin fibroblasts were normal. Serum iduronate sulfatase activities in the parents were in the normal range (father, 1.63 units/ml/h; mother, 1.25 units/ml/h). The results of analyses of restriction fragment length polymorphisms (RFLP) of DNA from cultured skin fibroblasts with the use of probes for loci extending from Xpter to Xq28 showed X chromosome heterozygosity and confirmed the paternal origin of one of the X chromosomes. Studies on sulfur-35 uptake in mixed fibroblast cultures showed cross-correction of [35S]-glycosaminoglycan accumulation between cells from the patient and normal cells or cells from a patient with Hurler disease; however, there was no cross-correction between cells from the patient and those from boys affected with classical Hunter disease. This represents only the second confirmed case of Hunter disease reported in a karyotypically normal girl.
Assuntos
Mucopolissacaridose II , Mucopolissacaridose I/genética , Sulfatases/deficiência , Cromossomo X/ultraestrutura , Arilsulfatases/metabolismo , Feminino , Genes Recessivos , Ligação Genética , Humanos , Iduronato Sulfatase/sangue , Lactente , Cariotipagem , Mucopolissacaridose I/diagnóstico , Mucopolissacaridose I/enzimologiaRESUMO
Gaucher disease (GD) is an inherited deficiency of beta-glucocerebrosidase (EC 3.1.2.45, gene symbol GBA). In type I GD, the CNS is not involved (nonneuronopathic), whereas in type II GD (acute neuronopathic) CNS involvement is early and rapidly progressive, while in type III GD (subacute neuronopathic) CNS involvement occurs later and is slowly progressive. The T6433C (L444P) substitution is prevalent in type GD II. It may occur alone as a single base-pair mutation but often is found as part of a complex allele containing additional GBA nucleotide substitutions, G6468C (A456P) and G6482C (V460V), without (recNciI) or with (recTL) G5957C (D409H). This complex allele is presumed to have formed by recombination (crossover, fusion) of the structural gene with the pseudogene, which contains the mutated sequences. Two complex alleles have never been demonstrated to coexist in any individual. We devised a selective PCR method for the specific amplification of the normal and/or fusion gene. Using this procedure we demonstrated the fusion gene in homozygous form for the first time, in a Macedonian/Ashkenazi Jewish GD type II fetus. Both parents were carriers of the recombination. This was confirmed by direct sequence analysis. A previous conceptus in this family was stillborn at 36 weeks, with features of severe type II GD. Neonates showing a severe clinical phenotype, analogous to the early neonatal lethal disease occurring in mice homozygous for a null allele produced by targeted disruption of GBA, have been described elsewhere, but the specific mutations in these cases have not yet been characterized.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Troca Genética , Doenças Fetais/genética , Doença de Gaucher/genética , Homozigoto , Animais , Sequência de Bases , Clonagem Molecular , Modelos Animais de Doenças , Morte Fetal/genética , Genótipo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Linhagem , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
The mechanism of profound generalized iduronate sulfatase (IDS) deficiency in a developmentally delayed female with clinical Hunter syndrome was studied. Methylation-sensitive RFLP analysis of DNA from peripheral blood lymphocytes from the patient, using MspI/HpaII digestion and probing with M27 beta, showed that the paternal allele was resistant to HpaII digestion (i.e., was methylated) while the maternal allele was digested (i.e., was hypomethylated), indicating marked imbalance of X-chromosome inactivation in peripheral blood lymphocytes of the patient. Similar studies on DNA from maternal lymphocytes showed random X-chromosome inactivation. Among a total of 40 independent maternal fibroblast clones isolated by dilution plating and analyzed for IDS activity, no IDS- clone was found. Somatic cell hybrid clones containing at least one active human X chromosome were produced by fusion of patient fibroblasts with Hprt- hamster fibroblasts (RJK88) and grown in HAT-ouabain medium. Methylation-sensitive RFLP analysis of DNA from the hybrids showed that of the 22 clones that retained the DXS255 locus (M27 beta), all contained the paternal allele in the methylated (active) form. No clone was isolated containing only the maternal X chromosome, and in no case was the maternal allele hypermethylated. We postulate from these studies that the patient has MPS II as a result of a mutation resulting in both the disruption of the IDS locus on her paternal X chromosome and unbalanced inactivation of the nonmutant maternal X chromosome.
Assuntos
Iduronato Sulfatase/genética , Mucopolissacaridose II , Mucopolissacaridose I/genética , Polimorfismo de Fragmento de Restrição , Aberrações dos Cromossomos Sexuais , Cromossomo X , Animais , Linhagem Celular , Células Cultivadas , Criança , Cricetinae , Cricetulus , Enzimas de Restrição do DNA , Feminino , Teste de Complementação Genética , Humanos , Células Híbridas/citologia , Hipoxantina Fosforribosiltransferase/genética , Cariotipagem , Masculino , Mucopolissacaridose I/patologia , Pele/patologiaRESUMO
Three new cases of Sandhoff disease are reported. One infant was the second affected child in a large family. The parents, who were cousins, were part of a large kindred from an isolated community in northern Saskatchewan. We assayed total and heat-stable hexosaminidases in 38 other members of the kindred and found two distinct cohorts. Sixteen individuals had low total and low heat-stable hexosaminidase and were diagnosed as carriers of Sandhoff disease. The values for the remainder were within normal limits. In a retrospective study of data from more than 14,000 Ashkenazi Jews, who were screened for Tay-Sachs disease, six were identified as Sandhoff carriers. Our data indicate that carrier detection requires measurement of both total and heat-stable enzyme activity.
Assuntos
Gangliosidoses/genética , Hexosaminidases/sangue , Consanguinidade , Feminino , Gangliosidoses/diagnóstico , Gangliosidoses/enzimologia , Heterozigoto , Temperatura Alta , Humanos , Lactente , Lipidoses/genética , Masculino , LinhagemRESUMO
Gaucher disease (GD), caused by inherited deficiency of beta-glucocerebrosidase (beta-Glc, EC 3.1.2.45), is classified type I if the CNS is not involved (non-neuronopathic), type II if CNS involvement is early and rapidly progressive (acute neuronopathic), and type III if CNS involvement occurs later and is slowly progressive (subacute neuronopathic). The clinical course is not predictable by measurement of residual beta-Glc activity. Patient classification by identification of specific mutations is more promising: homozygosity for the common A5841->G (N370S) mutation invariably predicts type I; homozygosity for the T6433->C (L444P) mutation usually indicates type III (Norbottnian). Type II disease patients often carry the T6433->C allele together with a complex allele derived in part from the downstream pseudogene by crossover or gene conversion, producing a T6433->C substitution, plus 2 or 3 additional single base substitutions (fusion gene). Employing selective PCR amplification of the structural gene, we detected homozygous T6433C (L444P) point mutations in a Caucasian boy, initially classified as having GD type I, who succumbed to severe visceral GD before age 3 years. A second novel PCR procedure for discriminating between the normal gene and the fusion gene confirmed the homozygous point mutation results. Post mortem neuropathological findings showed neuronal complex lipid accumulation consistent with late-onset type III disease. Although in Norbottnian patients it is generally accepted that onset of neurological findings is delayed, patients with the L444P/L444P genotype can only be initially classified as type III with this ancestry. Other patients described sporadically elsewhere are invariably considered type I until neurological findings arise.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Doença de Gaucher/genética , Mutação Puntual , Sequência de Bases , Pré-Escolar , Doença de Gaucher/classificação , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , FenótipoRESUMO
BACKGROUND AND METHODS: The prevention of Tay-Sachs disease (GM2 gangliosidosis, type 1) depends on the identification of carriers of the gene for this autosomal recessive disorder. We compared the enzyme-based test widely used in screening for Tay-Sachs disease with a test based on analysis of DNA. We developed methods to detect the three mutations in the HEXA gene that occur with high frequency among Ashkenazi Jews: two mutations cause infantile Tay-Sachs disease, and the third causes the adult-onset form of the disease. DNA segments containing these mutation sites were amplified with the polymerase chain reaction and analyzed for the presence of the mutations. RESULTS: Among 62 Ashkenazi obligate carriers of Tay-Sachs disease, the three specific mutations accounted for all but one of the mutant alleles (98 percent). In 216 Ashkenazi carriers identified by the enzyme test, DNA analysis showed that 177 (82 percent) had one of the identified mutations. Of the 177, 79 percent had the exon 11 insertion mutation, 18 percent had the intron 12 splice-junction mutation, and 3 percent had the less severe exon 7 mutation associated with adult-onset disease. The results of the enzyme tests in the 39 subjects (18 percent) who were defined as carriers but in whom DNA analysis did not identify a mutant allele were probably false positive (although there remains some possibility of unidentified mutations). In addition, of 152 persons defined as noncarriers by the enzyme-based test, 1 was identified as a carrier by DNA analysis (i.e., a false negative enzyme-test result). CONCLUSIONS: The increased specificity and predictive value of the DNA-based test make it a useful adjunct to the diagnostic tests currently used to screen for carriers of Tay-Sachs disease. Although some false positive results may be desirable on an enzyme-based test that is used in screening, the DNA test allows precise definition of the carrier state for the known mutations.