Postnatal and prenatal diagnosis of mucopolysaccharidosis type III (Sanfilippo syndrome) / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis (MPS) types IIIA, B, C, D are a group of autosomal recessive lysosomal storage disorders caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. MPSIIIA and MPSIIIB involve deficiencies of heparan N-sulfatase (SGSH) and alpha-N-acetylglucosaminidase (NAGLU). MPS IIIA and MPS IIIB are more common than MPS IIIC and IIID. The present study aimed to establish two enzyme assay methods for SGSH and NAGLU activities for carrying out postnatal and prenatal diagnosis of MPSIIIA and IIIB by means of SGSH and NAGLU activity assay on plasma, leukocyte, uncultured chorionic villi (CV) and cultured amniotic fluid cells (AF cell) using two newly synthesized substrates. Mutation analysis of SGSH gene was also performed.</p><p><b>METHODS</b>Two fluorigenic substrate (4-methylumbelliferyl-alpha-D-N-sulphoglucosaminide.Na and 4-methylumbelliferyl-alpha-N-acetylglucosaminide) were used for the assay of SGSH and NAGLU activity. SGSH activity in leukocyte was determined for diagnosis MPSIIIA proband. NAGLU activity was determined in plasma for diagnosis of MPSIIIB proband. Twelve cases with MPS III were enrolled in this study, 4 were female and 8 were male, age 3 - 10 years and were from 10 unrelated families. Eight exons of SGSH gene were amplified by PCR. The mutations of the patients were characterized by direct sequencing of the amplified DNA fragments. Prenatal diagnosis in 3 pregnancies at risk was carried out according to NAGLU activity on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation.</p><p><b>RESULTS</b>The SGSH activities in leukocyte of normal controls were 4.4 - 8.1 nmol/(17 h.mg protein). The NAGLU activity in plasma of normal controls was 33.3 - 62.4 nmol/(4 h.ml). The NAGLU activities were 44.9 - 91.7 nmol/(17 h.mg protein) and 53.2 - 82.2 nmol/(17 h.mg protein) in CV and cultured AF cells respectively. Five cases of MPS IIIB and 7 cases of MPS IIIA were diagnosed. The mutation analysis of SGSH gene showed 6 mutations (G191R, D235N, R377C, E447K, R233X and D219Wfs264X), only one of which (D219Wfs264X) has not been previously reported. Prenatal diagnosis was performed on 3 pregnancies at risk. NAGLU activity of one affected fetus was 1.5 nmol/(17 h.mg protein) in AF cell.</p><p><b>CONCLUSIONS</b>The method using synthesized fluorigenic 4-methylumbelliferyl-substrates were sensitive, rapid and convenient assay of SGSH and NAGLU activity and were reliable for early prenatal diagnosis. Mutation analysis on MPS IIIA patients suggests new possibilities for molecular diagnosis of the disease.</p>

Postnatal and prenatal diagnosis of mucopolysaccharidosis type II (Hunter syndrome) / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis type II (MPS II, Hunter syndrome, OMIM 309900) is an X-linked recessive lysosomal storage disease resulting from a deficiency of iduronte-2-sulphate sulphatase (IDS). The present study aimed to establish an enzyme assay method for IDS activity for carrying out postnatal and prenatal diagnosis of MPS II by means of IDS activity assay on plasma, uncultured chorionic villi (CV) and cultured amniotic fluid cells (AF cell) using a new synthesized substrate.</p><p><b>METHODS</b>A fluorigenic substrate (4-methylumbelliferyl-alpha-iduronate-2-sulphate, MU-alpha-Idu-2S) was used for the assay of IDS activity. IDS activity in plasma was determined for diagnosis of the proband. Prenatal diagnosis in 10 pregnancies at risk was carried out according to IDS activity on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation. At the same time, IDS activity was also determined in the maternal plasmas to observe the change of IDS activity in pregnancy. The fetal sex determination was performed by PCR amplification of the ZFX/ZFY genes.</p><p><b>RESULT</b>The IDS activity in plasma of normal controls and obligate heterozygotes were 240.2 - 668.2 nmol/(4 hxml) and 88.7 - 547.9 nmol/(4 hxml), respectively, while the enzyme activities in plasmas were in the range of 0.3 - 18.6 nmol/(4 hxml) in affected male. The IDS activities were 37.2 - 54.9 nmol/(4 hxmg protein) and 21.4 - 74.4 nmol/(4 hxmg protein) in CV and cultured AF cells respectively. Out of 50 suspected cases, 46 were diagnosed as having MPS II and 4 were excluded. Prenatal diagnosis was performed on 10 pregnancies at risk. Four of 5 male fetuses [IDS activity were 4.7, 1.8, 7.0 nmol/(4hxmg protein) in CV, 0.6 nmol/(4 hxmg protein) in AF cell] were diagnosed as having MPS II and the other 5 fetuses were normal females [IDS activity were: 48.7, 5.9, 25.2 nmol/(4 hxmg protein) in CV, 55.2, 40.9 nmol/(4 hxmg protein) in AF cell]. Increased IDS activity was observed in plasma of the pregnant women with unaffected fetuses, while the IDS activity decreased in pregnancies with affected fetuses. IDS activity of one female fetus was very low [5.9 nmol/(4 hxmg protein)], but the IDS activity in maternal plasmas increased, this fetus was a normal female.</p><p><b>CONCLUSIONS</b>The method using a synthesized fluorigenic 4-methylumbelliferyl-substrate was a sensitive, rapid and convenient assay of IDS activity and was reliable for early prenatal diagnosis. Determination of fetal sex would be helpful in excluding the female fetus with low IDS activity from being considered as an affected male fetus. It would be further helpful if IDS activity in maternal plasma was taken into account.</p>