Yu L, Heere-Ress E, Boucher B, Defesche J C, Kastelein J, Lavoie M A, Genest J
Cardiovascular Genetics Laboratory, Clinical Research Institute of Montréal, Québec, Canada.
Atherosclerosis. 1999 Sep;146(1):125-31. doi: 10.1016/s0021-9150(99)00109-4.
Familial hypercholesterolemia (FH) is an autosomal dominant lipoprotein disorder caused by defects in the low density lipoprotein (LDL) receptor (R) gene. We report a novel mutation of the LDL-R gene in a 38-year-old man with homozygous FH from the province of Trujilo in Northern Honduras. The patient presented with tendinous xanthomas over the extensor tendons as well as xanthelasmas at sites of surgical scars. He was diagnosed with severe coronary artery disease requiring revascularization at age 29. After an unsuccessful course of treatment with simvastatin, the patient has been treated with plasma apheresis and macromolecular plasma filtration bi-monthly. Haplotyping of the LDL-R gene revealed homozygosity for the rare 'J' allele and a loss of the EcoRV restriction cleavage site in exon 8. Single stranded conformational polymorphism of exons 3, 6, 7, 9, 10 and 8 reveals an abnormal migration pattern in exon 8. Direct sequencing of the promoter region, exons 1, 4, 8 and 13 revealed two RFLP's and a novel mutation in intron 7. This mutation consists of G-->C transposition at the acceptor splice site of exon 8 at the last nucleotide of intron 7 [LDL-R1061(-1)G-->C]. Reverse transcriptase (RT) PCR amplification of RNA from monocytes obtained from the patient reveals a decrease in LDL-R mRNA (52% of control) and skipping of exon 8 (approximately 38%, as assessed by densitometric scanning of the amplified fragments) to form a new RNA transcript that includes exons 7 and 9 without frameshift. Alternative RNA editing leads to a new cryptic acceptor splice site 17 bp downstream in exon 8 producing a frameshift mutation and a predicted premature stop codon 1138 bp from the transcriptional start site (approxiamtely 62%). Western blotting analysis using a monoclonal antibody (C7) directed at the amino terminus of the LDL-R protein reveals a marked reduction in LDL-R protein expressed in monocytes obtained from the patient. We conclude that LDL-R1061(-1)G-->C is a novel mutation of the LDL-R gene that results in marked decrease in LDL-R mRNA levels and protein expression by two alternate RNA editing mechanisms, that cause skipping of exon 8 or the use of a novel cryptic acceptor splice site in exon 8 with a frameshift and premature stop codon. The patient continues to do well on selective plasma filtration but developed bilateral severe carotid artery disease requiring surgical intervention.
家族性高胆固醇血症（FH）是一种常染色体显性脂蛋白紊乱疾病，由低密度脂蛋白（LDL）受体（R）基因缺陷引起。我们报告了一名来自洪都拉斯北部特鲁希略省的38岁纯合子FH男性患者的LDL-R基因新突变。该患者在伸肌腱处出现腱黄瘤，手术瘢痕部位出现睑黄瘤。他在29岁时被诊断为严重冠状动脉疾病，需要进行血管重建。在用辛伐他汀治疗失败后，该患者接受了血浆置换和大分子血浆过滤，每两个月一次。LDL-R基因单倍型分析显示，罕见的“J”等位基因纯合，外显子8中EcoRV限制性切割位点缺失。外显子3、6、7、9、10和8的单链构象多态性显示外显子8中有异常迁移模式。对启动子区域、外显子1、4、8和13进行直接测序，发现两个限制性片段长度多态性（RFLP）和内含子7中的一个新突变。该突变由内含子7最后一个核苷酸处外显子8的受体剪接位点的G→C转位组成[LDL-R1061（-1）G→C]。对从患者获得的单核细胞的RNA进行逆转录酶（RT）PCR扩增，发现LDL-R mRNA减少（为对照的52%），外显子8跳跃（通过对扩增片段进行光密度扫描评估，约为38%），形成一个新的RNA转录本，该转录本包含外显子7和9，没有移码。替代性RNA编辑导致外显子8下游17 bp处出现一个新的隐蔽性受体剪接位点，产生移码突变和距转录起始位点1138 bp处的一个预测的提前终止密码子（约62%）。使用针对LDL-R蛋白氨基末端的单克隆抗体（C7）进行的蛋白质印迹分析显示，从患者获得的单核细胞中表达的LDL-R蛋白明显减少。我们得出结论，LDL-R1061（-1）G→C是LDL-R基因的一个新突变，通过两种替代性RNA编辑机制导致LDL-R mRNA水平和蛋白表达显著降低，这两种机制导致外显子8跳跃或在外显子8中使用一个新的隐蔽性受体剪接位点，伴有移码和提前终止密码子。该患者在选择性血浆过滤治疗下情况持续良好，但出现双侧严重颈动脉疾病，需要手术干预。
