Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse. Marc1-/- mice were generated using Regeneron's (Tarrytown, NY) VelociGene® technology (ID #20561), and Wild-type (Marc1+/+) littermates were used as controls. All experiments were performed on 100% C57BL/6NTac background. Marc2 knockout mice (ID #20646) were generated similarly as Marc1 KO mouse using Regeneron VelociGene® technology, where second exon of Marc2 gene was replaced with LacZ reporter. Marc2 mice were bred on 100% C57BL/6NTac background. RNA-Seq was performed on RNA purified from wild-type and homozygous Marc1 or Marc2 knockout mice tissues (heart, kidney, liver, lung, and white adipose).近期的人类全基因组关联研究已在MARC1基因中鉴定出2个常见错义变异：p.Ala165Thr与p.Met187Lys，这些变异与较低的肝脏脂肪含量、肝脏酶水平降低以及对多数病因导致的肝硬化具有保护作用相关。本研究通过全外显子组关联研究，在来自5个祖先群体的54万名个体中重复验证了此前关于MARC1 p.Ala165Thr与p.Met187Lys变异的研究发现。本研究同时发现了MARC1基因中新型罕见的推定功能丧失变异，其表型与MARC1 p.Ala165Thr/p.Met187Lys变异相似。对重组人MARC1蛋白的体外研究显示，Ala165Thr位点的氨基酸替换会导致蛋白不稳定，并使其在肝细胞中出现异常定位，这提示抑制或敲除MARC1基因可能产生肝脏保护作用。基于这一假说，我们构建了Marc1基因敲除小鼠，并评估了Marc1基因敲除对小鼠肝脏表型的影响。出乎意料的是，本研究发现全身敲除Marc1基因的小鼠并不能抵抗肝脏甘油三酯蓄积、肝脏炎症或纤维化。为解释未观察到预期表型的原因，我们发现Marc1在小鼠肝脏中仅发挥微弱作用，而其旁系同源基因Marc2才是小鼠体内Marc家族的主要功能酶。本研究结果凸显了人类与小鼠MARC1基因生理功能之间的显著差异。本研究使用再生元制药（Regeneron，位于纽约州塔里敦）的VelociGene®技术（编号#20561）构建了Marc1基因敲除（Marc1-/-）小鼠，并以同窝野生型（Marc1+/+）小鼠作为对照。所有实验均在纯合C57BL/6NTac遗传背景小鼠中开展。Marc2基因敲除小鼠（编号#20646）的构建方法与Marc1敲除小鼠一致，同样采用Regeneron VelociGene®技术，将Marc2基因的第二外显子替换为LacZ报告基因。Marc2敲除小鼠均在纯合C57BL/6NTac遗传背景下繁育。本研究对野生型以及纯合Marc1或Marc2敲除小鼠的组织（心脏、肾脏、肝脏、肺脏与白色脂肪组织）中提取的RNA进行了RNA测序（RNA-Seq）。