Department of Lipidology, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8640, Japan.
Atherosclerosis. 2010 May;210(1):166-72. doi: 10.1016/j.atherosclerosis.2009.11.018. Epub 2009 Nov 20.
OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol trafficking by mediating degradation of cell-surface LDL receptors (LDLR). Gain-of-function PCSK9 mutations are known to increase plasma LDL-C levels. We attempted to find gain-of-function PCSK9 mutations in Japanese subjects and determine the frequency and impacts of these mutations, especially on circulating PCSK9 and LDL-C levels. METHODS: PCR-SSCP followed by direct sequence analysis was performed for all 12 exons and intronic junctions of the PCSK9 in 55 subjects with clinically diagnosed familial hypercholesterolaemia (clinical-FH), who were confirmed to have no LDLR mutations. Among the mutations detected, PCSK9 E32K was likely to be a gain-of-function mutation, and screening was performed by PCR-RFLP in clinical-FH and general Japanese controls. The levels of PCSK9 in plasma from subjects and in media of HepG2 cells transfected with PCSK9 constructs were measured by ELISA. RESULTS: We detected 7 PCSK9 variants, including E32K. The frequency of PCSK9 E32K in clinical-FH (6.42%) was significantly higher than that in controls (1.71%). Three cases representing homozygous FH phenotypes were double heterozygous for PCSK9 E32K and LDLR C183S, C292X or K790X. Two cases were true homozygous for PCSK9 E32K; to our knowledge, these are the first true homozygotes for gain-of-function PCSK9 mutations reported to date. The PCSK9 E32K mutant had over 30% increased levels of PCSK9 in plasma from the subjects and in media of transiently transfected HepG2 cells as compared with those in controls. Furthermore, LDL-C levels in the PCSK9 E32K true homozygotes and heterozygotes were 2.10- and 1.47-fold higher than those in controls with comparable circulating PCSK9 levels, respectively, suggesting enhanced function of PCSK9 E32K. CONCLUSIONS: We found 2 true homozygotes for PCSK9 E32K and 3 double heterozygotes for PCSK9 E32K and LDLR mutations associated with autosomal dominant hypercholesterolaemia. This study provided evidence that PCSK9 E32K significantly affects LDL-C levels via increased mass and function of PCSK9, and could exacerbate the clinical phenotypes of patients carrying LDLR mutations.
目的： 前蛋白转化酶枯草溶菌素 9（PCSK9）通过介导细胞表面 LDL 受体（LDLR）的降解来调节胆固醇的转运。已知功能获得性 PCSK9 突变会增加血浆 LDL-C 水平。我们试图在日本受试者中寻找功能获得性 PCSK9 突变，并确定这些突变的频率和影响，尤其是对循环 PCSK9 和 LDL-C 水平的影响。
方法： 在 55 例临床诊断为家族性高胆固醇血症（临床 FH）的患者中，对 PCSK9 的所有 12 个外显子和内含子连接处进行 PCR-SSCP 后直接测序，这些患者均未发现 LDLR 突变。在所检测到的突变中，PCSK9 E32K 可能是一种功能获得性突变，因此在临床 FH 和普通日本对照中通过 PCR-RFLP 进行筛选。通过 ELISA 测量受试者血浆中的 PCSK9 水平和转染 PCSK9 构建体的 HepG2 细胞培养基中的 PCSK9 水平。
结果： 我们检测到 7 种 PCSK9 变体，包括 E32K。PCSK9 E32K 在临床 FH（6.42%）中的频率明显高于对照组（1.71%）。3 例代表纯合 FH 表型的患者同时为 PCSK9 E32K 和 LDLR C183S、C292X 或 K790X 的双杂合子。2 例为 PCSK9 E32K 的真正纯合子；据我们所知，这是迄今为止报告的首例真正的功能获得性 PCSK9 突变纯合子。与对照组相比，PCSK9 E32K 突变体使受试者血浆中的 PCSK9 水平增加了 30%以上，瞬时转染 HepG2 细胞的培养基中的 PCSK9 水平也增加了 30%以上。此外，PCSK9 E32K 真正纯合子和杂合子的 LDL-C 水平分别比具有相似循环 PCSK9 水平的对照组高 2.10 倍和 1.47 倍，表明 PCSK9 E32K 的功能增强。
结论： 我们发现了 2 例 PCSK9 E32K 的真正纯合子和 3 例 PCSK9 E32K 和 LDLR 突变的双杂合子，这些突变与常染色体显性高胆固醇血症有关。这项研究提供的证据表明，PCSK9 E32K 通过增加 PCSK9 的质量和功能显著影响 LDL-C 水平，并可能加重携带 LDLR 突变的患者的临床表型。
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