Beg O U, Meng M S, Skarlatos S I, Previato L, Brunzell J D, Brewer H B, Fojo S S
Molecular Disease Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1990 May;87(9):3474-8. doi: 10.1073/pnas.87.9.3474.
The molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in the proband from a Bethesda kindred has been identified. The pre- and post-heparin plasma LPL mass in the proband was elevated when compared to controls; however, there was no detectable LPL activity, indicating the presence of a defective enzyme (termed LPLBethesda). Analysis of the patient's post-heparin plasma by heparin-Sepharose affinity chromatography demonstrated that the mutant LPL had an altered affinity for heparin. Southern blot hybridization of the gene for LPLBethesda revealed no major rearrangements. Northern blot analysis of LPLBethesda mRNA from patient monocyte-derived macrophages revealed normal-sized mRNAs (3.4 and 3.7 kilobases) as well as normal cellular mRNA levels when compared to control macrophages. Sequence analysis of polymerase chain reaction-amplified LPL cDNA revealed a G----A substitution at position 781 of the normal LPL gene that resulted in the substitution of an alanine for a threonine at residue 176 and the loss of an SfaNI site present in the normal LPL gene. Amplification of cDNA by the PCR followed by digestion with SfaNI established that the patient was a true homozygote for the mutation. Expression of LPL cDNA in COS-7 cells resulted in the synthesis of a nonfunctional LPL enzyme establishing that the Ala----Thr substitution was the mutation responsible for the inactive LPL. The identification of this mutation in the LPL gene defines a region of the LPL enzyme, at Ala-176, that is essential for normal heparin-binding and catalytic activity. We propose that an amino acid substitution in this critical region of LPLBethesda results in the synthesis of a nonfunctional enzyme that leads to the chylomicronemia syndrome expressed in this proband.
已确定导致来自贝塞斯达家族的先证者脂蛋白脂肪酶（LPL）活性缺乏的分子缺陷。与对照组相比，先证者肝素前和肝素后血浆中的LPL质量升高；然而，未检测到LPL活性，表明存在缺陷酶（称为LPL贝塞斯达）。通过肝素-琼脂糖亲和层析分析患者的肝素后血浆表明，突变型LPL对肝素的亲和力发生了改变。LPL贝塞斯达基因的Southern印迹杂交未发现主要重排。对患者单核细胞衍生巨噬细胞的LPL贝塞斯达mRNA进行Northern印迹分析，与对照巨噬细胞相比，显示出正常大小的mRNA（3.4和3.7千碱基）以及正常的细胞mRNA水平。聚合酶链反应扩增的LPL cDNA序列分析显示，正常LPL基因第781位发生了G→A取代，导致第176位残基处的苏氨酸被丙氨酸取代，正常LPL基因中存在的一个SfaNI位点丢失。通过PCR扩增cDNA，然后用SfaNI消化，确定患者是该突变的真正纯合子。LPL cDNA在COS-7细胞中的表达导致合成了一种无功能的LPL酶，证实丙氨酸→苏氨酸取代是导致LPL无活性的突变。LPL基因中该突变的鉴定确定了LPL酶在丙氨酸-176处的一个区域，该区域对于正常的肝素结合和催化活性至关重要。我们提出，LPL贝塞斯达这个关键区域的氨基酸取代导致合成了一种无功能的酶，从而导致该先证者出现乳糜微粒血症综合征。
