1. Iron, Inflammation, and Early Death in Adults With Sickle Cell DiseaseNovelty and Significance
by Eduard J. van Beers, Yanqin Yang, Nalini Raghavachari, Xin Tian, Darlene T. Allen, James S. Nichols, Laurel Mendelsohn, Sergei Nekhai, Victor R. Gordeuk, James G. Taylor, and Gregory J. Kato
Circulation Research
Volume 116(2):
January 16, 2015
Copyright © American Heart Association, Inc. All rights reserved.

2. A subgroup of sickle cell disease (SCD) patients cluster together in a group with high expression of iron-regulated genes.
A subgroup of sickle cell disease (SCD) patients cluster together in a group with high expression of iron-regulated genes. Peripheral blood mononuclear cells (PBMC) transcriptome profiling in 24 SCD patients and 10 healthy controls identified 3 clusters of subjects with high, low, and intermediate expression of iron-regulated genes. Patients (ClinicalTrials.gov identifiers: NCT ). The Heat map shows unsupervised 2-way hierarchical clustering of the predefined set of iron-regulated genes on the y axis (for a tabular version of this list, see Online Data Supplement) and the clustering of study participants on the x axis. None of the controls was found to be in the high iron cluster. C indicates control; and S, SCD patient.
Eduard J. van Beers et al. Circ Res. 2015;116:
Copyright © American Heart Association, Inc. All rights reserved.

3. Quantitative polymerase chain reaction (Q-PCR) results confirm the relation between iron-regulated genes and genes involved in inflammatory and anti-inflammatory pathways.
Quantitative polymerase chain reaction (Q-PCR) results confirm the relation between iron-regulated genes and genes involved in inflammatory and anti-inflammatory pathways. To validate the microaray results, we performed quantitative polymerase chain reaction (Q-PCR) normalized to the housekeeping gene RNA18S5 on the same samples. (ClinicalTrials.gov identifiers: NCT ). A, This diagram shows a good correlation (R=0.72, P=0.023) between the fold change as found by microarray and Q-PCR. B, Bars show the fold change in expression of the group of patients with highly expressed iron-regulated genes over the other groups. C, The number of PCR cycles to reach threshold compared with the housekeeping gene RNA18S5 (ΔCT) of FTL was highly correlated to TLR4 in both patients (R=0.930, P<0.001) as controls (R=0.943, P=0.026), although mean expression of both genes was much higher in patients than in controls (P<0.001). TLR4 was also highly correlated to ALDH1A1, GAPDH, SAT2, and HMOX expression (data not shown). To get a better impression of actual gene expression, x and y axis are reversed and originate 20 ΔCT. D, Bars show the fold change in expression of selected genes in sickle cell disease (SCD) patients compared with healthy controls. Black bars represent genes that were included in the predefined set of iron-regulated genes (see Online Table I). White bars represent genes that were identified as signifcantly differentially expressed between the 3 iron clusters or were included to validate the microarray data. ALDH1A1 indicates aldehyde dehydrogenase 1A1; ECE1, endothelin converting enzyme 1; FTL1, ferritin light chain 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMOX, heme oxygenase-1; IL6, interleukin-6; SAT2, spermidine/spermine N1-acetyltransferase family member 2; ST13, suppression of tumorigenicity 13; and TLR4, toll-like receptor 4.
Eduard J. van Beers et al. Circ Res. 2015;116:
Copyright © American Heart Association, Inc. All rights reserved.

4. Patients with the ferroportin Q248H mutation which is associated with high retention of intracellular iron have higher levels of C-reactive protein (CRP) and interleukin-6.
Patients with the ferroportin Q248H mutation which is associated with high retention of intracellular iron have higher levels of C-reactive protein (CRP) and interleukin-6. To assess relationship between intracellular iron and inflammation, we used available ferroportin Q248H mutation status. Patients were analyzed previously for ferroportin mutation status.29A and B, In the gene-expression part of the study (ClinicalTrials.gov identifier: NCT ), the 5 patients with a ferroportin Q248H mutation (GT and TT genotype) had a significant (P<0.05) higher plasma level of CRP, and log 10 interleukin-6 (IL-6) trended (P=0.08) to be higher compared with 19 patients with wild-type ferroportin (GG genotype). C and D, As a sensitivity analysis, we repeated this in a case–control study in selection of patients of the National Institutes of Health (NIH) sickle cell disease (SCD) cohort (ClinicalTrials.gov identifier: NCT ). Fourteen patients with Q248H mutation were matched on genotype and ferritin level. We found that the 14 patients with the mutation had a significant higher CRP and log10 il6 than the matched controls. Because the ferroportin Q248H is also associated with less sensitivity for hepcidin and hepcidin levels are strongly associated with ferritin levels in SCD, we decided to perform this last analysis only in patients with a ferritin level <1000 mg/dL. *P<0.05.
Eduard J. van Beers et al. Circ Res. 2015;116:
Copyright © American Heart Association, Inc. All rights reserved.

5. Kaplan–Meier curve showing a significant difference in survival between patients with low and high C-reactive protein (CRP).
Kaplan–Meier curve showing a significant difference in survival between patients with low and high C-reactive protein (CRP). Sickle cell disease (SCD) patients (n=412) of the National Institutes of Health (NIH) pulmonary hypertension screening cohort (ClinicalTrials.gov identifier: NCT ) were divided in a low, intermediate, and high CRP group at the 50th and 75th percentile. Median follow-up time was 47 months (interquartile range, 24–82; range 2–132). After 100 months, patients were censored. Log-rank test showed that survival of patients between CRP groups was significantly different (P=0.0017). Five-year mortality percentages for the low, intermediate, and high CRP groups were, respectively, 12.4%, 20.5%, and 25.8%. In a multivariate analysis, CRP was an independent predictor of mortality (see Table 2).
Eduard J. van Beers et al. Circ Res. 2015;116:
Copyright © American Heart Association, Inc. All rights reserved.