Parson Walther, Kraft Hans Georg, Niederstätter Harald, Lingenhel Arnulf W, Köchl Silvano, Fresser Friedrich, Utermann Gerd
Institute of Forensic Medicine, Medical University of Innsbruck, Innsbruck, Austria.
Hum Mutat. 2004 Dec;24(6):474-80. doi: 10.1002/humu.20101.
LPA, the gene coding for apolipoprotein(a) [apo(a)], is the major determinant of lipoprotein(a) [Lp(a)] plasma levels, which are associated with risk for coronary heart disease (CHD) and stroke. It is not completely understood how variation in LPA relates to Lp(a) concentrations. One type of variation related to Lp(a) levels is the number of Kringle (K) IV-2 (g.61C>T; GenBank L14005.1) repeats in LPA, but sequence variation may also contribute. Human apo(a) contains from two to >40 nearly identical K IV-2 repeats of genomic size 5.5 kb, which makes it difficult to detect mutations. To elucidate the genetic variation of the apo(a) K IV-2 domain, we isolated a single "nonexpressing" apo(a) allele with 26 K IV-2 repeats, followed by PCR, cloning and sequencing of 96 clones, resulting in an average coverage of each K IV-2 repeat of approximately four-fold. The previously described K IV types 2A and 2B (K IV-2A and K IV-2B) were detected in 74% of the clones. In addition, a new type designated 2C (K IV-2C) was present. A nonsense mutation in the first exon of K IV-2 (g.61C>T) predicted to result in a truncated protein (p.R21X) was found in nine clones on a K IV-2A background. The presence of this mutation was confirmed by analysis of genomic DNA and was shown to represent the rare allele (frequency 0.02) of a SNP. Immunoblot analysis of apo(a) from plasma confirmed the presence of a truncated apo(a) isoform in the index individual and family members. Our data show that SNPs affecting Lp(a) plasma concentrations also exist in the apo(a) K IV-2 domain.
脂蛋白A(LPA)基因编码载脂蛋白(a) [apo(a)],是血浆脂蛋白(a) [Lp(a)]水平的主要决定因素,而Lp(a)水平与冠心病(CHD)和中风风险相关。目前尚不完全清楚LPA的变异如何与Lp(a)浓度相关。与Lp(a)水平相关的一种变异类型是LPA中kringle(K)IV-2(g.61C>T;GenBank L14005.1)重复序列的数量,但序列变异也可能起作用。人类apo(a)包含2至40多个基因组大小为5.5 kb的几乎相同的K IV-2重复序列,这使得检测突变变得困难。为了阐明apo(a) K IV-2结构域的遗传变异,我们分离出一个具有26个K IV-2重复序列的单一“非表达性”apo(a)等位基因,随后对96个克隆进行PCR、克隆和测序,每个K IV-2重复序列的平均覆盖度约为四倍。在74%的克隆中检测到了先前描述的K IV-2A和K IV-2B类型。此外,还存在一种新的2C型(K IV-2C)。在K IV-2A背景的9个克隆中发现了K IV-2第一个外显子中的一个无义突变(g.61C>T),预计会导致截短蛋白(p.R21X)。通过对基因组DNA的分析证实了该突变的存在,并表明它代表了一个单核苷酸多态性(SNP)的罕见等位基因(频率为0.02)。对血浆中apo(a)的免疫印迹分析证实了索引个体及其家庭成员中存在截短的apo(a)异构体。我们的数据表明,影响Lp(a)血浆浓度的单核苷酸多态性也存在于apo(a) K IV-2结构域中。