中华儿科杂志
中華兒科雜誌
중화인과잡지
Chinese Journal of Pediatrics
2008年
6期
407-410
,共4页
张为民%施惠平%孟岩%李贝特%邱正庆%刘俊涛
張為民%施惠平%孟巖%李貝特%邱正慶%劉俊濤
장위민%시혜평%맹암%리패특%구정경%류준도
黏多糖累积病Ⅲ型%诊断,鉴别%产前诊断%突变
黏多糖纍積病Ⅲ型%診斷,鑒彆%產前診斷%突變
점다당루적병Ⅲ형%진단,감별%산전진단%돌변
Mucopolysaccharidosis type Ⅲ%Diagnosis,differential%Prenatal diagnosis%MUtation
目的 建立以人工合成荧光底物测定乙酰肝素N-硫酸酯酶(SGSH),α-N-乙酰葡萄糖苷酶(NAGLU)活性的方法,应用这两种方法对黏多糖贮积症Ⅲ型中的A、B亚型(MPSⅢA和ⅢB)病例进行鉴别诊断,并对MPSⅢB高危妊娠的孕妇进行了产前诊断,同时从分子遗传学方面对MPSⅢA患儿进行了SGSH基因分析.方法 采用人工合成的荧光底物(4-甲基伞形酮-α-D-N-硫酸葡萄糖苷4-methylumbelliferyl-α-D-N-sulphoglucosaminide.Na,4-甲基伞形酮-N-乙酰-α-葡萄糖苷4-methylumbelliferyl-α-N-acetylglucosaminide)测定疑似患儿白细胞和血浆中SGSH及NAGLU活性,在收集的12例患儿中,女4例,男8例,年龄3~10岁.来自10个无相关的家庭.用MPSⅢA的患儿外周血白细胞DNA进行SGSH基因的外显子PCR扩增,并通过的序列测定确定突变位置.用绒毛或经培养的羊水细胞为检材,对MPSⅢB高危妊娠的孕妇进行产前诊断.结果 获得中国正常人白细胞中SGSH活性正常值4.4～8.1 nmot/(17 h·mg蛋白),各种组织中NAGLU活性正常值:血浆中为33.3～62.4 nmoL/(4 h·ml),绒毛组织为44.9~91.7 nmol/(17 h ·mg蛋白),经培养的羊水细胞为53.2-82.2 nmol/(17 h·mg蛋白);在12例疑似患儿中确诊了7例MPSⅢA(其中两例为同胞),5例MPS ⅢB患者(其中2例为同胞),SGSH基因分析发现6种突变类型,已知突变5种(G191R,D235N,R377C,E447K和R233X),插入突变1种(D219Wfs264X),此插入突变为新生突变.3例MPSⅢB产前诊断中,2例胎儿正常,1例胎儿受累.结论 应用荧光法测定SGSH和NAGLU酶活性是敏感、可靠、快速的方法,可用于MPSⅢA和ⅢB病例的鉴别诊断及产前诊断;SGSH基因分析方法成本低,突变检出率高,可用于MPSⅢA的诊断和产前诊断.
目的 建立以人工閤成熒光底物測定乙酰肝素N-硫痠酯酶(SGSH),α-N-乙酰葡萄糖苷酶(NAGLU)活性的方法,應用這兩種方法對黏多糖貯積癥Ⅲ型中的A、B亞型(MPSⅢA和ⅢB)病例進行鑒彆診斷,併對MPSⅢB高危妊娠的孕婦進行瞭產前診斷,同時從分子遺傳學方麵對MPSⅢA患兒進行瞭SGSH基因分析.方法 採用人工閤成的熒光底物(4-甲基傘形酮-α-D-N-硫痠葡萄糖苷4-methylumbelliferyl-α-D-N-sulphoglucosaminide.Na,4-甲基傘形酮-N-乙酰-α-葡萄糖苷4-methylumbelliferyl-α-N-acetylglucosaminide)測定疑似患兒白細胞和血漿中SGSH及NAGLU活性,在收集的12例患兒中,女4例,男8例,年齡3~10歲.來自10箇無相關的傢庭.用MPSⅢA的患兒外週血白細胞DNA進行SGSH基因的外顯子PCR擴增,併通過的序列測定確定突變位置.用絨毛或經培養的羊水細胞為檢材,對MPSⅢB高危妊娠的孕婦進行產前診斷.結果 穫得中國正常人白細胞中SGSH活性正常值4.4～8.1 nmot/(17 h·mg蛋白),各種組織中NAGLU活性正常值:血漿中為33.3～62.4 nmoL/(4 h·ml),絨毛組織為44.9~91.7 nmol/(17 h ·mg蛋白),經培養的羊水細胞為53.2-82.2 nmol/(17 h·mg蛋白);在12例疑似患兒中確診瞭7例MPSⅢA(其中兩例為同胞),5例MPS ⅢB患者(其中2例為同胞),SGSH基因分析髮現6種突變類型,已知突變5種(G191R,D235N,R377C,E447K和R233X),插入突變1種(D219Wfs264X),此插入突變為新生突變.3例MPSⅢB產前診斷中,2例胎兒正常,1例胎兒受纍.結論 應用熒光法測定SGSH和NAGLU酶活性是敏感、可靠、快速的方法,可用于MPSⅢA和ⅢB病例的鑒彆診斷及產前診斷;SGSH基因分析方法成本低,突變檢齣率高,可用于MPSⅢA的診斷和產前診斷.
목적 건립이인공합성형광저물측정을선간소N-류산지매(SGSH),α-N-을선포도당감매(NAGLU)활성적방법,응용저량충방법대점다당저적증Ⅲ형중적A、B아형(MPSⅢA화ⅢB)병례진행감별진단,병대MPSⅢB고위임신적잉부진행료산전진단,동시종분자유전학방면대MPSⅢA환인진행료SGSH기인분석.방법 채용인공합성적형광저물(4-갑기산형동-α-D-N-류산포도당감4-methylumbelliferyl-α-D-N-sulphoglucosaminide.Na,4-갑기산형동-N-을선-α-포도당감4-methylumbelliferyl-α-N-acetylglucosaminide)측정의사환인백세포화혈장중SGSH급NAGLU활성,재수집적12례환인중,녀4례,남8례,년령3~10세.래자10개무상관적가정.용MPSⅢA적환인외주혈백세포DNA진행SGSH기인적외현자PCR확증,병통과적서렬측정학정돌변위치.용융모혹경배양적양수세포위검재,대MPSⅢB고위임신적잉부진행산전진단.결과 획득중국정상인백세포중SGSH활성정상치4.4～8.1 nmot/(17 h·mg단백),각충조직중NAGLU활성정상치:혈장중위33.3～62.4 nmoL/(4 h·ml),융모조직위44.9~91.7 nmol/(17 h ·mg단백),경배양적양수세포위53.2-82.2 nmol/(17 h·mg단백);재12례의사환인중학진료7례MPSⅢA(기중량례위동포),5례MPS ⅢB환자(기중2례위동포),SGSH기인분석발현6충돌변류형,이지돌변5충(G191R,D235N,R377C,E447K화R233X),삽입돌변1충(D219Wfs264X),차삽입돌변위신생돌변.3례MPSⅢB산전진단중,2례태인정상,1례태인수루.결론 응용형광법측정SGSH화NAGLU매활성시민감、가고、쾌속적방법,가용우MPSⅢA화ⅢB병례적감별진단급산전진단;SGSH기인분석방법성본저,돌변검출솔고,가용우MPSⅢA적진단화산전진단.
Objective Mucopolysaceharidosis(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 reqtIired for the lysosomal degradation of heparan sulfate.MPSⅢA and MPSⅢB involve deficiencies of heparan N-sulfatase(SGSH)and α-N-acetylglucosaminidase(NAGLU).MPSⅢA and MPSⅢB are more common than MPSⅢC and Ⅲ D.The present study aimed to establish two enzyme assay methods for SGSHand 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 eell)using two newly synthesized substrates.Mutation analysis of SGSH gene was also performed.Methods Two fluorigenic substrate(4-methylumbelliferyl-α-D-N-sulphoglucosaminide.Na and 4-methylumbelliferyl-α-N-acetylglueosaminide)were used for the assay of SGSH and NAGLU activity.SGSH activitv in leukocyte was determined for diagnosis MPSⅢA proband.NAGLU activity was determinedin 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 amolified by PCR The mutations of the patients were characterized by direct sequencing of theamplified DNA fragments.Prenatal diagnosis in 3 pregnancies at risk was carried out according to NAGLU activitv on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation.Results The respectively.Five cases of MPSⅢB and 7 cases of MPSⅢA were diagnosed.The mutation analysis of SGSH gene showed 6 mutations(G191R,D235N,R377C,E447K,R233X and D219Wfs264X),only oneof which (D219Wlfs264X) has not been previously reported.Prenatal diagnosis Was performed on 3Conclusions 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 MPSⅢA patients suggests new possibilities for molecular diagnosis of the disease.
