RUNX1 mutations mitigate quiescence to promote transformation of hematopoietic progenitors in Fanconi anemia.

Many inherited bone marrow failure syndromes (IBMFSs) present a high risk of transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). During transformation of IBMFSs, hematopoietic stem and progenitor cells (HSPCs) with poor fitness gain ectopic, dysregulated self-renewal secondary to somatic mutations via undefined mechanisms. Here, in the context of the prototypical IBMFS Fanconi anemia (FA), we performed multiplexed gene editing of mutational hotspots in MDS-associated genes in human induced pluripotent stem cells (iPSCs) followed by hematopoietic differentiation. We observed aberrant self-renewal and impaired differentiation of HSPCs with enrichment of RUNX1 insertions and deletions (indels), generating a model of IBMFS-associated MDS. We observed that compared to the failure state, FA MDS cells show mutant RUNX1-mediated blunting of the G1/S cell cycle checkpoint that is normally activated in FA in response to DNA damage. RUNX1 indels also lead to activation of innate immune signaling, which stabilizes the homologous recombination (HR) effector BRCA1, and this pathway can be targeted to abrogate viability and restore sensitivity to genotoxins in FA MDS. Together, these studies develop a paradigm for modeling clonal evolution in IBMFSs, provide basic understanding of the pathogenesis of MDS, and uncover a therapeutic target in FA-associated MDS.

Nicotinamide N-methyltransferase expression decreases in iron overload, exacerbating toxicity in mouse hepatocytes.

Iron overload causes the generation of reactive oxygen species that can lead to lasting damage to the liver and other organs. The goal of this study was to identify genes that modify the toxicity of iron overload. We studied the effect of iron overload on the hepatic transcriptional and metabolomic profile in mouse models using a dietary model of iron overload and a genetic model, the hemojuvelin knockout mouse. We then evaluated the correlation of nicotinamide N-methyltransferase (NNMT) expression with body iron stores in human patients and the effect of NNMT knockdown on gene expression and viability in primary mouse hepatocytes. We found that iron overload induced significant changes in the expression of genes and metabolites involved in glucose and nicotinamide metabolism and that NNMT, an enzyme that methylates nicotinamide and regulates hepatic glucose and cholesterol metabolism, is one of the most strongly down-regulated genes in the liver in both genetic and dietary iron overload. We found that hepatic NNMT expression is inversely correlated with serum ferritin levels and serum transferrin saturation in patients who are obese, suggesting that body iron stores regulate human liver NNMT expression. Furthermore, we demonstrated that adenoviral knockdown of NNMT in primary mouse hepatocytes exacerbates iron-induced hepatocyte toxicity and increases expression of transcriptional markers of oxidative and endoplasmic reticulum stress, while overexpression of NNMT partially reversed these effects. Conclusion: Iron overload alters glucose and nicotinamide transcriptional and metabolic pathways in mouse hepatocytes and decreases NNMT expression, while NNMT deficiency worsens the toxic effect of iron overload. For these reasons, NNMT may be a drug target for the prevention of iron-induced hepatotoxicity. (Hepatology Communications 2017;1:803-815).

Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia.

The prognostic value of the serum ferritin in a southern Brazilian cohort of patients with Gaucher disease.

The clinical utility of serum ferritin as a biomarker of disease severity and prognosis in Gaucher disease (GD) is still debated. Here, we aimed to evaluate ferritin and its relation to clinicolaboratory parameters of GD patients seen at the Reference Center for Gaucher Disease of Rio Grande do Sul, Brazil, so as to gather evidence on the utility of ferritin as a biomarker of this condition. A retrospective chart review was performed collecting pre-and posttreatment data from GD patients. Eighteen patients with ferritin levels available before and after treatment were included in the study. Nine of these participants were males, and seventeen had type I GD. All patients were given either enzyme replacement (n = 16) or substrate reduction therapy (n = 2), and ferritin was found to decrease from 756 [318-1441] ng/mL at baseline to 521 [227-626] ng/mL (p=0.025) after 28.8 month soft treatment. Serum ferritin levels did not correlate with measures of disease severity, but showed an association with age at onset of treatment (ρ= 0.880; n = 18; p < 0.001). In conclusion, although serum ferritin did not correlate with disease severity, after a median 28.8 months of treatment, clinical outcomes had clearly improved, and ferritin levels had decreased.

Serum ß2-microglobulin is frequently elevated in type 1 Gaucher patients.

ß2-Microglobulin is the major prognostic factor in multiple myeloma, a known comorbidity of Gaucher disease. We evaluated herein serum ß2-microglobulin levels of 31 type 1 Gaucher patients; for 8/31 patients, pre- and post-treatment comparisons were made. Thirteen patients (on treatment = 6) had high levels of ß2-microglobulin, and showed higher chitotriosidase activity and Severity Score Index, and lower concentration of platelets, than patients with normal levels. Levels of ß2-microglobulin correlated with chitotriosidase activity (ρ = 0.65; p < 0.01), platelets (ρ = - 0.42; p = 0.02) and α1- (ρ = 0.43; p = 0.02) and α2-protein bands (ρ = - 0.40; p = 0.03). Regarding pre- and post-treatment values, median ß2-microglobulin levels decreased after treatment (pre- = 2931 ng/mL; post- = 1970 ng/mL; p < 0.01). Our data suggest that levels of serum ß2-microglobulin are frequently elevated in type 1 Gaucher patients, correlate with severity of the disease and decrease after treatment.

Pitfalls in the prenatal diagnosis of mucolipidosis II alpha/beta: A case report.

Mucolipidosis II alpha/beta is an autosomal recessive disorder caused by deficient activity of GlcNAc-1-phosphotransferase. We report the prenatal diagnosis of a fetus who was found to exhibit normal levels of lysosomal enzymes in the amniotic fluid but low levels in amniocytes, and who was found to be heterozygous for the most common GNPTAB mutation. As in some carriers of Mucolipidosis II biochemical abnormalities may hinder prenatal diagnosis, we suggest DNA analysis should be performed whenever possible.