| ObjectiveLysosomal storage diseases(LSDs)can manifest as non-immune hydrops fetalis(NIHF),isolated fetal ascites,fetal growth restriction(FGR),fetal liver and/or spleen enlargement and skeletal dysplasia in uterus.Although LSDs are known as rare genetic metabolic diseases,making a clear diagnosis is of great value considering their obvious risk of recurrence.Therefore,it is necessary to establish a screening method to detect then in the clinic practice of prenatal diagnosis.Fourteen kinds of LSDs are strongly related to NIHF,the most common of which are mucopolysaccharidosis(MPS),Gaucher disease,GM1 Gangliosidosis and metachromatic leukodystrophy(MLD).Screening these LSDs from idiopathic NIHF is a feasible way to reduce costs and increase the detection rate.In the invasive prenatal diagnosis of NIHF,amniotic fluid is the most common specimen.These LSDs can be observed by detecting L-?-iduronidase,galactose-6-sulfatase,?-glucuronidase,?-glucosidase,arylsulfatase A and ?-galactose in amniotic cells.Therefore,we aim at discussing the application of enzyme activity detection and making more clear diagnosis of NIHF by establishing the detecting method and normal reference value of lysosomal enzyme activity in our laboratory.Material and MethodsA total number of 68 amniotic fluid samples between 16 and 22 weeks of gestation were included in this study,from women who got amniocentesis due to high risk of chromosomal aneuploidy.None of these women had any history of adverse pregnancy and family history,and fetal ultrasound and amniotic fluid chromosome results showed no abnormalities.All amniotic cells were cultured to expand,harvested with a cell scraper after 21 to 28 days,and stored at-80?.When detecting the enzyme activity,amniotic cells were quickly thawed,which membrane was lysed with double-distilled water,and furtherly made into the homogenate by ultrasonic pulverizer.The protein concentration of the cell homogenate was determined by BCA protein kit.The synthetic fluorescent substrate was used to was used in determination of the activity of L-?-iduronidase,galactose-6-sulfatase,?-glucuronidase,?-glucosidase and ?-galactosidase in human amniotic cells,while colorimetric method was used in determination of the activity of arylsulfatase A.Enzyme activity is a kind of continuous measurement data.Considering the small size of this study,Q-Q chart and scatter chart are used to check whether the data conforms to the normal distribution.The result is expressed as the average±standard deviation(x±s)and 95%confidence interval of enzyme activity is calculated.ResultsThe activities of L-?-iduronidase,galactose-6-sulfatase,?-glucuronidase,?-glucosidase and arylsulfatase A in amniocytes were detected,while ?-galactose was not.We established the normal reference range of activities of five lysosomal enzymes in our laboratory.L-?-iduronidase 61.95±4.93(52.00?71.91)nmol/mg·pro/h N=41 galactose-6-sulfatase 114.63±17.53(97.10?132.16)nmol/mg·pro/17h N=43?-glucuronidase 66.86±4.47(57.92?75.79)nmol/mg·pro/h N=62?-glucosidase 95.94±8.23(79.45?112.44)nmol/mg·pro/h N=56 arylsulfatase A 570.04±59.18(449.17?690.90)nmol/mg·pro/17h N=31ConclusionsOur laboratory established the normal reference range of activities of five lysosomal enzymes,including L-?-iduronidase,galactose-6-sulfatase,?-glucuronidase,?-glucosidase and arylsulfatase A in amniocytes,which provides a new method in the prenatal diagnosis and etiological analysis of NIHF.The activity of lysosomal enzymes varies greatly in the general population,thus lysosomal storage disease(LSD)can not be diagnosed by activity slightly below the reference range.But detection of lysosomal enzyme activities can be viewed as an diagnosis of exclusion in the research of NIHF.Detection of glycosaminoglycan(GAGs)in amniotic fluid and further molecular genetic diagnosis should be conducted if necessary. |
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