NPC1L1基因多形性對血中膽固醇濃度的影響

Abstract

中文摘要 目的： 本篇研究目的在找出國人NPC1L1基因的多形性，並進ㄧ步探討這些基因多形性變異對血脂濃度變化的影響。 背景： NPC1L1的生理功能為協助小腸細胞對於膽固醇的吸收，然而國人於NPC1L1序列上的基因多形性變異，以及它們對於人體的影響目前仍不清楚。 方法: 首先，本實驗隨機收集50個DNA檢體，藉由DHPLC以及DNA定序，針對NPC1L1的啟動子以及密碼區域序列進行基因多形性變異偵測。之後，進ㄧ步利用luciferase assay針對NPC1L1的啟動子序列多形性變異進行功能性分析。此外，本實驗進一步收集224個案，進行NPC1L1基因多形性變異與血脂濃度變化的相關研究。 結果： 本篇實驗總共發現11個基因多形性變異分布於NPC1L1基因序列內。其中，以g.-762T>C (啟動子區域多形性變異)以及g.1679C>G (L272L，同質多形性變異)在國人較為常見，比例分別為34% 以及 36% 。這兩個基因多形性變異呈現高度連鎖性的關係(D’ 值 = 0.7459，P 值 < 0.00001)。針對g.-762T>C (啟動子多形性變異)，藉由進行luciferase assay分析的結果顯示，-762C對偶基因比-762T具有較高的啟動子活性(1.30 ± 0.22 vs 0.37 ± 0.06，3.5倍，P < 0.05)。本實驗也證實，-762C以及-762T兩種基因型的建構質體，其NPC1L1的啟動子活性都會受到膽固醇濃度變化加以調控。此外，相關的研究中也顯示，在隱性基因遺傳模式下，-762C對偶基因與血脂中總膽固醇以及低密度脂蛋白-膽固醇的濃度變化呈現顯著相關(LDL-C數值 = 131.2 ± 8.1 mg/dL vs 116.4 ± 2.2 mg/dL; total cholesterol 數值 = 214.7 ± 9.0 mg/dL vs 196.9 ± 2.6, P 值 < 0.05, n = 224)。 結論: 在國人的NPC1L1基因序列內，-762C對偶基因是相當常見的。-762C對偶基因具有較高的啟動子活性，另外也會使血脂中總膽固醇以及低密度脂蛋白-膽固醇呈現較高濃度的情況。

Abstract Objective： The aims of this study were to identify genetic polymorphisms of the Niemann-Pick C1-like 1 (NPC1L1) gene and to investigate whether these polymorphisms were associated variations in serum cholesterol levels. Background： NPC1L1 is responsible for intestinal cholesterol absorption while gene variations in NPC1L1 gene and their effects on serum cholesterol levels remain unclear. Methods： We screened the promoter and coding regions of the NPC1L1 gene for genetic polymorphisms by DHPLC and direct DNA sequencing of genomic DNA from 50 individuals. Functional studies on promoter polymorphisms were performed using luciferase assay. Association between the polymorphisms and serum cholesterol levels was investigated in 224 individuals. Result： There were totally 11 single nucleotide polymorphisms identified. Among them, a promoter polymorphism g.-762T>C and a synonymous polymorphism g.1679C>G were common (34% and 36% respectively). These two polymorphisms were highly linked (D’ value = 0.7459, P value < 0.00001). For the g.-762T>C promoter polymorphism, luciferase assay in HepG2 cell line demonstrated that the -762C allele had a significantly higher promoter activity than the -762T allele (1.30 ± 0.22 vs 0.37 ± 0.06, 3.5-fold, p<0.05). We also showed that NPC1L1 promoter activity was regulated by cholesterol contents in both genotypes. When association studies were performed, we found that the -762C allele was associated with significantly higher serum total cholesterol and LDL-cholesterol levels in a recessive model (LDL-C value = 131.2 ± 8.1 mg/dL vs 116.4 ± 2.2 mg/dL; total cholesterol value = 214.7 ± 9.0 mg/dL vs 196.9 ± 2.6, P value < 0.05, n = 224). Conclusion： The C allele at -762 position of the NPC1L1 gene was common in Chinese. The -762C allele had a higher promoter activity and was associated with higher serum total cholesterol and LDL-C levels.