Steyrer E, Haubenwallner S, Hörl G, Giessauf W, Kostner G M, Zechner R
Department of Medical Biochemistry, University of Graz, Austria.
Hum Genet. 1995 Jul;96(1):105-9. doi: 10.1007/BF00214196.
We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.
我们已经在三名奥地利血统的兄弟姐妹中，对卵磷脂胆固醇酰基转移酶（LCAT）基因中导致LCAT缺乏症（LCAT-D）的分子缺陷进行了特征分析。这些患者表现出典型症状，包括角膜混浊、溶血性贫血和肾功能障碍。这三名患者血浆中的LCAT活性无法检测到。对所有六个LCAT外显子的聚合酶链反应（PCR）扩增DNA进行DNA序列分析，发现LCAT基因外显子IV中有一个新的点突变，即密码子140处的G到A替换，导致精氨酸变为组氨酸。该突变导致外显子IV内限制性内切酶HhaI的切割位点丢失：用HhaI消化629 bp的外显子IV PCR产物后，发现患者为该突变的纯合子。11名家庭成员中有8名被鉴定为杂合子。用含有野生型或突变型LCAT cDNA的质粒对COS-7细胞进行转染研究发现，与含有野生型酶的细胞培养基相比，表达突变蛋白后未检测到酶活性。这有力证明了所观察到的突变是受影响个体中LCAT缺乏症的病因，并支持精氨酸140对具有酶活性的LCAT蛋白结构至关重要的结论。
