Hemochromatosis - Epidemiology and Molecular Mechanisms

描述(申请人提供)：本项目的目的是为了更好地了解铁稳态的调节。铁稳态失调会导致铁超载或缺铁。铁稳态的主要调节者是由25个氨基酸组成的多肽海普西丁。HFE、TfR2和HJV基因突变引起的血色素沉着症主要是由于海普西丁转录失调所致。我们最近发现，Tmprss 6基因编码的一种血清蛋白水解酶是海普西丁的一种强有力的下调因子。只有在完好的动物身上才能观察到对铁的强烈转录反应。为了了解铁对海普西丁的正常转录调节，也为了深入了解参与其调节的已知基因产物的作用，我们将研究肝脏中的海普西丁转录，这是野生型小鼠和缺乏这些基因的突变小鼠合成的主要部位。在双管齐下的方法中，我们将：1)研究从铁刺激的小鼠制备的核提取物；2)在我们设计的体内系统中研究海普西丁启动子报告结构的反应。经典的体外技术包括EMSA、芯片分析、DNA亲和纯化和质量分光光度鉴定将被用来鉴定核提取液中的转录因子。这些研究将揭示哪些转录因子参与铁的反应，以及这种反应在突变动物中是如何改变的。在C282Y HFE突变纯合子的患者中，血色沉着症表型的表达有显著的变异性。到目前为止，研究还没有确定遗传或环境因素，除了很小一部分已知的变异性外，其他因素都无法解释。我们将根据前人对铁的经验，在小鼠身上调查是否存在铁吸收的跨代表观遗传修饰。接受缺铁饮食或含铁过剩饮食的小鼠将被培育，它们的后代将断奶以相同的饮食或正常的饮食。那些断奶后饮食不足或铁过量的人将繁殖出更多的后代。如果发生了跨代表观遗传修饰，那么转移到正常饮食的缺铁小鼠的铁负担将比来自铁重祖细胞的小鼠更大。公共卫生相关性：通过研究铁超载和铁缺乏的各种小鼠模型，我们将了解身体如何调节铁。这将为铁超载、慢性炎症贫血以及血色素沉着症本身的治疗打开大门。如果我们能够证明铁吸收的调节是表观遗传的，这一发现不仅将解释血色病的可变外显性，而且将极大地改变我们对自然选择如何在仅几代人的时间内发挥作用的看法。

DESCRIPTION (provided by applicant): The purpose of this project is to better understand the regulation of iron homeostasis. Dysregulation of iron homeostasis results in iron overload or iron deficiency. The major regulator of iron homeostasis is the 25 amino acid peptide hepcidin. Hemochromatosis caused by genetic mutations in HFE, TfR2 and HJV is due primarily to dysregulation of hepcidin transcription. We have recently discovered that a serum protease encoded by the Tmprss6 gene is a potent downregulator of hepcidin. A robust transcriptional response to iron is only observed in intact animals. To understand normal transcriptional regulation of hepcidin by iron and also to gain insight into the roles of the products of the genes known to be involved in its regulation, we will investigate hepcidin transcription in liver, the primary site of synthesis in wildtype mice and mutant mice lacking these genes. In a two-pronged approach we will: 1) study nuclear extracts prepared from mice stimulated with iron; and 2) investigate the response of hepcidin promoter reporter constructs in an in vivo system that we have devised. Classical ex vivo techniques including EMSA, ChIP assays, DNA affinity purification and mass spectrophotometric identification will be employed to identify transcription factors in nuclear extracts. These studies will reveal which transcription factors are involved in the response to iron and how this response is modified in mutant animals. There is marked variability in the expression of the hemochromatosis phenotype in patients homozygous for the C282Y HFE mutation. Studies to date have failed to identify either genetic or environmental factors that account for any but a very small portion of the known variability. We will investigate, in mice, whether there is transgenerational epigenetic modification of iron absorption, based on the experience of prior generations with iron. Mice receiving iron-deficient diets or diets containing iron excess will be bred and their offspring will either be weaned to the same diet or to a normal diet. Those weaned to a deficient or iron excess diet will be bred for additional generations. If transgenerational epigenetic modification occurs, then the iron burden of iron deficient mice moved to normal diet will be greater than that of mice derived from iron-heavy progenitors. PUBLIC HEALTH RELEVANCE: By studying various mouse models of iron overload and iron deficiency, we will gain an understanding how the body regulates iron. This will open the door to treatments of iron overload, the anemia of chronic inflammation, as well as of hemochromatosis itself. If we are able to demonstrate that the regulation of iron absorption is inherited epigenetically, this finding would not only explain the variable penetrance of hemochromatosis, but would greatly alter our perception of how natural selection functions over the span of only a few generations.