RESUMO
Subcellular fractions were isolated by differential centrifugation from pure suspensions of human blood lymphocytes incubated with and without phytohemagglutinin (PHA). Between 30 and 120 min after addition of PHA to intact cells, redistribution of acid hydrolases (beta glucuronidase, acid phosphatase), from a 20,000 g x 20 min granular fraction into the corresponding supernatant, was observed. No increase in total acid hydrolase activity was found at these times. The mitochondrial marker enzyme, malate dehydrogenase, did not undergo redistribution. Granules derived from PHA-treated cells became more fragile upon subsequent incubation with membrane-disruptive agents in vitro (streptolysin S, filipin). These changes were associated with an increase in the over-all permeability of the stimulated cell to substances in the surrounding medium, such as neutral red. Augmentation of dye entry into lymphocytes required intact metabolism as judged by response to temperature and inhibitors (cyanide, antimycin A, 2,4-dinitrophenol). PHA, however, did not release enzyme activity from hydrolase-rich granules in vitro or render them more susceptible to subsequent challenge with membrane-disruptive agents. These studies suggest that PHA induces early changes in the surface of lymphocytes. The consequent redistribution of acid hydrolases may play a role in remodeling processes of the stimulated cells.
Assuntos
Hidrolases/metabolismo , Lectinas/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Lisossomos/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Antimicina A/farmacologia , Corantes , Cianetos/farmacologia , Dinitrofenóis/farmacologia , Glucuronidase/metabolismo , Humanos , Malato Desidrogenase/metabolismo , Proteínas/metabolismo , TemperaturaRESUMO
A technique has been developed for the detection of inborn errors by multiple enzyme analysis of lymphocytes stimulated by phytohemagglutinin. Its practicality has been demonstrated in Pompe's disease in which there is a deficiency of acid alpha-1,4-glucosidase (E.C.3.2.1.20).
Assuntos
Glucosidases/sangue , Heterozigoto , Linfócitos/enzimologia , Erros Inatos do Metabolismo/diagnóstico , Ensaios Enzimáticos Clínicos , Técnicas de Cultura , Humanos , Lectinas , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genéticaRESUMO
Cystathionase activity in a lymphoid cell line extracts from a vitamin B6-responsive patient with cystathioninuria was increased strikingly by pyridoxal phosphate. Immunodiffusion with antiserum to human hepatic cystathionase showed identity between this cystathionase protein and cystathionase from an extract of normal lymphoid cells. Neither an increase in cystathionase activity nor immunochemical identity was found using extract of cells from a B6-unresponsive patient.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Cistationina gama-Liase/deficiência , Liases/deficiência , Piridoxina/uso terapêutico , Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Linhagem Celular , Reações Cruzadas , Cistationina/urina , Cistationina gama-Liase/imunologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Mutação , Fosfato de Piridoxal/farmacologiaRESUMO
Different clinical expressions of acid alpha-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid alpha-glucosidase (EC 3.2.1.20) was purified from normal and from adult acid alpha-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, alpha-methylglucoside and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid alpha-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid alpha-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid alpha-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzymes. The findings indicate that this case of infantile acid alpha-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells.
Assuntos
Glucosidases/deficiência , Doença de Depósito de Glicogênio Tipo II/enzimologia , Doença de Depósito de Glicogênio/enzimologia , alfa-Glucosidases/deficiência , Adulto , Formação de Anticorpos , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Eletroforese em Gel de Amido , Feminino , Fibroblastos/enzimologia , Genes , Humanos , Concentração de Íons de Hidrogênio , Imunodifusão , Lactente , Masculino , Maltose , Mutação , Placenta/enzimologia , Gravidez , Pele/enzimologia , alfa-Glucosidases/isolamento & purificaçãoRESUMO
The metabolism of (14)C-labeled testosterone by cultured human fibroblasts and amniotic fluid cells was investigated. Radiolabeled testosterone was incubated with the cultured cells for 48 hr, and the labeled metabolites present in the medium were subsequently identified. The major metabolic products of testosterone formed by cultured fibroblasts were Delta(4)-androstenedione, dihydrotestosterone, androsterone, and androstanediol. The amount of testosterone metabolized through each of two pathways was calculated and used to form a ratio designated the 17beta-hydroxyl/17-ketonic ratio. Fibroblasts from normal male and female children and adult females had high 17beta-hydroxyl/17-ketonic ratios indicating testosterone metabolism occurred primarily through the 17beta-hydroxyl pathway. There was change in the pattern of testosterone metabolism with age in males, i.e., adult males had much lower 17beta-hydroxyl/17-ketonic ratios than did male children. The testosterone metabolism of fibroblast cultures derived from three children with testicular feminization and their mothers was compared to normal age and sexmatched controls. Fibroblasts of children with testicular feminization metabolized testosterone predominantly through the 17-ketonic pathway and manifested a pattern of testosterone metabolism distinctly different from their sex and age matched controls. The mothers of children with testicular feminization could be distinguished from normal females by their much lower 17beta-hydroxyl/17-ketonic ratios. The much lower amounts of dihydrotestosterone and androstanediol produced by fibroblasts from patients with testicular feminization as compared with normals suggests there is a decrease in testosterone 5alpha-reductase activity in these patients. Cultured amniotic fluid cells metabolized testosterone to the same four major metabolites found in fibroblast cultures, but their activity was much lower than that of fibroblasts. Most of the amniotic fluid cell cultures metabolized testosterone largely through the 17beta-hydroxyl pathway as did fibroblasts from normal children.
Assuntos
Líquido Amniótico/metabolismo , Células Cultivadas/metabolismo , Fibroblastos/metabolismo , Testosterona/metabolismo , 17-Cetosteroides/biossíntese , Líquido Amniótico/citologia , Síndrome de Resistência a Andrógenos/diagnóstico , Síndrome de Resistência a Andrógenos/metabolismo , Androstanos/biossíntese , Androsterona/biossíntese , Isótopos de Carbono , Criança , Pré-Escolar , Cromatografia em Camada Fina , Di-Hidrotestosterona/biossíntese , Feminino , Humanos , Técnicas In Vitro , Masculino , Oxigenases de Função Mista/metabolismo , Gravidez , Pele/citologia , Esteróis/biossíntese , Testosterona/isolamento & purificaçãoRESUMO
Permanent cell lines have been established from a spleen nodule and lymph node of a male Hodgkin's disease (HD) patient whose father has the same disease. Th in vitro growth pattern morphological and cytogenetic characteristics of these lines maintained continuously for over 2 years are described. The cultures contain a population of mixed cell types that grow in suspension. Between 5 and 10% of the cells have surface immunoglobulins M and D. B-cell alloantigens are also detectable. While the cultures are predominantly lymphoid, some of the large cells, by light and electron microscopy, resemble the Reed-Sternberg and Hodgkin's cells of the original biopsies. Although the cells maintain the human diploid karyotype, they are heterotransplantable in nude mice. After 14 months of culture, chromosome rearrangement and losses, commonly seen in leukemic bone marrow, occurred. Close to 100% of the cells are Epstein-Barr nuclear antigen positive, but they lack Epstein-Barr viral (EBV) capsid antigen and EBV-induced early antigen. Nucleic acid hybridization tests indicated that there were no more than two EBV genome equivalents per cell. Tests with HD sera free of anti-EBV were negative. Electron microscope examination of the cells revealed the presence of intracellular as well as extracellular rare pleomorphic particles ranging from 400 to 1200 A. The nature of these particles, which increased in number after the cultures were treated with halogenated pyrimidines but not with dimethyl sulfoxide, remains questionable. The cultures derived from the mouse-passaged HD cells, however, had reverse transcriptase activity and readily identifiable type C particles which were probably of murine origin. These cultures have some unique features that make them useful in studying the perplexing pathological entity of HD.
Assuntos
Linhagem Celular , Doença de Hodgkin/patologia , Adolescente , Animais , Antígenos Virais/isolamento & purificação , Aberrações Cromossômicas , DNA Viral/isolamento & purificação , Herpesvirus Humano 4/imunologia , Doença de Hodgkin/etiologia , Doença de Hodgkin/metabolismo , Humanos , Corpos de Inclusão Viral , Linfonodos/patologia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , DNA Polimerase Dirigida por RNA/metabolismo , Baço/patologia , Transplante HeterólogoRESUMO
Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.
Assuntos
Membrana Celular/enzimologia , Doença de Gaucher/enzimologia , Glucosidases/metabolismo , Fibroblastos/enzimologia , Heterozigoto , Homozigoto , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Cinética , Leucócitos/enzimologia , Linfa/enzimologia , Polietilenoglicóis/farmacologia , Ácido Taurocólico/farmacologiaRESUMO
Progressive muscle weakness in acid maltase deficiency (AMD) is associated with intralysosomal accumulation of glycogen and altered myofibrillar morphology. A rapid fall in circulating branched chain amino acids after protein ingestion in a child with AMD suggested that increased net muscle protein catabolism may play a part in the pathogenesis of this condition. To reduce this muscle catabolism, the patient was treated with a high-protein diet for 12 months. This has reversed the weakness and wasting, with improvement in muscle function, exercise tolerance, and growth.
Assuntos
Proteínas Alimentares/administração & dosagem , Glucana 1,4-alfa-Glucosidase/deficiência , Glucosidases/deficiência , Aminoácidos de Cadeia Ramificada/metabolismo , Pré-Escolar , Glicogênio/metabolismo , Humanos , Masculino , Músculos/enzimologia , Músculos/ultraestrutura , Doenças Musculares/dietoterapiaRESUMO
Two siblings, 9 and 4 1/2 years old, had alpha-L-fucosidase deficiency, angiokeratoma, progressive psychomotor retardation, neurologic signs, coarse facila features, and dysostosis multiplex. It appears that genetic heterogeneity is present in fucosidosis; there are at least two types. In type 1, patients have no vascular lesions, but have rapid psychomotor regression, severe and rapidly progressing neurologic signs, elevated sodium and chloride excretion in the sweat, and fatal outcome before the sixth year. In type 2, patients have angiokeratoma, milder psychomotor retardation and neurologic signs, longer survival, and normal salinity in the sweat. Quantitative studies on erythrocytes and in saliva disclosed severely increased expressions of Lea and Leb. Biopsies of skin and gingiva showed alterations as seen in angiokeratoma. There was also evidence of lysosomal storage in vascular endothelium, eccrine sweat gland epithelium, and fibroblasts of the skin.
Assuntos
Dissacaridases/metabolismo , Mucopolissacaridoses/genética , alfa-L-Fucosidase/metabolismo , Adulto , Criança , Pré-Escolar , Doença de Fabry/complicações , Feminino , Heterozigoto , Humanos , Masculino , Mucopolissacaridoses/complicações , Mucopolissacaridoses/diagnósticoRESUMO
Cytogenetic analysis of the products of conception in a pregnancy at risk for Bloom's syndrome (BS) documented the karyotype 49,XX, +2, +8, +11. Autosomal triple trisomy has previously been reported in abortuses but is exceedingly rare. Other interesting but previously unreported observations made during the present study were the following: BS in a Brazilian individual, the first instance of BS diagnosed in South America; transmission of the BS mutation in Jews that are non-Ashkenazi; a medulloblastoma in the propositus, the first malignant brain tumor reported in BS; and, as in all previously examined pregnancies at risk for BS, non-homozygosity for the BS mutation.
Assuntos
Síndrome de Bloom/genética , Trissomia , Aborto Espontâneo , Adulto , Síndrome de Bloom/diagnóstico , Síndrome de Bloom/etnologia , Brasil , Neoplasias Cerebelares/genética , Pré-Escolar , Córion/ultraestrutura , Feminino , Humanos , Judeus/genética , Cariotipagem , Meduloblastoma/genética , Gravidez , Diagnóstico Pré-Natal , Troca de Cromátide IrmãRESUMO
Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring alpha-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent alpha-L-fucosidase activity were obtained; the pH optimum was 4.5. alpha-L-Fucosidase activity (mean +/- SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 +/- 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.
Assuntos
alfa-L-Fucosidase , Medula Óssea/patologia , Células Cultivadas , Criança , Pré-Escolar , Fibroblastos/enzimologia , Doença de Gaucher/enzimologia , Humanos , Leucodistrofia Metacromática/enzimologia , Manosidases/análise , Mucopolissacaridoses/enzimologia , alfa-Manosidase , beta-Galactosidase/análiseRESUMO
Larsen syndrome is a heterogeneous (autosomal dominant or recessive) disorder of characteristic facial changes, multiple joint dislocations, and bone deformities. Few data on the adult presentation of the recessive form of this disorder have been reported; thus, we set out to describe two sibs thought to be affected with autosomal recessive Larsen syndrome who were evaluated as infants and later as adults. Aside from secondary joint changes and the presence of cataracts, changes described in children with autosomal recessive Larsen syndrome were noted. Three years after evaluation, the sister gave birth to a daughter with Larsen syndrome. This occurrence raises the possibility of germ-line mosaicism as the mode of inheritance in this family. Thus, germ-line mosaicism must be considered in the genetic counseling of families with Larsen syndrome in which neither parent appears affected. These patients also illustrate that despite the severe skeletal and joint deformities, the prognosis can be good with careful orthopedic management.
Assuntos
Face/anormalidades , Mutação em Linhagem Germinativa , Artropatias/congênito , Luxações Articulares/congênito , Luxações Articulares/genética , Mosaicismo , Anormalidades Múltiplas/genética , Adulto , Doenças Ósseas/congênito , Doenças Ósseas/genética , Feminino , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Luxação Congênita de Quadril/genética , Humanos , Lactente , Artropatias/genética , Masculino , Escoliose/genética , SíndromeRESUMO
A patient with distal arthrogryposis, congenital dislocations of the hips, a prominent forehead, epicanthal folds, thin lips, and a poorly defined philtrum was found to have a deletion of 15q and a duplication of 16q. Her mother, maternal grandmother, and great grandmother had a balanced t(15q-, 16q+). The gene for adenine phosphoribosyl transferase was assignable to the 16q22----16qter area that was duplicated.
Assuntos
Adenina Fosforribosiltransferase/genética , Artrogripose/genética , Deleção Cromossômica , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 16 , Família Multigênica , Pentosiltransferases/genética , Translocação Genética , Amniocentese , Bandeamento Cromossômico , Mapeamento Cromossômico , Feminino , Humanos , Lactente , Cariotipagem , Linhagem , GravidezRESUMO
Mesomelic dysplasia is a form of short-limb dwarfism characterized by disproportionate shortness of the middle segment of all limbs. Included in this category of skeletal disorders is the Nievergelt syndrome, which typically manifests a rhomboidal shape of the tibiae and fibulae, an unusual foot deformity, radioulnar synostosis, and dysplasia of the elbow and knee joints. We describe a patient with mesomelic dysplasia with findings suggestive of the Nievergelt syndrome and with absence of fibulae and hexadactyly.
Assuntos
Anormalidades Múltiplas/diagnóstico , Nanismo/diagnóstico , Fíbula/anormalidades , Dedos do Pé/anormalidades , Anormalidades Múltiplas/genética , Nanismo/diagnóstico por imagem , Nanismo/genética , Humanos , Lactente , Masculino , Radiografia , SíndromeRESUMO
We report on a 4(1/2)-year-old girl, who presented with multiple minor anomalies consistent with trisomy for 4p. GTG-banding identified a de novo terminal inversion duplication of distal 4p, dup(4)(p16.3p15.3). Fluorescence in situ hybridization (FISH) with a wcp4 probe confirmed the chromosome 4 origin of the additional material. FISH with a 4p subtelomere probe, D4F26, showed no signal on the dup(4) chromosome identifying a deletion of this region. Molecular analysis of 4p STS loci confirmed the subtelomeric deletion and showed loss of the paternal allele in this region. The paternal origin of the deleted region and homozygosity for one of the two paternal alleles within the region of the duplication suggests that a sister chromatid rearrangement on the paternal chromosome 4 was involved in the formation of the dup(4) chromosome. To date, the best characterized mechanisms of formation of chromosome duplications are terminal inversion duplications of 8p, which were shown to be derived from rearrangements at maternal meiosis-I. Our data show that mechanisms other than a maternal meiosis-I rearrangement can lead to the formation of terminal inversion duplications. FISH analysis with the appropriate subtelomeric probes is warranted in terminal inversion duplications to check for associated deletions.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 4/genética , Telômero/genética , Pré-Escolar , Bandeamento Cromossômico , Deleção Cromossômica , Inversão Cromossômica , Mapeamento Cromossômico , DNA/genética , Saúde da Família , Feminino , Duplicação Gênica , Genótipo , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Repetições de Microssatélites , LinhagemRESUMO
Uniparental disomy (UPD) for several chromosomes has been associated with disease phenotypes. Maternal UPD for chromosome 14 has been described and has a characteristic abnormal phenotype. Paternal UPD14 is rare and only three previous cases have been reported. We describe a new case of paternal UPD for chromosome 14 in an infant with a 45,XX,der(13q;14q) karyotype, which was confirmed by molecular analysis. The proposita had findings similar to those of the previous cases of patUPD14 and we conclude that there is a characteristic patUPD14 syndrome most likely due to imprinting effects. Couples with Robertsonian translocations involving chromosome 14 should be counseled as to the possibility of UPD14 and the option of prenatal diagnosis when indicated.
Assuntos
Anormalidades Múltiplas/genética , Aneuploidia , Cromossomos Humanos Par 14 , Adulto , Mapeamento Cromossômico , Feminino , Marcadores Genéticos , Impressão Genômica , Humanos , Lactente , Linfócitos , Masculino , Linhagem , Polimorfismo Genético , Diagnóstico Pré-Natal , Translocação GenéticaRESUMO
From February 1969 to August 1976, we studied 1,048 amniotic fluids. Of these, 958 (91.4%) were primarily for prenatal cytogenetic diagnosis. Cytogenetic studies were attempted in 1,021 cases; the diagnosis was successful in 1,000 of these. The failure rate of obtaining a diagnosis from the amniotic fluid cell culture of the first amniocentesis was 5% (50 cases); 29 cases had a repeat tap and successful diagnosis was achieved in all. In 21 cases, a repeat tap was refused. Thus, the overall failure rate of obtaining a final cytogenetic diagnosis was 2.06% (21/1,021). There were 32 fetal losses after amniocentesis including 16 spontaneous second trimester abortions, 7 fetal deaths in utero and 9 stillbirths. In two additional cases, fetal death had occurred before amniocentesis. This number of fetal losses does not exceed the number that would be expected in the same maternal age group without amniocentesis. In our series, the frequencies of trisomy in maternal age groups 40 years and over, 37-39 years, 35-36 years, and under 35 years were 4.5, 3.14, 0 and 0% respectively. These frequencies are comparable to those reported from other prospective prenatal studies and higher than those of retrospective live born studies. Various problems and pitfalls in prenatal cytogenetic diagnosis are discussed.
Assuntos
Amniocentese , Cromossomos Humanos 6-12 e X , Síndrome de Down/diagnóstico , Idade Materna , Gravidez de Alto Risco , Trissomia , Adulto , Amniocentese/efeitos adversos , Bandeamento Cromossômico/métodos , Feminino , Morte Fetal/etiologia , Humanos , Cariotipagem , Mosaicismo , GravidezRESUMO
The following is a summary of presentations given during an ancillary meeting to the 1993 American Society of Human Genetics Meeting in New Orleans, LA. This ancillary meeting, entitled "Recent Research on Chromosome 4p Syndromes and Genes," reviewed the history of the Wolf-Hirschhorn syndrome (WHS), the natural history of patients with WHS, and the smallest region of deletion associated with the WHS. The proximal 4p deletion syndrome and the duplication 4p syndrome were also described and advice was offered regarding detection of chromosome 4p deletions, duplications, and rearrangements. The current status of the physical map of chromosome 4p with emphasis on the genes that map to the 4p16 region was presented along with a preliminary phenotypic map of 4p16. The goal of this format was to provide a comprehensive review of the clinical presentations, diagnostic capabilities, and genetic mapping advances involving chromosome 4p.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 4 , Anormalidades Múltiplas/genética , Deleção Cromossômica , Mapeamento Cromossômico , Rearranjo Gênico , Genética , Humanos , Sociedades Científicas , Síndrome , Estados UnidosRESUMO
Within the past 24 months, we have performed prenatal diagnostic studies in 4 pregnancies known to be at risk for well-described genetic syndrome involving renal abnormalities, ie, Meckel syndrome, Roberts syndrome, and bilateral renal agenesis. The diagnostic techniques utilized were ultrasonographic scanning (B-mode and grey scale), biochemical assays, and radiographic evaluation. The ultrasound finding common to the 3 affected cases was extreme oligohydramnios, which we considered indirect evidence that renal anomalies were present. The ultrasound scans of the fetuses affected with Meckel and Roberts syndrome demonstrated anechoic cystic spaces in the abdomen, representing the enlarged dysplastic cystic kidneys. An encephalocele was well demonstrated by B-mode scan in the fetus with Meckel syndrome. The absence of normal limbs in the Roberts syndrome was evident on serial grey scale scans of the fetus. Biochemical and radiographic studies provided results consistent with the suspected diagnoses. The importance of providing genetic counseling and prenatal diagnosis to families at risk is emphasized.
Assuntos
Doenças Genéticas Inatas/diagnóstico , Rim/anormalidades , Diagnóstico Pré-Natal , Líquido Amniótico/metabolismo , Feminino , Aconselhamento Genético , Humanos , Masculino , Gravidez , UltrassonografiaRESUMO
The lysosomal enzyme acid alpha glucosidase (GAA) or acid maltase is deficient in glycogen storage disease type II. We sought to determine the molecular basis for the disease in an adult-onset patient, unusual for very low enzyme activity similar to that seen with the infantile-onset form and with a previously reported defect in phosphorylation. We constructed cDNA and genomic DNA libraries from the patient's cell line (GM 1935) and determined the nucleotide sequence of the coding region. There were three base-pair substitutions in one allele (C1935 to A; G2446 to A and C2780 to T), all predicting amino acid changes (Asp-645 to Glu; Val-816 to Ile and Thr-927 to Ile). To determine which of the three base-pair substitutions resulted in loss of enzyme activity, we next utilized primer-directed mutagenesis and transient gene expression in an SV40-immortalized GAA-deficient fibroblast cell line. Only the construct containing the G2446 to A mutation (Val-816 to Ile) lost GAA enzyme activity, while the other two substitutions (including the Thr-927 to Ile change that predicts a loss of a potential site for N-linked glycosylation and mannose phosphorylation) each resulted in enzyme activity equal to the control. Analysis of RFLPs in genomic DNA, as well as sequence analysis for the three base-pair alterations, indicated that the patient was a genetic compound. We next digested PCR-amplified cDNA (reverse-transcribed from RNA) with Aat II to detect the base-pair 1935 substitution and found that virtually all of the mRNA was derived from the allele with the three base-pair substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)