Reduced iron export associated with hepcidin resistance can explain the iron overload spectrum in ferroportin disease.

BACKGROUND & AIMS: Ferroportin disease (FD) and hemochromatosis type 4 (HH4) are associated with variants in the ferroportin-encoding gene SLC40A1. Both phenotypes are characterized by iron overload despite being caused by distinct variants that either mediate reduced cellular iron export in FD or resistance against hepcidin-induced inactivation of ferroportin in HH4. The aim of this study was to assess if reduced iron export also confers hepcidin resistance and causes iron overload in FD associated with the R178Q variant. METHODS: The ferroportin disease variants R178Q andA77D and the HH4-variant C326Y were overexpressed in HEK-293T cells and subcellular localization was characterized by confocal microscopy and flow cytometry. Iron export and cytosolic ferritin were measured as markers of iron transport and radioligand binding studies were performed. The hepcidin-ferroportin axis was assessed by ferritin/hepcidin correlation in patients with different iron storage diseases. RESULTS: In the absence of hepcidin, the R178Q and A77D variants exported less iron when compared to normal and C326Y ferroportin. In the presence of hepcidin, the R178Q and C326Y, but not the A77D-variant, exported more iron than cells expressing normal ferroportin. Regression analysis of serum hepcidin and ferritin in patients with iron overload are compatible with hepcidin deficiency in HFE hemochromatosis and hepcidin resistance in R178Q FD. CONCLUSIONS: These results support a novel concept that in certain FD variants reduced iron export and hepcidin resistance could be interlinked. Evasion of mutant ferroportin from hepcidin-mediated regulation could result in uncontrolled iron absorption and iron overload despite reduced transport function.

Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea.

Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

Lipocalin-2 ensures host defense against Salmonella Typhimurium by controlling macrophage iron homeostasis and immune response.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages.

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor.

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a (59)Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.

Synonymous mutation in adenosine triphosphatase copper-transporting beta causes enhanced exon skipping in Wilson disease.

Wilson disease (WD) is caused by biallelic pathogenic variants in adenosine triphosphatase copper-transporting beta (ATP7B); however, genetic testing identifies only one or no pathogenic ATP7B variant in a number of patients with WD. Synonymous single-nucleotide sequence variants have been recognized as pathogenic in individual families. The aim of the present study was to evaluate the prevalence and disease mechanism of the synonymous variant c.2292C>T (p.Phe764=) in WD. A cohort of 280 patients with WD heterozygous for a single ATP7B variant was investigated for the presence of c.2292C>T (p.Phe764=). In this cohort of otherwise genetically unexplained WD, the allele frequency of c.2292C>T (p.Phe764=) was 2.5% (14 of 560) compared to 7.1 × 10-6 in the general population (2 of 280,964 in the Genome Aggregation Database; p < 10-5 ; Fisher exact test). In an independent United Kingdom (UK) cohort, 2 patients with WD homozygous for p.Phe764= were identified. RNA analysis of ATP7B transcripts from patients homozygous or heterozygous for c.2292C>T and control fibroblasts showed that this variant caused high expression of an ATP7B transcript variant lacking exon 8. Conclusion: The synonymous ATP7B variant c.2292C>T (p.Phe764=) causes abnormal messenger RNA processing of ATP7B transcripts and is associated with WD in compound heterozygotes and homozygotes.

Increased serum lipoprotein(a) concentrations and low molecular weight phenotypes of apolipoprotein(a) are associated with symptomatic peripheral arterial disease.

BACKGROUND: Increased concentrations of lipoprotein(a) [Lp(a)] have been considered a genetically determined risk factor for coronary artery and cerebrovascular disease. Only 2 small and conflicting studies have investigated the possibility of an association of peripheral arterial disease (PAD) with high serum Lp(a) concentrations and low molecular weight (LMW) phenotypes of apolipoprotein(a) [apo(a)]. METHODS: We measured serum concentrations of Lp(a) and apo(a) phenotypes in 213 patients with symptomatic PAD and 213 controls matched for sex, age (within 2 years), and presence of diabetes. RESULTS: Patients with PAD showed significantly higher median serum concentrations of Lp(a) (76 vs 47 mg/L; P = 0.003) and a higher frequency of LMW apo(a) phenotypes (41% vs 26%; P = 0.002) than controls. After adjustment for several potential confounders, increased Lp(a) concentrations (>195 mg/L, i.e., 75th percentile of the entire study sample) and LMW apo(a) phenotypes were significant predictors of PAD, with odds ratios of 3.73 (95% CI 2.08-6.67; P <0.001) and 2.21 (95% CI 1.33-3.67; P = 0.002), respectively. CONCLUSIONS: In this study sample, both increased serum concentrations of Lp(a) and the presence of LMW apo(a) phenotypes were associated with the presence of symptomatic PAD independent of traditional and nontraditional cardiovascular risk factors. Because PAD is considered an indicator of systemic atherosclerotic disease, our results suggest a possible role of Lp(a) as a genetically determined marker for systemic atherosclerosis.