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Serum hepcidin concentration is regulated by iron status, inflammation, erythropoiesis and numerous other factors, but underlying processes are incompletely understood. We studied the association of common and rare single nucleotide variants (SNVs) with serum hepcidin in one Italian study and two large Dutch population-based studies. We genotyped common SNVs with genome-wide association study (GWAS) arrays and subsequently performed imputation using the 1000 Genomes reference panel. Cohort-specific GWAS were performed for log-transformed serum hepcidin, adjusted for age and gender, and results were combined in a fixed-effects meta-analysis (total N 6,096). Six top SNVs (p<5x10-6) were genotyped in 3,821 additional samples, but associations were not replicated. Furthermore, we meta-analyzed cohort-specific exome array association results of rare SNVs with serum hepcidin that were available for two of the three cohorts (total N 3,226), but no exome-wide significant signal (p<1.4x10-6) was identified. Gene-based meta-analyses revealed 19 genes that showed significant association with hepcidin. Our results suggest the absence of common SNVs and rare exonic SNVs explaining a large proportion of phenotypic variation in serum hepcidin. We recommend extension of our study once additional substantial cohorts with hepcidin measurements, GWAS and/or exome array data become available in order to increase power to identify variants that explain a smaller proportion of hepcidin variation. In addition, we encourage follow-up of the potentially interesting genes that resulted from the gene-based analysis of low-frequency and rare variants., Competing Interests: DWS is an employee of Radboudumc, that offers hepcidin measurements to the scientific, medical and pharmaceutical communities at a fee-for-service bases via the www.hepcidinanalysis.com initiative. The remaining authors declare that they have no competing interests. The competing interests do not alter our adherence to PLOS ONE policies on sharing data and materials.

Low iron stores may protect from malaria infection, therefore improving iron stores in early pregnancy in line with current recommendations could increase malaria susceptibility. To test this hypothesis we compared iron biomarkers and red cell indices in nulliparae and primigravidae who participated in a randomized controlled trial of long-term weekly iron supplementation. Cross-sectional and longitudinal data analysis from a randomized controlled trial of long-term weekly iron supplementation in rural Burkina Faso. Malaria parasitaemia was monitored and biomarkers and red cell indices measured at study end-points: plasma ferritin, transferrin receptor (sTfR), zinc protoporphyrin, hepcidin, sTfR/log 10 ferritin ratio, body iron, haemoglobin, red cell distribution width; mean corpuscular haemoglobin concentration/volume, and C-reactive protein. Correlation coefficients between biomarkers and red cell indices were determined. A regression correction approach based on ferritin was used to estimate iron body stores, allowing for inflammation. Body iron differences were compared between nulliparae and primigravidae, and the association determined of iron biomarkers and body iron stores with malaria. Iron and haematological indices of 972 nulliparae (mean age 16.5 years) and 314 primigravidae (median gestation 18 weeks) were available. Malaria prevalence was 54.0% in primigravidae and 41.8% in nulliparae (relative risk 1.28, 95% CI 1.13–1.45, P < 0.001), anaemia prevalence 69.7% and 43.4% (P < 0.001), and iron deficient erythropoiesis (low body iron) 8.0% and 11.7% (P = 0.088) respectively. Unlike other biomarkers the sTfR/log 10 ferritin ratio showed no correlation with inflammation as measured by CRP. Most biomarkers indicated reduced iron deficiency in early pregnancy, with the exception of haemoglobin. Body iron increased by 0.6–1.2 mg/kg in early gestation, did not differ by malaria status in nulliparae, but was higher in primigravidae with malaria (6.5 mg/kg versus 5.0 mg/kg; relative risk 1.53, 95% CI 0.67–2.38, P < 0.001). In primigravidae, early pregnancy haemoglobin was not a good indicator of requirement for iron supplementation, which could be detrimental given the association of better iron status with increased malaria infection. clinicaltrials.gov:NCT01210040. Until placed in a public repository, data relating to the current study can be requested from the corresponding author and will be made available following an end user data agreement and sponsor approval. • Iron deficiency prevalence low using several biomarkers. • sTfR/log 10 ferritin ratio not correlated with inflammation. • Body iron increased in early pregnancy. • Early first pregnancy haemoglobin not a good indicator of iron need. • Malaria prevalence higher with better iron status. [ABSTRACT FROM AUTHOR]

The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Despite its promise as a biomarker, the availability of high-sensitive hepcidin assays is still limited. We developed and validated a RadioImmunoAssay (RIA) to measure hepcidin quantitatively in human serum. This assay exhibited a very low detection limit (0·02 μg/l), low imprecision (coefficient of variation-range 4·4–6·2%) and good linearity and recovery (range: 81–105%). Hepcidin levels of samples of controls and patients with iron deficiency and inflammation showed an excellent correlation with our previously described quantitative time-of-flight mass spectrometry assay (range 2·5–266·8 μg/l, r = 0·92, P < 0·0001). The RIA detected: (i) differences in mean hepcidin levels between men ( n = 29) and women ( n = 35), (ii) differences between individuals of different HFE-genotypes ( n = 60) and (iii) daily increases in hepcidin levels ( n = 64). The assay (i) is easy to perform and many samples can be processed within one assay-run, (ii) shows accurate, reproducible and high-sensitive measurements and (iii) is anticipated to be particularly useful to study the effects of pathological and physiological stimuli on hepcidin levels in the lower range. [ABSTRACT FROM AUTHOR]