Pagani F, Zagato L, Merati G, Paone G, Gridelli B, Maier J A
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Hum Mol Genet. 1994 Sep;3(9):1605-9. doi: 10.1093/hmg/3.9.1605.
The genetic defect causing cholesteryl ester storage disease (CESD) has been investigated in an 11 year old patient. Lysosomal acid lipase (LAL) activity in cultured skin fibroblasts and peripheral lymphocytes was reduced to approximately 3% and approximately 4% of controls, respectively. The parents had low acid lipase activity in white blood cells. Using the polymerase chain reaction followed by ribonuclease protection assay, we examined the LAL mRNA from the liver of the affected patient to identify small deletion, abnormal splicing or missense mutation. Using this technique we identified a LAL mRNA cytosine to thymidine transition in position 923, predicting a missense substitution of tyrosine for histidine in codon 274. By differential oligonucleotide hybridization on an amplified white blood cell mRNA, the cytosine to thymidine transition was investigated in the family members and in the population. No normal mRNA coding for cytosine in position 923 was detectable in the propositus and mRNA from the phenotypically normal parents coded for both cytosine and thymidine. This can only be accounted for by assuming that the propositus is homozygote for the mutation. The mutation, segregated in the family with levels of acid lipase activity in white blood cells, was not detected in mRNA from 60 normal subjects. These data provide evidence that the cytosine to thymidine transition in position 923 in LAL mRNA causes the clinical expression of CESD in this patient. The predicted substitution of tyrosine for histidine in codon 274 suggests that this amino acid is involved in the structure-function of the lysosomal acid lipase enzyme.
对一名11岁的胆固醇酯贮积病（CESD）患者的基因缺陷进行了研究。培养的皮肤成纤维细胞和外周淋巴细胞中的溶酶体酸性脂肪酶（LAL）活性分别降至对照组的约3%和约4%。其父母白细胞中的酸性脂肪酶活性较低。我们采用聚合酶链反应，随后进行核糖核酸酶保护分析，检测了该患病患者肝脏中的LAL mRNA，以确定是否存在小缺失、异常剪接或错义突变。利用该技术，我们在第923位鉴定出LAL mRNA胞嘧啶到胸腺嘧啶的转变，预测密码子274中的组氨酸被酪氨酸错义替代。通过对扩增的白细胞mRNA进行差异寡核苷酸杂交，研究了该家族成员和人群中的胞嘧啶到胸腺嘧啶的转变。在先证者中未检测到编码第923位胞嘧啶的正常mRNA，而表型正常的父母的mRNA同时编码胞嘧啶和胸腺嘧啶。这只能通过假设先证者为该突变的纯合子来解释。该突变在家族中与白细胞中酸性脂肪酶活性水平共分离，在60名正常受试者的mRNA中未检测到。这些数据证明，LAL mRNA第923位胞嘧啶到胸腺嘧啶的转变导致了该患者CESD的临床表型。密码子274中预测的组氨酸被酪氨酸替代表明，该氨基酸参与了溶酶体酸性脂肪酶的结构-功能。
