Identification of Differentially Expressed Genes in Mice with Nutritional or Genetic Causes of Iron Overload

Iron overload causes the generation of reactive oxygen species, which can lead to lasting organ damage, particularly to the liver. In patients with hereditary hemochromatosis, transfusion-dependent anemias, and hemoglobinopathies, iron overload is a major cause of mortality. A deeper understanding of iron regulation and the biological pathways involved in maintaining homeostasis may reveal new therapeutic targets for patients with iron overload disorders. We designed this study to discover genes that are differentially expressed in nutritional and genetic models of iron overload. For the nutritional iron overload study, 5-week old male C57BL/6 mice were placed on a soy-free diet (AIN-93G) containing different amounts of iron per kilogram of food: iron-deficient (2.5 mg/kg, n=3), iron-sufficient (37.5 mg/kg, n=3), and iron-excess (750 mg/kg, n=3). In the second study, 5-week old male C57BL/6 mice that were either wild type or HJV knockout mice that exhibited severe early onset iron overload secondary to homozygous deficiency of the bone morphogenic protein coreceptor, hemojuvelin (HJV), were maintained on the iron-deficient (2.5 mg/kg iron) diet (n=2 per group). For both studies animals were sacrificed after 50 days and liver RNA was extracted and sequenced at 40-50 million reads per sample. The RNA integrity number (RIN) for each sample was >6 and assessments of read duplication, base call frequency, and read quality indicated excellent quality of the data. For the HJV knockout mice, we used a false discovery rate <0.05 and a mean-fold change >2, to reveal genes that were differentially expressed compared to wild type mice. For the dietary iron study, genes were grouped by self-organizing maps to identify transcripts whose level of expression trended with increased or decreased dietary iron intake. The resulting analysis identified 148 genes in nutritionally iron-overloaded mice and 688 genes in HJV knockout mice that exhibited significant changes in expression. Of these, 28 genes were differentially regulated in both nutritionally iron overloaded and HJV knockout mice, including expected genes, such as transferrin receptor, HAMP (hepcidin), and bone morphogenic protein 6, and unexpected genes such as cytochrome P450 17a1 (cyp17a1), an enzyme that catalyzes critical steps in steroid synthesis, and nicotinomide N-methyltransferase (nnmt), an enzyme that regulates drug metabolism and DNA methylation. We clustered the 688 differentially expressed genes from the HJV knockout mice into functional pathways using the Functional Analysis tool from DAVID Bioinformatics Resources 6.7 (NIAID). Clusters were considered significant if there were >2 genes in the pathway and the Benjamini-Hochberg P-value was <0.05. We found that the expression of genes involved with PPAR signaling (P=0.0086) was decreased, while expression of transcripts involved with Huntington’s disease (P=0.038) was increased in HJV knockout mice compared to wild-type mice. Our RNA sequencing analysis identified a variety of novel pathways that were differentially regulated in dietary and genetic models of iron overload. Further studies are underway to characterize the potential roles of these genes in iron homeostasis.