Maternal Liver Disease and Fatty Acid Oxidation Defects

线粒体三功能蛋白(TFP)催化长链脂肪酸β-氧化螺旋的最后3步，由4个α和4个β亚基组成。长链3-羟基酰基辅酶A脱氢酶(LCHAD)位于α-亚基。阿尔法亚单位的突变，如流行的G1528C突变，会导致“孤立的”LCHAD缺陷。其他突变导致TFP完全缺乏(所有3种酶都缺乏)。最近，我们记录了一种胎儿和母亲的相互作用，导致携带孤立LCHAD缺乏症胎儿的杂合子妇女发生母体肝病。这引发了几个问题。首先，这种母胎相互作用的机制是什么？第二，高脂饮食、禁食等环境因素对易感杂合子母体肝病的发生有何影响？我们的假设是，由于受影响的胎儿或易感的杂合子在氧化应激条件下产生的肝毒性脂肪酸代谢物积累，杂合子妇女会患上急性妊娠脂肪肝(AFLP)或HELLP(溶血、肝酶升高和血小板降低)综合征。为了验证这一假设，我们提出了以下研究。1)采用常规的和可诱导的Cre/lox P策略，建立和鉴定了两种TFP完全缺陷(零突变)和孤立LCHAD缺陷(G1528C突变)的基因敲除小鼠模型。将进行临床、生化、组织学和分子分析。组织特异性和发育阶段特异性的基因表达也将在这些小鼠中表现出来。脂肪酸代谢物累积的差异将与基因类型和表型相关，以阐明脂肪酸代谢物在这些疾病的基因-表型相关性中的作用。2)采用植入前基因分型和胚胎移植的方法，在基因敲除小鼠中独立研究胎儿和母体基因对母体肝病发生的影响。怀孕的水坝将接受监测，以寻找肝病的证据。将测量胎儿和母体血清、胎儿和母体肝脏以及胎盘中的脂肪酸代谢物，并将其与胎儿/母体基因型和母体表型相关联，以确定对母体肝脏有潜在毒性的脂肪酸代谢物。3)在基因敲除小鼠中进行饮食研究，以阐明高脂饮食和禁食对怀孕的杂合子在怀未受影响胎儿时的影响。将对四种不同的高脂肪饮食进行研究，以阐明脂肪含量、脂肪酸结构和蛋白质/碳水化合物含量的影响。

Mitochondrial trifunctional protein (TFP) catalyzes the last 3 steps in the beta-oxidation spiral of long chain fatty acids and consists of 4 alpha and 4 beta subunits. Long chain 3- hydroxyacyl Co-A dehydrogenase (LCHAD) resides in the alpha- subunit. Mutations in the alpha-subunit such as the prevalent G1528C mutation cause "isolated" LCHAD deficiency. Other mutations cause complete TFP deficiency (all the 3 enzymes are deficient). Recently, we have documented a fetal-maternal interaction that causes maternal liver disease in heterozygote women who carry fetuses with isolated LCHAD deficiency. This raises several questions. First, what is the mechanism of this fetal-maternal interaction? Second, what is the effect of environmental factors such as high fat diet and fasting on the development of maternal liver disease in the susceptible heterozygotes? Our hypothesis is that heterozygote women develop acute fatty liver of pregnancy (AFLP) or HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome due to accumulation of hepato-toxic fatty acid metabolites generated by either the affected fetus or the susceptible heterozygote under conditions of oxidative stress. To test this hypothesis we propose the following studies. 1) To use conventional and inducible Cre/lox P strategies to generate and characterize two knockout mice models for complete TFP deficiency (null mutation) and isolated LCHAD deficiency (G1528C mutation). Clinical, biochemical, histological, and molecular analyses will be performed. Tissue-specific and developmental stage-specific gene expression will also be characterized in these mice. Differences in the accumulated fatty acid metabolites will be correlated to the genotypes and phenotypes to elucidate the role of fatty acid metabolites in the genotype-phenotype correlations in these disorders. 2) To employ preimplantation genotyping and embryo transfer to independently study the effects of fetal and maternal genotypes on development of maternal liver disease in knockout mice. Pregnant dams will be monitored for evidence of liver disease. Fatty acid metabolites will be measured in fetal and maternal sera, fetal and maternal livers, and placentas, and will be correlated to the fetal/maternal genotypes and maternal phenotypes to identify the fatty acid metabolites that are potentially toxic to the maternal liver. 3) To conduct dietary studies in knockout mice to elucidate the effects of high fat diet and fasting on pregnant heterozygotes while carrying unaffected fetuses. Four different high fat diets will be studied to elucidate the effects of fat content, fatty acid configuration, and protein/carbohydrate contents.