Ferroportin Q248H Mutation Research Articles

Ferroportin Q248H Mutation Leads to a Moderate Iron Load in Mutant Slc40a1Q248H/Q248H Mice

BACKGROUND: In Africans, ferroportin (Scl40a1 or FPN) Q248H mutation is associated with increased iron load. FPN Q248H has high allele frequency (2.2-13.4%) and is linked to reduction of inflammation and reduced anemia in Africa. We previously showed that FPN Q248H resists degradation by physiologic hepcidin concentrations and thus may facilitate higher iron uptake. OBJECTIVES: to analyze the effect of FPN Q248H mutation in vivo in a knock-in Slc40a1Q248H mouse model on normal and high iron diet. METHODS: The study was approved by Howard University IACUC committee. The Slc40a1Q248H/WT mice were generated by Flp- FRT recombination on the C57BL/6 background and homozygous Slc40a1Q248H/Q248H mutant mice were produced in Howard University animal facility by breeding. Twenty-three weeks old homozygous and wild type C57BL/6 (WT) females were fed normal or 2% carbonyl iron diet. Total iron concentration and ferritin were measured in serum by ELISA. Expression of FPN, transferrin receptor (TFR), ferritin heavy chain (FHC), divalent metal transporter 1 (DMT1), duodenal cytochrome B (Dcytb), hepcidin, and IL-1β, IL-6, and IL-18 cytokines were measured by real-time RT-PCR. Iron deposition was determined by Perl's staining. Hepatic oxidative stress was measured by malondialdehyde ELISA. RESULTS: On normal iron diet, Slc40a1Q248H/Q248H mice grew normally and showed a tendency to increased serum ferritin, decreased serum iron and reduced hematocrit compared to WT mice. Levels of FPN,Dcytb and DMT1 mRNA were increased in duodenum of mutant mice. In the spleen of Slc40a1Q248H/Q248H mice, FPN,FTH1, TFR and DMT1 mRNAs were also elevated. In contrast, in the liver of Slc40a1Q248H/Q248H mice, levels of FPN and TFR mRNA were decreased, but hepcidin levels were increased compared to WT mice. There was a trend towards hepatic FTH1 level increase in the mutant mice. Hepatic expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA was similar between mutant and WT mice. No iron accumulation was found in the liver, and iron accumulation in the spleen was comparable between WT and Slc40a1Q248H/Q248H mice. On high iron diet, Slc40a1Q248H/Q248H mice showed significant increase in serum ferritin compared to WT mice on normal diet. Levels of FPN mRNA remained increased whereas Dcytb and DMT1 mRNA levels were decreased in duodenum of the mutant mice. In the liver, FPN mRNA levels were further decreased in the mutant mice, and FTH1 levels were equally increased in WT and Slc40a1Q248H/Q248H mice. In the spleen, FPN mRNA levels were equally increased for WT and Slc40a1Q248H/Q248H mice. Spleen FTH1 mRNA levels were further elevated in mutant mice compared to WT mice. Higher iron accumulation was found in the liver and spleen of Slc40a1Q248H/Q248H mice compared to WT mice. Elevated levels of hepatic oxidative stress were observed in the liver of Slc40a1Q248H/Q248H mice. Expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA remained low in the liver of Slc40a1Q248H/Q248H mice. CONCLUSION: Upregulation of duodenal FPN, Dcytb and DMT1 and splenic FPN,FTH1, TFR and DMT1 expression indicated increased iron absorption and recycling in female Slc40a1Q248H/Q248H mice. The mutantmice showed tendency to mild iron load in tissues as indicated by increased serum ferritin and higher ferritin levels in the spleen and liver. High iron diet led to significant iron accumulation in the liver and increased hepatic stress compared to WT mice. Surprisingly, hepatic levels of pro-inflammatory cytokines were reduced in the mutant mice. Taken together, our study of the first in vivo model of FPNQ248H mutation indicates that it alters iron metabolism and might reduce liver inflammation. ACKNOWLEDGEMENTS: This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685.

Ferroportin Q248H mutation was not found to be protective against malaria and anemia in children under 5 years living in South Kivu/Democratic Republic of Congo, an endemic area of Plasmodium infection

BackgroundFerroportin (FPN) is known as an iron exporter and its effect on RBC iron could therefore hamper the growth of malaria parasites, since parasites are in need of iron. The aim of this study was to examine the prevalence of FPN Q248H in South Kivu/DRC and to evaluate its role in Plasmodium infected children and to explore its relationship with anemia. Materials and methodsWe conducted a cross-sectional study in the health zone of Miti Murhesa in South Kivu/DRC. 1088 children aged under five years were included. The FPN Q248H mutation was analyzed by PCR (N = 1071). Allele frequency was calculated based on Hardy-Weinberg equation. Plasmodium infection was assessed by LAMP malaria assay (N = 1057). Statistical analysis was done using Medcalc® software. P-values < 0.05 were considered significant. ResultsWe found 11.4% FPN Q248H mutation. T allele frequency was estimated to be 0.0588 ± 0.0052. No significant differences for frequencies of anemia and malaria were observed between FPN Q248H mutation and FPN wild type. However, Plasmodium infected carriers of the FPN Q248H mutation had lower hemoglobin values than wild type children. ConclusionEven though FPN Q248H mutation is associated with lower hemoglobin values in Plasmodium infected children, it was not found to be protective against malaria and anemia in children under 5 years living in malaria endemic area of South Kivu/Democratic Republic of Congo.

Similar Ferroportin Q248H polymorphism prevalence in patients with Plasmodium falciparum malaria and control subjects in the low-endemic setting of Botswana

IntroductionRecent evidence suggests that ferroportin (FPN) Q248H may confer a survival advantage against malaria by reducing erythrocytic intracellular iron in Africans. We investigated if FPN Q248H mutation, that is prevalent in Batswana, is a factor in limiting the susceptibility to Plasmodium falciparum malaria. Methods264 archived dried blood spot samples (183 P. falciparum malaria cases and 81 controls, matched for geographical region and season for equal exposure) were genotyped. Human and P. falciparum DNA was extracted using Chelex-100 resin and P. falciparum molecular confirmation performed. Ferroportin Q248H mutation was identified by restriction fragment length polymorphism. The prevalence of the FPN Q248H mutation and allele frequency and the accompanying 95% confidence interval were calculated. A qPCR method was employed to estimate P. falciparum parasitaemia. Association between FPN and malaria susceptibility was tested using Pearson Chi-square test and Mood's median test was used to compare P. falciparum parasitaemias according to FPN Q248H mutation. ResultsAll samples were successfully genotyped. The FPN Q248H allele frequency was 0.08 (95% CI: 0.05–0.11) in cases and 0.08 (95% CI: 0.02–0.14) in controls, consistent with Hardy-Weinberg equilibrium. The prevalence of FPN Q248H phenotype was comparable in patients with P. falciparum malaria and in un-infected individuals, 16.4% (95% CI: 11.0–21.8) vs 14.8% (95% CI: 7.1–22.5), P = 0.746. In addition, no association of presence of FPN Q248H with reduced parasitaemia was recorded, P = 0.837. ConclusionIn this small study, FPN Q248H polymorphism prevalence was comparable between patients with P. falciparum malaria and control subjects in the low-endemic setting of Botswana.

Ferroportin Q248H Mutation is Not Protective Against Malaria, But is Associated with Anemia in Children Under 5 Years in South Kivu/Democratic Republic of Congo with Malaria

Ferroportin Q248H Mutation is Not Protective Against Malaria, But is Associated with Anemia in Children Under 5 Years in South Kivu/Democratic Republic of Congo with Malaria

Abstract PO-147: Kidney iron overload in African American renal cancer patients with the ferroportin Q248H mutation

Abstract Iron plays a role in cellular replication, metabolism, and growth. Although iron is needed, the imbalance of iron in the body can result in adverse health effects, including cancer. Interestingly, cancer cells have frequently been observed to express increased levels or activity of proteins that are associated with iron metabolism. Ferroportin (FPN), the only known mammalian cell iron exporter, is essential for iron homeostasis. Germline mutations in FPN occur more frequently in African and African American populations. A glutamine to histidine amino acid substitution at position 248 (Q248H) is the first and most prevalent gain-of-function mutation observed in FPN, with an allele frequency range of 2.2% to 13.4% in African and African Americans. However, it is unknown whether FPN Q248H is involved in cancer-associated reprogramming of iron metabolism, especially in individuals of African descent where the FPN Q248H mutation is enriched. The Q248H mutation traditionally renders FPN resistant to degradation. As a result, iron release is increased and aberrant uptake of iron across epithelial barriers of tissues can occur to cause iron overload, a risk factor for cancer. We hypothesize that patients with FPN Q248H have improper regulation of iron and thus increased unbound iron that will result in subsequent systemic and kidney-specific iron overload. We leveraged the repository of kidney cancer patients and banked samples of the Urologic Oncology Branch at the NIH to determine the association of FPN Q248H with kidney cancer. In our study, Black males with kidney cancer that expressed FPN Q248H had earlier onset of cancer compared to those without the variant. There were higher iron levels (p&amp;lt;0.01) and transferrin saturation (p=0.01), a measure for blood iron transport, in serum form Black males with FPN Q248H compared to Black males without. Additionally, serum levels of the iron storage protein ferritin were elevated in association with FPN Q248H. In the kidney, both normal and tumor tissue had distinct levels of iron, as well as protein expression of ferritin and FPN between patients with and without FPN Q248H. In all, these results highlight a potential novel mechanism for global iron-associated cancer risk populations. This work will provide a foundation for future studies that include pre-cancer individuals and other cancer outcomes where the relationship between FPN Q248H and iron overload can be further examined to expand our knowledge of cancer risk. Citation Format: Samira Brooks, Cathy Vocke, Deliang Zhang, Victor Gordeuk, Tracey Rouault, Marston Linehan. Kidney iron overload in African American renal cancer patients with the ferroportin Q248H mutation [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-147.

Disease correction by intraparenchymal or cisternal delivery of a modified AAV8 capsid expressing codon optimized NAGLU for mucopolysaccharidosis type IIIB mice

The ferroportin Q248H mutation is relatively common in sub-Saharan Africa. No previous study examined its relationship with atypical diabetes mellitus (DM) in this area.To determine the potential interactions between ferroportin Q248H mutation, hyperferritinemia and DM in South Kivu (RDC).Presence of ferroportin Q248H mutation and iron status were investigated in diabetic patients (n = 179, age (mean) 57.7 years, CRP (median) 0.16 mg/L) and non-diabetic subjects (n = 86, age 44.5 years, CRP 0.07 mg/L) living in the city of Bukavu. Hyperferritinemia was considered for values greater than 200 and 300 μg/L in women and in men, respectively.The prevalence of ferroportin Q248H mutation [12.1%] was non-significantly higher in diabetics than non-diabetics [14.0% vs. 8.1%, p = 0.17]. Similarly, hyperferritinemia frequency was higher in diabetic patients with Q248H mutation [44.0% vs. 14.3%, p = 0.16] and in mutation carriers [37.0% vs 16.5%, p = 0.001] than in the control groups, respectively. The association between Q248H mutation and DM was nevertheless not significant [adjusted OR 1.70 (95% CI: 0.52–5.58), p = 0.37], whereas hyperferritinemia [OR 2.72 (1.24–5.98), p = 0.01] showed an independent effect after adjustment for age and metabolic syndrome.The present work suggests a potential association between abnormal iron metabolism, ferroportin Q248H mutation and atypical DM in Africans, which may be modulated by environmental factors.

Control of HIV-1 Infection By Ferroportin Q248H Mutation

BACKGROUND: We recently showed that patients with Sickle Cell Disease (SCD), a hereditary hemolytic disorder, have low incidence of HIV-1 infection [1] and reduced ex vivo HIV-1 infection [2]. PBMC from SCD patients exhibited increased expression of iron export protein, ferroportin and reduced cellular iron levels leading to CDK2 inhibition, reduced SAMHD1 phosphorylation and increased expression of IkBα. Ferroportin expression is regulated by liver-produced hepcidin that facilitates ferroportin internalization and degradation. Ferroportin Q248H mutation has an allele frequency of 2.2-13.4% in African populations. We previously reported reduced sensitivity of ferroportin Q248H mutant to physiologic hepcidin concentrations in patients with sickle cell disease [3]. OBJECTIVES: To analyze the effect of ferroportin Q248H mutation on HIV-1 infection in vitro and in disease progression among a cohort of HIV-1 infected African-American women. METHODS: HEK293 cells were used to express ferroportin Q248H mutant and test cellular ferritin and intracellular labile iron using calcein-AM. Confocal microscopy was used to visualize ferroportin expression. HIV-1 transcription was measured in 293T cells transfected with HIV-1 LTR-Luciferase vector and Tat expressing vector. Ex vivo infection was analyzed in monocyte-derived macrophages infected with VSVg-pseudotyped HIV-1 virus. Ferroportin Q248H mutation was genotyped using Thermo Fisher probe (C_25753769_10) and genotyping services at University of Utah. RESULTS: We observed reduced intracellular iron in ferroportin Q248H expressing cells compared to WT ferroportin even when the cells were treated with hepcidin. In the absence of hepcidin, both WT ferroportin and Q248H ferroportin efficiently inhibited HIV-1 transcription and replication. Hepcidin induced HIV-1 transcription and replication in the cells with WT ferroportin but not Q248H mutant ferroportin. HIV-1 replication was reduced in primary macrophages obtained from patients with ferroportin Q248H mutation. To test whether expression of ferroportin Q248H offered protection from HIV-1 infection, we analyzed a cohort of HIV-1 infected women (WIHS). We genotyped 970 African-American subjects of whom 628 were HIV-1 infected and 342 were non-infected. The prevalence of Q248H hetero or homozygote mutations was 7.0% in non-infected and 11.8% among HIV-1 infected individuals (Odds Ratio=1.77, p=0.02). Analysis of HIV viral load showed significant lower viral load in the subjects with ferroportin Q248H mutation compared to WT. CONCLUSIONS: Our findings point to the contribution of iron metabolism in HIV-1 restriction and the potential role of the ferroportin Q248H mutation in the regulation of HIV-1 infection in vivo. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597 and P30AI087714). We thank Women's Interagency HIV-1 study (WIHS) for sharing DNA samples and providing access to the clinical data. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. REFERENCES: Nouraie M, Nekhai S, Gordeuk VR. Sickle cell disease is associated with decreased HIV but higher HBV and HCV comorbidities in U.S. hospital discharge records: a cross-sectional study. Sex Transm Infect. 2012;88(7):528-533. Kumari N, Ammosova T, Diaz S, et al. Increased iron export by ferroportin induces restriction of HIV-1 infection in sickle cell disease. Blood Adv. 2016;1(3):170-183. Nekhai S, Xu M, Foster A, et al. Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica. 2013;98(3):455-463. Disclosures Anastos: NINR: Research Funding; NHGRI: Research Funding; NICHD: Research Funding; NIMH: Research Funding; NHLBI: Research Funding; NCI: Research Funding; NIAID: Research Funding; NINDS: Research Funding; NIDCR: Research Funding; NIMHD: Research Funding; NLM: Research Funding; Fogarty: Research Funding; NIDDK: Research Funding; NIA: Research Funding; NIAAA: Research Funding; NIDA: Research Funding.

The association between fructosamine-3 kinase 900C/G polymorphism, transferrin polymorphism and human herpesvirus-8 infection in diabetics living in South Kivu

Prevalences of human herpesvirus-8 (HHV-8) infection and diabetes mellitus are very common in certain parts of Africa, containing iron-rich soils. We hypothesized that some genetic factors could have a link with susceptibility to HHV-8 infection. We focused on ferroportin Q248H mutation (rs11568350), transferrin (TF) polymorphism and fructosamine-3 kinase (FN3K) 900C/G polymorphism (rs1056534). The study population consisted of 210 type 2 diabetic adults and 125 healthy controls recruited in Bukavu (South Kivu). In the whole study population (diabetics+healthy controls), ferroportin Q248H mutation was detected in 47 subjects (14.0%) with 43 heterozygotes and 4 homozygotes. TF phenotype frequencies were 88.1% (CC), 10.4% (CD) and 1.5% (BC). Genotype frequencies of FN3K 900C/G polymorphism were respectively 9,3% (CC), 43.3% (GC) and 47.4% (GG). Prevalence of HHV8-infection in the study population was 77.3%. HHV-8 infection rate and HHV-8 IgG antibody titer were significantly higher in diabetics then in controls (p<0.0001). Significant differences were observed in HHV-8 infection rate and in HHV-8 IgG antibody titer according to FN3K rs1056534 (p<0.05 and p<0.05, respectively) and TF polymorphism (p<0.05 and p=0.005, respectively). No significant differences in HHV-8 infection rate and in HHV-8 IgG antibody titer were observed in the ferroportin Q248H mutation carriers (rs11568350) in comparison with ferroportin wild type. In a multiple regression analysis, FN3K rs1056534, TF polymorphism and presence of diabetes mellitus were predictors for HHV-8 infection. In contrast to these findings, ferroportin Q248H mutation (rs11568350) did not influence the susceptibility for an HHV-8 infection in sub-Saharan Africans.

Serum ferritin concentration is affected by ferroportin Q248H mutation in Africans

BackgroundFerroportin Q248H mutation is common in populations with African ancestry. Studies have reported that the mutation does not alter the ferroportin–hepcidin axis, but there is evidence suggesting that the mutation may lead to hyperferritinemia. We report on the relationship of ferroportin Q248H mutation on serum ferritin (SF) in health adults. Subjects and methodsA total of 174 apparently healthy adults from Botswana were studied. SF was measured using an enzyme immunoassay and ferroportin Q248H mutation was identified by polymerase chain reaction and restriction enzyme digestion. Independent sample Mann–Whitney U test was used to correlate the presence of the mutation with SF. ResultsFerroportin Q248H mutation was identified in 30 individuals (17.2%) (one homozygote, 29 heterozygotes) and was absent in 144 individuals (82.8%), with Q248H allele frequency of 8.9%. In males, SF was significantly higher in ferroportin Q248H heterozygotes compared to wild types, p=0.029, but the relationship between ferroportin Q248H mutation and iron stores was blunted in females. ConclusionOur study of healthy adults provides further evidence that ferroportin Q248H mutation affects SF concentration in Africans.

Ferroportin Q248H mutation, hyperferritinemia and atypical type 2 diabetes mellitus in South Kivu

BackgroundThe ferroportin Q248H mutation is relatively common in sub-Saharan Africa. No previous study examined its relationship with atypical diabetes mellitus (DM) in this area. ObjectiveTo determine the potential interactions between ferroportin Q248H mutation, hyperferritinemia and DM in South Kivu (RDC). MethodologyPresence of ferroportin Q248H mutation and iron status were investigated in diabetic patients (n=179, age (mean) 57.7 years, CRP (median) 0.16mg/L) and non-diabetic subjects (n=86, age 44.5 years, CRP 0.07mg/L) living in the city of Bukavu. Hyperferritinemia was considered for values greater than 200 and 300μg/L in women and in men, respectively. ResultsThe prevalence of ferroportin Q248H mutation [12.1%] was non-significantly higher in diabetics than non-diabetics [14.0% vs. 8.1%, p=0.17]. Similarly, hyperferritinemia frequency was higher in diabetic patients with Q248H mutation [44.0% vs. 14.3%, p=0.16] and in mutation carriers [37.0% vs 16.5%, p=0.001] than in the control groups, respectively. The association between Q248H mutation and DM was nevertheless not significant [adjusted OR 1.70 (95% CI: 0.52–5.58), p=0.37], whereas hyperferritinemia [OR 2.72 (1.24–5.98), p=0.01] showed an independent effect after adjustment for age and metabolic syndrome. ConclusionsThe present work suggests a potential association between abnormal iron metabolism, ferroportin Q248H mutation and atypical DM in Africans, which may be modulated by environmental factors.

Ferroportin Q248H Mutation Protects From HIV-1 Infection in Vitro

AbstractAbstract 993Ferroportin is the only iron exporter expressed in mammalian cells, and hepcidin produced by the liver binds to ferroportin leading to its internalization and degradation by lysosomes. We recently reported that expression of ferroportin in 293T cells transfected with HIV-1 LTR-LacZ and Tat expression vector led to decreased HIV transcription, possibly by reducing availability of intracellular iron, and that exposure to hepcidin restored HIV transcription1. The Q248H mutation in ferroportin has an allele frequency of 2.2–13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. The ferroportin Q248H mutation was reported to associate with lower hepcidin levels in HIV-1 infected Rwandese women2. We also recently showed that ferroportin Q248H mutant has reduced sensitivity to physiologic hepcidin concentrations. We expressed WT and Q248H mutant ferroportin in 293T cells that express very low levels of endogenous ferroportin. We also expressed ferroportin C326Y, a mutant that is not sensitive to hepcidin. We analyzed the effect of ferroportin Q248H on cellular Intracellular ferritin levels which reflect the amount of iron stored within the cells. 293T cells were transfected with ferroportin expressing vectors, incubated with ferric ammonium citrate as a source of iron, pretreated with cycloheximide to stop de-novo protein synthesis and then treated with 30 nM hepcidin. Ferritin levels increased significantly in the cells expressing WT ferroportin and treated with hepcidin (Fig.1A). In contrast, ferritin levels remained the same in untreated and hepcidin treated cells expressing ferroportin Q248H or C326Y (Fig.1A). This observation suggests continuing iron export by ferroportin Q248H with low dose hepcidin. HIV-1 transcription can be induced in 293T cells by co-expression of HIV-1 LTR reporter construct and HIV-1 Tat expression vector (Fig.1B, lane 2). HIV-1 Tat binds to TAR RNA located in the beginning of HIV-1 transcript and facilitates a recruitment of a host cell transcription elongation factor, CDK9/cyclin T1, inducing efficient elongation of HIV-1 transcription. Expression of ferroportin WT, Q248H or C326Y mutant inhibited Tat –induced HIV-1 transcription in comparison to non-relevant control (Fig.1B, lanes 3, 4, 6 and 8). Treatment with physiological hepcidin concentrations reversed the inhibition of Tat-induced HIV-1 transcription by WT but not the Q248H or C326Y mutant ferroportin (Fig.1B, lanes 5, 7 and 9). In this experiment, we utilized c-myc tagged ferroportin expression vectors as in our previous study1. We also obtained very similar results with EGFP-fused ferroportin expression, which also allowed an easier detection of reduction in ferroportin expression in the presence of hepcidin. Finally, we also isolated monocytes from two subjects, one with heterozygote and one with homozygote ferroportin Q248H. Monocytes were infected ex-vivo with pseudotyped HIV-1 virus expressing luciferase. HIV-1 replication was reduced in primary monocytes with heterozygote and homozygote ferroportin Q248H as compared to a control. Ferroportin glutamine 248 is located within the intracellular loop (residues 228–307), in close proximity to lysine residues 229–269 which ubiquitination promotes ferroportin internalization3. Future studies should address the details of ubiquitination of human ferroportin Q248H compared to WT ferroportin. An added protection value could be observed lower hepcidin expression levels in HIV-1 infected individuals with the ferroportin Q248H2. Further studies are needed to uncover a mechanism of this reduced hepcidin expression. Further molecular analysis is needed to understand the mechanism of ferroportin Q248H internalization. Taken together, our study shows that the ferroportin Q248H that has a reduced sensitivity to hepcidin may offer an additional protection from HIV-1. [Display omitted] [Display omitted] Acknowledgments.This work was supported NIH Research Grants SC1GM082325, R25 HL003679, 2G12RR003048, 8G12MD007597, K25GM097501 and 1P30HL107253. Disclosures:No relevant conflicts of interest to declare.

Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Ferroportin Q248H mutation has an allele frequency of 2.2-13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. Some investigators have reported that ferroportin Q248H is degraded after exposure to hepcidin in exactly the same manner as wild-type ferroportin, but supraphysiological concentrations of hepcidin were used. The aim of our study was to determine whether ferroportin Q248H may have reduced sensitivity to physiological concentrations of hepcidin. The sensitivity of ferroportin Q248H to hepcidin was determined in 293T cells transiently expressing ferroportin using immunoblotting and fluorescence analysis. Ferritin concentrations were measured in these cells and also in human primary monocytes derived from humans with different ferroportin genotypes. The effect of Q248H on serum iron measures was examined in patients with sickle cell anemia. Immunoblotting and fluorescence analysis showed decreased sensitivity of ferroportin Q248H to physiological concentrations of hepcidin. Lower ferritin concentrations were observed after incubation with iron and hepcidin in 293T cells expressing ferroportin Q248H and in primary monocytes from ferroportin Q248H subjects. In sickle cell anemia, ferroportin Q248H heterozygotes had lower serum ferritin concentrations than wild-type subjects, consistent with enhanced iron release by macrophage ferroportin Q248H. A clinical benefit of ferroportin Q248H was suggested by lower echocardiographic estimates of pulmonary artery pressure in patients carrying mutant alleles. In conclusion, our results suggest that ferroportin Q248H protein is resistant to physiological concentrations of hepcidin and that this mutation has discernible effects on iron metabolism-related clinical complications of sickle cell anemia. They provide a mechanistic explanation for the effect of ferroportin Q248H on iron status in individuals of African descent and suggest that these changes in iron metabolism may be beneficial under certain disease-specific circumstances. (ClinicalTrials.gov Identifier:NCT00011648).

Ferroportin (SLC40A1) Q248H mutation is associated with lower circulating serum hepcidin levels in Rwandese HIV-positive women

The Q248H mutation in the gene SLC40A1 which encodes for the cellular iron exporter ferroportin is relatively common in Africa. This mutation has been associated with resistance to hepcidin and therefore we hypothesized that iron-related parameters and the prevalence of opportunistic infections in HIV might be influenced by the Q248H mutation. We conducted a cross-sectional study among 200 HIV-positive women in the Butare University Teaching Hospital in Rwanda. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the Q248H mutation. Physical examination was carried out and WHO HIV disease stage classification, complete blood count, CD4 count, indirect measures of iron status, serum hepcidin, and C-reactive protein concentrations were determined. The prevalence of ferroportin Q248H mutation was 6%. Subjects with ferroportin Q248H mutation had significantly higher values for serum ferritin (P = 0.001) and significantly lower values for serum hepcidin (P = 0.001) and transferrin (P = 0.01). Among the 12 HIV + Q248H heterozygotes, 8 suffered from at least one opportunistic infection. There was significantly higher prevalence of pulmonary TB (P = 0.01) and Pneumocystis jiroveci pneumonia (P = 0.02) in subjects with ferroportin Q248H mutation. Low hepcidin levels were found in ferroportin Q248H heterozygotes with HIV infection, notwithstanding the absence of anemia and the higher prevalence of some opportunistic infections. Hepcidin seems to be regulated in a different way in Q248H heterozygotes than is known thus far.

Ferroportin ( SLC40A1) Q248H mutation is associated with lower circulating plasma tumor necrosis factor-α and macrophage migration inhibitory factor concentrations in African children

Background Iron deficiency and the Q248H mutation in the gene, SLC40A1, that encodes for the cellular iron exporter, ferroportin, are both common in African children. The iron status of macrophages influences the pro-inflammatory response of these cells. We hypothesized that Q248H mutation may modify the inflammatory response by influencing iron levels within macrophages. Methods The Q248H mutation and circulating concentrations of ferritin, C-reactive protein and selected pro-inflammatory cytokines (interleukin-12, interferon-γ, TNF-α, and macrophage migration inhibitory factor) and anti-inflammatory cytokines (interleukin-4 and interleukin-10) were measured in 69 pre-school children recruited from well-child clinics in Harare, Zimbabwe. Results In multivariate analysis, both ferroportin Q248H and ferritin < 10 ug/L were associated with significantly lower circulating concentrations of tumor necrosis factor-α. Ferroportin Q248H but not low iron stores was associated with lower circulating macrophage migration inhibitory factor as well. Anti-inflammatory cytokine levels were not significantly associated with either ferroportin Q248H or iron status. Conclusions Ferroportin Q248H and low iron stores are both associated with lower circulating tumor necrosis factor-α, while only ferroportin Q248H is associated with lower circulating macrophage migration inhibitory factor. Whether the reduced production of tumor necrosis factor-α observed in ferroportin Q248H heterozygotes may be of significance in anemia of chronic disease is yet to be determined.

Ferropotin Inhibits HIV‐1 Transcription

HIV‐1 transcription is induced by viral transcriptional activator, Tat protein that recruits transcriptional co‐activators, including CDK9/cyclin T1 to the HIV‐1 promoter. We recently showed that Tat is a substrate for CDK2 and that mutations in the Ser16 and Ser 46 residues of Tat that were phosphorylated by CDK2, prevented HIV‐1 transcription and viral replication. We also recently showed that iron chelators ICL670 and 311 inhibited on Tat‐induced HIV‐1 transcription by inhibiting cellular activities of CDK2 and CDK9. Thus our previous studies suggest that a decrease in cellular iron might have a protective effect against HIV‐1. Here, we analyzed the effect of iron exporter, ferroportin on HIV‐1 transcription and viral replication. Expression of ferroportin in 293T cells dramatically inhibited HIV‐1 transcription. Induction of ferroportin expression in THP‐1 cells inhibited one round of replication of pseudotyped HIV‐1. Previously, ferroportin Q248H mutation was shown to have a potential protective effect against iron deficiency in children exposed to repeated inflammatory conditions. We analyzed the effect of hepcidin on the expression of ferroportin and found that the Q248H mutant was less sensitive to lower concentrations of hepcidin. This finding suggested that Q248H mutation might be protective against HIV‐1 infection under conditions of mild inflammation. We are currently investigating the direct effect of hepcidin and ferroportin Q248H mutation on HIV‐1 transcription.

Ferroportin (Q248H) mutations in African families with dietary iron overload

Dietary iron overload found in sub-Saharan Africa might be caused by an interaction between dietary iron and an iron-loading gene. Caucasian people with ferroportin gene mutations have iron overload histologically similar to that found in African patients with iron overload. Ferroportin is also implicated in the hypoferremic response to inflammation. The prevalence of the ferroportin Q248H mutation, unique to African people, and its association with dietary iron overload, mean cell volume (MCV) and C-reactive protein (CRP) were examined in 19 southern African families. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the Q248H mutation. Statistical analysis was carried out to correlate the presence of the mutation with markers of iron overload and inflammation. We identified three (1.4%) Q248H homozygotes and 53 (24.1%) heterozygotes in the families examined in the present study. There was no increased prevalence of the mutation in index subjects or their families. Logistic regression showed significantly higher serum ferritin concentrations with the mutation. The mean cell volume (MCV) was significantly lower, and the serum CRP significantly higher in subjects who carried the mutation. The present study of 19 families with African iron overload failed to show evidence that the ferroportin (Q248H) mutation is responsible for the condition. Logistic regression, correcting for factors influencing iron status, did show increased ferritin levels in individuals with the mutation. The strong association with low MCV suggests the possibility that the ferroportin (Q248H) mutation might interfere with iron supply, whereas the elevated serum CRP might indicate that the ferroportin mutation influences the inflammatory response in African populations.