RESUMO
Iduronate 2-sulphate sulphatase (EC 3.1.6.-) was found in human placenta in three forms which could be separated by elution from DEAE-cellulose using an NaCl gradient. Form C, most firmly bound to DEAE-cellulose, was 40% larger than the other two (forms A and B in order of ease of elution from the ion exchanger). Forms B and C contained sialic acid which could be removed by neuraminidase digestion. After removal of sialic acid form B became indistinguishable from form A. The enzyme forms found in placenta were compared with those from other human tissues and fluids by means of DEAE-cellulose chromatography and gel chromatography. Serum and amniotic fluid contained only form C, urine and cultured fibroblasts contained the less-anionic forms as well, and kidney contained appreciable amounts only of form A. Pre- and post-natal diagnosis of the Hunter syndrome both involve measurements on the enzyme which is present in form C. This is not accompanied by less-anionic forms which constitute the bulk of the enzyme as it is isolated from easily available sources such as urine.
Assuntos
Líquidos Corporais/enzimologia , Iduronato Sulfatase/isolamento & purificação , Isoenzimas/isolamento & purificação , Placenta/enzimologia , Sulfatases/isolamento & purificação , Líquido Amniótico/enzimologia , Cromatografia DEAE-Celulose , Feminino , Fibroblastos/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Rim/enzimologia , Masculino , Peso Molecular , Mucopolissacaridose II/enzimologia , Neuraminidase/farmacologia , GravidezRESUMO
We have studied the carrier state of the Hunter syndrome using a series of obligate carriers, females at high genetic risk, and normal control women. Specific odds of a female being a carrier of Hunter syndrome were based on serum levels of iduronate 2-sulphate sulphatase activity. These, together with the prior genetic odds, may be used in calculating the overall odds of a woman being a carrier. Iduronate 2-sulphate sulphatase levels were found to increase significantly with age. Obligate carriers from families of severe cases had significantly lower enzyme levels compared with those from families of mild cases. In contrast, enzyme levels in sera of mild and severe cases were not significantly different. With the accumulation of more data the effect of age of the potential carrier and the severity of the disease may have to be taken into consideration in the risk calculation. Hair-root analysis was more reliable in the detection of carriers than estimation of serum enzyme levels, but some individuals could not be classified with confidence by hair-root analysis alone. Carrier detection was most reliable when hair-root analysis and serum enzyme levels were taken together.
Assuntos
Triagem de Portadores Genéticos/métodos , Mucopolissacaridose II , Mucopolissacaridose II/genética , Adolescente , Adulto , Idoso , Feminino , Ligação Genética , Cabelo/enzimologia , Hexosaminidases/metabolismo , Humanos , Iduronato Sulfatase/sangue , Iduronato Sulfatase/metabolismo , Masculino , Pessoa de Meia-Idade , Mucopolissacaridose II/enzimologia , Cromossomo X , beta-N-Acetil-HexosaminidasesRESUMO
A more sensitive assay procedure has been developed for the enzyme iduronate 2-sulphate sulphatase which is deficient in the Hunter syndrome. The substrate is the same as previously described by Lim et al. [1], O-(alpha-L-idopyranosyluronic acid 2-sulphate)-(1leads to 4)-2,5 anhydro-D-[3H-1]mannitol 6-sulphate, but, after incubation, it is separated from the product by ion-exchange chromatography on a micro-column of Dowex 1 x 2 (Cl-1) instead of high voltage electrophoresis or ECTEOLA cellulose chromatography. Since the blank correction is then much smaller, a shorter incubation time can be used and conversion of the substrate reduced from approximately 50% down to levels where complications resulting from substrate depletion and product inhibition are minimal. Using whole serum the apparent Km for the substrate is 0.2 mmol/l. With an incubation time of 20 min, sera from heterozygotes exhibited approximately 35% of the normal levels of iduronate 2-sulphate sulphatase (0.11-0.61, mean 0.34 nmol.h-1.mg-1 protein for carriers; 0.24-2.35, mean 0.94 nmol.h-1.mg-1 protein for 37 normal females). Serum analyses can thus be used to supplement those on hair roots in the detection of carriers of the Hunter syndrome.
Assuntos
Iduronato Sulfatase/sangue , Mucopolissacaridose II/diagnóstico , Sulfatases/sangue , Cromatografia por Troca Iônica/métodos , Ensaios Enzimáticos Clínicos , Triagem de Portadores Genéticos , Cabelo/enzimologia , Homozigoto , Humanos , CinéticaRESUMO
A study has been undertaken to determine the efficiency of current methods in providing an adequate amount of chorionic villus DNA for antenatal diagnosis using recombinant DNA techniques or enzyme assay. Chorionic biopsies were obtained from 40 women undergoing elective first trimester termination of pregnancy (8-12 weeks) under general anaesthesia. The villus tissue was isolated from maternal tissue under a dissection microscope and the presence of any remaining contamination was ascertained by conventional histology and immuno-cytochemical examinations. A high level of success was achieved in obtaining a pure fetal sample. In the first 20 samples the DNA yield obtained using the method of Williamson et al [1] was found to be 0.5 +/- 0.5 micrograms/mg wet weight of villus tissue (mean +/- 1 SD). In the subsequent 20 biopsies using a modified procedure, the yield was significantly improved to 1.0 +/- 0.65 (p less than 0.002). A normal range for the enzyme iduronate sulphatase, which is deficient in Hunter's syndrome (mucopolysaccharidosis II), is also reported. It is suggested that as little as 20 mg of chorionic villi may be used to provide sufficient material for a reliable study using recombinant DNA or biochemical methods.
Assuntos
Vilosidades Coriônicas/análise , Ensaios Enzimáticos Clínicos , DNA/análise , Iduronato Sulfatase/análise , Placenta/análise , Diagnóstico Pré-Natal , Sulfatases/análise , Líquido Amniótico/citologia , Líquido Amniótico/enzimologia , Biópsia , Vilosidades Coriônicas/citologia , Vilosidades Coriônicas/enzimologia , DNA Recombinante , Feminino , Triagem de Portadores Genéticos , Doenças Genéticas Inatas/diagnóstico , Humanos , Mucopolissacaridose II/diagnóstico , Distrofias Musculares/diagnóstico , Gravidez , Primeiro Trimestre da GravidezRESUMO
Sixteen pregnancies at risk for Hunter syndrome have been monitored by amniocentesis. Iduronate 2-sulphate sulphatase levels were measured in amniotic fluid, cultured amniotic fluid cells and cord blood. Thirteen of the pregnancies resulted in normal livebirths, two are continuing and one affected pregnancy was terminated. Reduced enzyme levels were observed in either amniotic fluid, cells or cord blood for four female fetuses. Such fetuses are likely to be carriers expressing reduced enzyme levels. The affected male fetus had reduced enzyme activity in amniotic fluid; insufficient cells were cultured for enzyme estimation, however no enzyme activity was detected in fetal liver after termination. Eight cord blood enzyme estimations have been performed, five confirming normal male infants.