MOLECULAR DEFECTS IN GENETIC DISORDERS OF LIPOPROTEIN METABOLISM

卵磷脂胆固醇酰基转移酶（LCAT）是一种63 Kd的酶， 负责酯化几乎所有的游离胆固醇， 等离子体LCAT功能缺陷的患者存在严重的 低脂蛋白血症确立了LCAT在HDL中的重要作用 新陈代谢.我们已经确定了一个新的突变LCAT基因的一个 法国患者出现角膜混浊和HDL-C水平较低 超过10 mg/dl。DNA测序和RFLP分析显示，患者 是一个C到T和C到A突变的复合杂合子，导致 Arg 99被替换为Cys，Tyr 83被替换为Stop。在 两种突变LCAT的体外表达建立了功能性的 两种基因缺陷的重要性。 胆固醇酯转移蛋白（CETP）负责转移 中性脂质之间的B-含脂蛋白和HDL。虽然 CETP缺乏是日本高HDL的常见原因，遗传基础 这种疾病的特征还没有完全确定。我们研究了一 患者的TC、HDL-C和apoA-I水平分别为300、236和233 mg/dl，完全不存在CETP活性和质量， 等离子体对患者基因的序列分析显示， 供体共有GT在内含子10中被GG取代，在内含子10中被AT取代 14.用Nde Ⅰ和Mae Ⅲ进行限制性酶切， 是两种基因缺陷的复合杂合子RT-PCR测序 从巨噬细胞RNA扩增的DNA显示异常剪接， 外显子10的缺失以及天然AG位点的选择性剪接 位于内含子13中正常剪接受体5'端的31个核苷酸。 该缺陷导致在外显子13之间插入31 bp片段 和外显子14以及引入符合读码框的终止密码子。的 异常剪接的mRNA的存在被进一步证实， 使用CETP特异性引物扩增患者RT-PCR DNA， 确定这些缺陷的功能意义。

Lecithin cholesteryl acyltransferase (LCAT) is a 63 Kd enzyme that is responsible for the esterification of virtually all free cholesterol in plasma. Patients with functional defects in LCAT present with profound hypoalphalipoproteinemia establishing an important role for LCAT in HDL metabolism. We have identified a novel mutation in the LCAT gene of a French patient presenting with corneal opacities and HDL-C levels of less than 10 mg/dl. DNA sequencing and RFLP analysis revealed that the patient is a compound heterozygote for a C to T and C to A mutation resulting in the substitution of Arg 99 to Cys and Tyr 83 to stop, respectively. In vitro expression of the two mutant LCAT established the functional significance of both gene defects. Cholesteryl ester transfer protein (CETP) is responsible for the transfer of neutral lipids between the B-containing lipoproteins and HDL. Although CETP deficiency is a common cause of high HDL in Japan, the genetic basis of this disorder has not been fully characterized. We have studied a patient presenting with TC, HDL-C and apoA-I levels of 300, 236 and 233 mg/dl, respectively and total absence of CETP activity and mass in plasma. Sequence analysis of the patients's gene revealed that the splice donor consensus GT was substituted by GG in intron 10 and by AT in intron 14. Restriction digestion using NdeI and MaeIII established that the pt was a compound heterozygote for both gene defects. Sequencing of RT-PCR amplified DNA from macrophage RNA demonstrated abnormal splicing with deletion of exon 10 as well as alternative splicing at a native AG site located 31 nucleotides 5' of the normal splice acceptor in intron 13. This defect results in the insertion of a 31 bp fragment between exon 13 and exon 14 as well as the introduction of an in frame stop codon. The presence of abnormally spliced mRNA was further confirmed by amplification of patient RT-PCR DNA using CETP specific primers thus establishing the functional significance of these defects.