Choy F Y, Wei C, Levin D
Centre for Environmental Health, Department of Biology, University of Victoria, British Columbia, Canada.
Am J Med Genet. 1996 Oct 28;65(3):184-9. doi: 10.1002/(SICI)1096-8628(19961028)65:3<184::AID-AJMG3>3.0.CO;2-Q.
Gaucher disease is an inherited sphingolipidosis resulting from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1 as non-neuronopathic, type 2 as acute neuronopathic, and type 3 as subacute neuronopathic. We recently identified a rare mutation (G-->A at glucocerebrosidase cDNA nucleotide position 1604) [Choy et al., 1994a, Am J Med Genet 51:156-160] and a novel mutation (T-->G at glucocerebrosidase cDNA nucleotide position 1366) in two type 1 Gaucher patients by sequence analysis of the entire glucocerebrosidase coding region [Choy et al., 1994a, 1994b, Hum Mol Genet 3:821-823]. To demonstrate that these are deleterious and not neutral mutations, we cloned the full-length glucocerebrosidase cDNA of patients and of a normal control in the plasmid vector pAcUW1, recombined the human gene into the Baculovirus genome downstream of its polyhedron p10 promoter, and expressed the inserted gene in cultured cells of Spodoptera frugiperda transfected by recombinant Baculovirus. The levels of residual glucocerebrosidase activity determined in transfected cells with the Gaucher G1604A and T1366G alleles are 6.9% and 2.9% of that expressed by the normal allele (normal = 352.0 nmol/hr/mg protein or 100%). By comparison, the enzyme-specific activity expressed in transfected cells by 2 known Gaucher alleles, A1226G and T1448C, that are prevalent in type 1 and type 2 Gaucher disease are 23.4% and 3.3% of normal. No endogeneous glucocerebrosidase activity was detected in cultured cells transfected by either the wild-type Baculovirus or Baculovirus with the pAcUW1 plasmid vector without the glucocerebrosidase cDNA insert. These findings show that the Baculovirus expression system in cultured Spodoptera frugiperda cells is a suitable system for the functional expression and characterization of the normal and mutant glucocerebrosidase alleles. Moreover, the use of this expression system demonstrates that the G1604A and T1366G mutations are both deleterious mutations resulting in profoundly deficient glucocerebrosidase activity and subsequent Gaucher disease.
戈谢病是一种由于葡萄糖脑苷脂酶活性缺乏导致的遗传性鞘脂贮积病。戈谢病有三种临床类型：1型为非神经病变型，2型为急性神经病变型，3型为亚急性神经病变型。我们最近通过对整个葡萄糖脑苷脂酶编码区进行序列分析，在两名1型戈谢病患者中鉴定出一种罕见突变（葡萄糖脑苷脂酶cDNA核苷酸位置1604处的G→A）[Choy等人，1994年a，《美国医学遗传学杂志》51:156 - 160]以及一种新突变（葡萄糖脑苷脂酶cDNA核苷酸位置1366处的T→G）[Choy等人，1994年a，1994年b，《人类分子遗传学》3:821 - 823]。为了证明这些是有害而非中性突变，我们将患者和正常对照的全长葡萄糖脑苷脂酶cDNA克隆到质粒载体pAcUW1中，将人类基因重组到杆状病毒基因组多面体p10启动子下游，并在被重组杆状病毒转染的草地贪夜蛾培养细胞中表达插入基因。用戈谢病G1604A和T1366G等位基因在转染细胞中测定的残余葡萄糖脑苷脂酶活性水平分别为正常等位基因表达活性的6.9%和2.9%（正常 = 352.0 nmol/小时/毫克蛋白或100%）。相比之下，在1型和2型戈谢病中普遍存在的2种已知戈谢病等位基因A1226G和T1448C在转染细胞中表达的酶比活性分别为正常的23.4%和3.3%。在用野生型杆状病毒或携带无葡萄糖脑苷脂酶cDNA插入片段的pAcUW1质粒载体的杆状病毒转染的培养细胞中未检测到内源性葡萄糖脑苷脂酶活性。这些发现表明，草地贪夜蛾培养细胞中的杆状病毒表达系统是用于正常和突变葡萄糖脑苷脂酶等位基因功能表达及特性分析的合适系统。此外，使用该表达系统证明G1604A和T1366G突变均为有害突变，导致葡萄糖脑苷脂酶活性严重缺乏及随后的戈谢病。
