Regulation of ApoB Secretion in Inbred Mouse Strains

描述(由申请人提供)：血浆载脂蛋白B(Apo B)和低密度脂蛋白(LDL)水平升高与动脉粥样硬化性冠心病(CHD)的风险增加相关，动脉粥样硬化性冠心病(CHD)是工业化世界中死亡的主要原因。血浆载脂蛋白B水平升高和载脂蛋白B过度产生也是家族性混合性高脂血症(FCHL)的主要特征，FCHL是一种在普通人群中普遍存在的疾病。这种流行疾病的遗传基础是异质性的，未知的。调节血浆载脂蛋白B水平的基因是FCHL表型基因的合理候选者。载脂蛋白B是肝脏分泌极低密度脂蛋白所必需的，是血浆极低密度脂蛋白和低密度脂蛋白的必需蛋白质成分。血浆载脂蛋白B水平在一定程度上受含载脂蛋白B颗粒的分泌速度控制。利用人载脂蛋白B转基因小鼠模型(HuBTg)，我们发现C57BL/6(B6)和129/Sv(129)背景的HuBTg小鼠血浆载脂蛋白B水平的差异主要是由于肝脏载脂蛋白B的转录后调节分泌速率所致。进一步对两个小鼠品系的遗传学研究发现，6号和4号染色体上有两个新的数量性状基因座(QTL)，它们对血浆载脂蛋白B水平有主要影响。这些基因被命名为载脂蛋白B调节基因(ABRG)。我们的精细定位分析已经将Abrg1定位在6号染色体上约2.9Mb的区间，将Abrg2定位在4号染色体上12 cM的区间。本研究的目的是鉴定和鉴定6号染色体上的Abrg1基因。具体目的是：1.定位克隆6号染色体上调控血浆载脂蛋白B水平的Abrg1基因。将采取几种平行的方法来确定ABRGL的候选基因。这些方法包括逆转录-聚合酶链式反应、实时定量聚合酶链式反应和DNA测序分析。还将设计研究以确定表达变体的等位基因变异对转录的影响。2.ABRG基因在同基因小鼠中的特征及其候选基因的功能研究我们将通过体外和体内的过度表达研究，对候选变异基因进行功能测试，包括Copsla基因(一种表达变体)。Abrg1基因的最佳候选者，无论是Cops7a还是另一个待鉴定的基因，将通过敲入和/或敲除基因靶向技术进行验证。将进行研究，以确定Abrg1基因的功能作用。总体而言，已发现的基因将是一个新的调节因子，影响包含载脂蛋白B的脂蛋白的组装和分泌途径。作为载脂蛋白B分泌速率的调节者，这个新基因将是导致FCHL患者亚群中载脂蛋白B过度产生的基因之一的有力候选者。它也将成为普通人群中高脂血症患者药物发现和药物干预的潜在靶点。

DESCRIPTION (provided by applicant): Elevated plasma levels of apolipoprotein B (apo B) and low density lipoprotein (LDL) are associated with a higher risk for atherosclerotic coronary heart disease (CHD), a leading cause of mortality in the industrialized world. Elevated plasma apo B levels and overproduction of apo B are also major characteristics of familial combined hyperlipidemia (FCHL), a prevalent disorder in the general population. The genetic basis of this prevalent disease is heterogeneous and unknown. Genes that regulate plasma apo B levels are logical candidates for genes which contribute to the FCHL phenotype. Apo B is required for the secretion of very low density lipoproteins (VLDL) from the liver and is the mandatory protein constituent of both plasma VLDL and LDL. Plasma apo B levels are controlled, in part, by secretion rates of apo B-containing lipoprotein particles. Using the human apo B transgenic mouse model (HuBTg), we have shown that HuBTg mice of C57BL/6 (B6) and 129/Sv (129) background differ in their plasma apo B levels mainly due to hepatic apo B secretion rates via post-transcriptional regulation. Further genetic studies of two mouse strains identified two novel quantitative trait loci (QTL) on chromosomes 6 and 4 which have major effects on plasma apo B levels. These genes are designated Apo B regulatory genes (Abrg). Our fine-mapping analyses have localized the Abrgl to an interval of approximately 2.9 Mb on chromosome 6 and the Abrg2 to a 12-cM interval on chromosome 4. The goals of the current proposal are to identify and characterize the Abrgl gene on chromosome 6. The specific aims are: 1. Positional cloning of the Abrgl on chromosome 6 that regulates plasma apo B levels. Several parallel approaches will be taken to identify candidate genes for the Abrgl. These approaches include RT-PCR, quantitative real-time PCR assays, and DNA sequencing analysis. Studies will also be designed to determine the effects of allelic variations of expression variants on transcription. 2. Characterization of the Abrg genes in congenic mice and functional studies of the Abrgl candidate genes. We will perform functional tests on candidate variant genes, including the Copsla gene (an expression variant), by over expression studies in vitro and in vivo. The best candidate for the Abrgl gene, either Cops7a or another gene to be identified, will then be verified via knock-in and/or knock-out gene-targeting techniques. Studies will be designed to characterize the functional roles of the Abrgl gene. Overall, the gene identified will be a novel regulator affecting the pathways involved in the assembly and secretion of apo B containing lipoproteins. As a regulator of apo B secretion rates, this novel gene will be a strong candidate for one of the genes causative for overproduction of apo B in subsets of FCHL patients. It will also be a potential target for drug discovery and pharmacological intervention in hyperlipidemic patients in the general population.