α-半乳糖苷酶A活性检测与Fabry病诊断筛查

Value of α-galactosidase A activity assay in diagnosis and screening of Fabry's disease

目的:建立α-半乳糖苷酶A(α-GalA)活性的检测方法,为Fabry病的诊断和筛查提供依据。方法:分别采用外周血粒细胞和干燥血滴滤纸片(干血片)检测11个家系15例Fabry病患者和30名健康者的α-GalA活性,检测采用底物荧光法。比较Fabry病患者组与健康对照组间、干血片法不同保存时间组间的酶活性,并建立酶活性正常参考范围,计算2种检测酶活性方法的灵敏度。结果:正常人干血片酶活性为(3.68±0.98)nmol/(ml·h),粒细胞酶活性为(46.21±8.56)nmol/(h·mg),两者正常参考范围分别为≥1.68nmol/(ml·h)、≥25.45nmol/(h·mg)。男性半合子患者酶活性显著下降(P<0.001),干血片酶活性值<0.15nmol/(ml·h)、粒细胞酶活性值<1nmol/(h·mg),酶活性的2种检测方法在男性半合子患者中的灵敏度为100%。在女性杂合子患者酶活性部分下降,4例女性杂合子患者中3例干血片检测酶活性的结果、2例检测外周血粒细胞酶活性的结果处于正常范围,检测女性杂合子干血片和粒细胞酶活性的灵敏度分别为25%和50%。干血片在室温下保存3、14和28d后酶活性值分别为(3.63±0.96)nmol/(ml·h)、(3.60±0.88)nmol/(ml·h)和(3.06±0.68)nmol/(ml·h),残余酶活性值大于基线值80%,显著高于男性半合子酶活性值(P<0.001)。结论:干血片检测酶活性更简易、快速、经济,便于保存运输,可用于高危人群筛查,外周血粒细胞酶活性测定对女性杂合子灵敏度更高,测定值更精确,适于家系筛查研究。酶活性检测可有效诊断和筛查男性半合子Fabry病患者。

Objective To set up enzymatic assay methods of α-galactosidase A （α-Gal A） for diagnosis and screening of Fabry＇s disease （FD）. Methods Fifteen patients with FD and 30 healthy controls were recruited, α-Gal A enzymatic activity in dried blood spots on filter paper （DBFP） and in peripheral leukocytes was measured by fluorogenic substrate,4-methylumbelliferyl-α-D-galactopyranoside in the two groups. Difference of data between FD patients and normal controls was analyzed, normal reference value range was defined and the sensitivity of both assay methods was evaluated. Results Enzymatic activity in DBFP and peripheral leukocytes was （3.68±0.98）nmol/（ml-h） and （46.21±8.56）nmol/（h .mg）. Normal reference value of enzymatic activity is over 1.68 nmol/（ml·h） in DBSP and over 25.45 nmol/（h·mg） in leukocytes. All the hemizygotes showed low enzymatic activity with both methods, showing sensitivity of 100% in hemizygotes. 3/4 of heterozygotes had the enzymatic activity within normal range in DBFP （sensitivity：25%）, while 1/2 within normal range in leukocyte （sensitivity：50%）. Conclusions Enzymatic diagnosis in dried blood is easier, faster and less expensive than the leukocyte assay and it appears to be a reasonable approach for the screening high risk populations of FD, while leukocyte assay has more accuracy and higher sensitivity in heterozygotes. Enzymatic activity in DBFP and peripheral leukocytes is of significant importance in diagnosis and screening of FD.