Pla2g12b and Hpn are Genes Identified by Mouse ENU Mutagenesis that affect HDL cholesterol. Mus musculus

Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: For line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 17 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3′ splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism. Overall design: Mutant mice were generated as part of The Jackson Laboratory’s Heart, Lung, Blood, and Sleep Disorder Mutagenesis Program by treating male C57BL/6J (B6) mice with N-ethyl-N-nitrosourea (ENU). Third generation (G3) mice were phenotyped to ensure capture of both dominant and recessive mutations. Two unique G3 animals with low HDL cholesterol levels were then used to establish new inbred lines (Hlb218 and Hlb320) by mating them with B6 mice and intercrossing the offspring with low HDL cholesterol for 7 generations. Livers from 3 Hlb218, 3 Hlb320 males, and 6 B6 male controls were obtained for gene expression analysis. The samples were randomized over Illumina Mouse-6 Expression 1.1 BeadChips .

尽管学界在解析影响高密度脂蛋白（High-Density Lipoprotein, HDL）颗粒、其功能及胆固醇含量的相关基因方面已取得长足进展，但迄今已鉴定的基因仅能解释一小部分遗传变异。我们利用小鼠N-乙基-N-亚硝基脲（N-ethyl-N-nitrosourea, ENU）诱变技术，发掘影响HDL胆固醇水平的新型基因。成功构建两株HDL胆固醇水平低下的突变品系（Hlb218与Hlb320）。通过连锁分析对这两个品系的致突变位点进行定位：Hlb218的致突变位点定位于10号染色体12 Mbp的区域内，而Hlb320的则定位于7号染色体17 Mbp的区域内。对Hlb218的肝脏RNA进行高通量测序，鉴定到Pla2g12b基因存在一处突变：G向A的碱基转换导致半胱氨酸突变为酪氨酸，极有可能造成二硫键缺失。对Hlb320的肝脏RNA进行微阵列分析显示，Hpn基因的表达量下调了7倍；测序发现其第8号外显子的3'剪接位点存在一处突变。Northern印迹（Northern blot）实验证实Hlb320的mRNA表达水平更低，且未检测到剪接差异，提示该突变仅影响剪接效率。除影响HDL胆固醇水平外，这两个突变基因还会导致血清非HDL胆固醇及甘油三酯水平降低。尽管这两个突变品系的小鼠HDL胆固醇水平低下，但与野生型对照小鼠相比，其动脉粥样硬化病灶面积并无显著差异。这些新型突变小鼠模型可为进一步研究这些基因的功能、其对HDL胆固醇水平的调控作用及代谢过程提供宝贵的实验工具。
实验整体设计：本研究依托杰克逊实验室（The Jackson Laboratory）心脏、肺、血液与睡眠障碍诱变项目，通过对雄性C57BL/6J（B6）小鼠施加N-乙基-N-亚硝基脲（ENU）诱变处理，构建突变小鼠。对第三代（G3）小鼠进行表型分型，以确保捕获显性与隐性突变。随后选取两株HDL胆固醇水平低下的独特G3个体，分别与B6小鼠交配，并将携带低HDL胆固醇表型的后代连续互交7代，从而构建两个近交系（Hlb218与Hlb320）。收集3只Hlb218雄性小鼠、3只Hlb320雄性小鼠及6只B6雄性对照小鼠的肝脏组织，用于基因表达分析。所有样本均随机分配至Illumina Mouse-6 Expression 1.1 BeadChips进行检测。