Effectiveness of ferritin-guided donation intervals in whole-blood donors in the Netherlands (FIND'EM): a stepped-wedge cluster-randomised trial.

BACKGROUND: Whole-blood donors are at increased risk for iron deficiency and anaemia. The current standard of haemoglobin monitoring is insufficient to ensure the maintenance of proper iron reserves and donor health. We aimed to determine the effects of ferritin-guided donation intervals for blood donor health and blood supply in the Netherlands. METHODS: In this stepped-wedge cluster-randomised trial (FIND'EM), the 138 fixed and mobile donation centres in the Netherlands are organised into 29 geographical clusters and the clusters were randomly assigned to four treatment groups, with two groups being further split into two per a protocol amendment. Eligible donors were whole-blood donors who consented for use of their leftover material in the study. Each group was sequentially crossed over from the existing policy (haemoglobin-based screening; control) to a ferritin-guided donation interval policy over a 3-year period. In the intervention groups, in addition to the existing haemoglobin screening, ferritin was measured in all new donors and at every fifth donation in repeat donors. Subsequent donation intervals were extended to 6 months if ferritin concentrations were 15-30 ng/mL and to 12 months if they were less than 15 ng/mL. Outcomes were measured cross-sectionally across all donation centres at four timepoints. Primary outcomes were ferritin and haemoglobin concentrations, iron deficiency, and haemoglobin-based deferrals. We assessed all outcomes by sex and menopausal status and significance for primary outcomes was indicated by a p value of less than 0·0125. This trial is registered in the Dutch trial registry, NTR6738, and is complete. FINDINGS: Between Sept 11, 2017, and Nov 27, 2020, 412 888 whole-blood donors visited a donation centre, and we did measurements on samples from 37 621 donations from 36 099 donors. Over 38 months, ferritin-guided donation intervals increased mean ferritin concentrations (by 0·18 log10 ng/mL [95% CI 0·15-0·22; p<0·0001] in male donors, 0·10 log10 ng/mL [0·06-0·15; p<0·0001] in premenopausal female donors, and 0·17 log10 ng/mL [0·12-0·21; p<0·0001] in postmenopausal female donors) and mean haemoglobin concentrations (by 0·30 g/dL [95% CI 0·22-0·38; p<0·0001] in male donors, 0·12 g/dL [0·03-0·20; p<0·0074] in premenopausal female donors, and 0·16 g/dL [0·05-0·27; p<0·0044] in postmenopausal female donors). Iron deficiency decreased by 36-38 months (odds ratio [OR] 0·24 [95% CI 0·18-0·31; p<0·0001] for male donors, 0·49 [0·37-0·64; p<0·0001] for premenopausal female donors, and 0·24 [0·15-0·37; p<0·0001] for postmenopausal female donors). At 36-38 months, haemoglobin-based deferral decreased significantly in male donors (OR at 36-38 months 0·21 [95% CI 0·10-0·40, p<0·0001]) but not significantly in premenopausal or postmenopausal female donors (0·81 [0·54-1·20; p=0·29] and 0·50 [95% CI 0·25-0·98; p=0·051], respectively). INTERPRETATION: Ferritin-guided donation intervals significantly improved haemoglobin and ferritin concentrations and significantly decreased iron deficiency over the study period. Haemoglobin-based deferrals decreased significantly for male donors, but not female donors. Although this intervention is overall beneficial for maintenance of iron and haemoglobin concentrations in donors, increased efforts are needed to recruit and retain donors. FUNDING: The Sanquin Research Programming Committee.

Iron homeostasis during anemia of inflammation: a prospective study of patients with tuberculosis.

Anemia of inflammation is a hallmark of tuberculosis. Factors controlling iron metabolism during anemia of inflammation and its resolution are uncertain. Whether iron supplements should be given during antituberculosis treatment to support hemoglobin (Hb) recovery is unclear. Before and during treatment of tuberculosis, we assessed iron kinetics, as well as changes in inflammation and iron metabolism indices. In a 26-week prospective study, Tanzanian adults with tuberculosis (N = 18) were studied before treatment and then every 2 weeks during treatment; oral and intravenous iron tracers were administered before treatment and after intensive phase (8/12 weeks) and complete treatment (24 weeks). No iron supplements were given. Before treatment, hepcidin and erythroferrone (ERFE) were greatly elevated, erythrocyte iron utilization was high (∼80%), and iron absorption was negligible (<1%). During treatment, hepcidin and interleukin-6 levels decreased ∼70% after only 2 weeks (P< .001); in contrast, ERFE did not significantly decrease until 8 weeks (P< .05). ERFE and interleukin-6 were the main opposing determinants of hepcidin (P< .05), and greater ERFE was associated with reticulocytosis and Hb repletion (P< .01). Dilution of baseline tracer concentration was 2.6-fold higher during intensive phase treatment (P< .01), indicating enhanced erythropoiesis. After treatment completion, iron absorption increased ∼20-fold (P< .001), and Hb increased ∼25% (P< .001). In tuberculosis-associated anemia of inflammation, our findings suggest that elevated ERFE is unable to suppress hepcidin, and iron absorption is negligible. During treatment, as inflammation resolves, ERFE may remain elevated, contributing to hepcidin suppression and Hb repletion. Iron is well absorbed only after tuberculosis treatment, and supplementation should be reserved for patients remaining anemic after treatment. This trial was registered at www.clinicaltrials.gov as #NCT02176772.

Iron deficiency.

Iron deficiency is one of the leading contributors to the global burden of disease, and particularly affects children, premenopausal women, and people in low-income and middle-income countries. Anaemia is one of many consequences of iron deficiency, and clinical and functional impairments can occur in the absence of anaemia. Iron deprivation from erythroblasts and other tissues occurs when total body stores of iron are low or when inflammation causes withholding of iron from the plasma, particularly through the action of hepcidin, the main regulator of systemic iron homoeostasis. Oral iron therapy is the first line of treatment in most cases. Hepcidin upregulation by oral iron supplementation limits the absorption efficiency of high-dose oral iron supplementation, and of oral iron during inflammation. Modern parenteral iron formulations have substantially altered iron treatment and enable rapid, safe total-dose iron replacement. An underlying cause should be sought in all patients presenting with iron deficiency: screening for coeliac disease should be considered routinely, and endoscopic investigation to exclude bleeding gastrointestinal lesions is warranted in men and postmenopausal women presenting with iron deficiency anaemia. Iron supplementation programmes in low-income countries comprise part of the solution to meeting WHO Global Nutrition Targets.

Menstrual cycle affects iron homeostasis and hepcidin following interval running exercise in endurance-trained women.

PURPOSE: Menstrual cycle phase affects resting hepcidin levels, but such effects on the hepcidin response to exercise are still unclear. Thus, we investigated the hepcidin response to running during three different menstrual cycle phases. METHODS: Twenty-one endurance-trained eumenorrheic women performed three identical interval running protocols during the early-follicular phase (EFP), late-follicular phase (LFP), and mid-luteal phase (MLP). The protocol consisted of 8 × 3 min bouts at 85% of the maximal aerobic speed, with 90-s recovery. Blood samples were collected pre-exercise and at 0 h, 3 h and 24 h post-exercise. RESULTS: Data presented as mean ± SD. Ferritin were lower in the EFP than the LFP (34.82 ± 16.44 vs 40.90 ± 23.91 ng/ml, p = 0.003), while iron and transferrin saturation were lower during the EFP (58.04 ± 19.70 µg/dl, 14.71 ± 5.47%) compared to the LFP (88.67 ± 36.38 µg/dl, 22.22 ± 9.54%; p < 0.001) and the MLP (80.20 ± 42.05 µg/dl, 19.87 ± 10.37%; p = 0.024 and p = 0.045, respectively). Hepcidin was not affected by menstrual cycle (p = 0.052) or menstrual cycle*time interaction (p = 0.075). However, when comparing hepcidin at 3 h post-exercise, a moderate and meaningful effect size showed that hepcidin was higher in the LFP compared to the EFP (3.01 ± 4.16 vs 1.26 ± 1.25 nMol/l; d = 0.57, CI = 0.07-1.08). No effect of time on hepcidin during the EFP was found either (p = 0.426). CONCLUSION: The decrease in iron, ferritin and TSAT levels during the EFP may mislead the determination of iron status in eumenorrheic athletes. However, although the hepcidin response to exercise appears to be reduced in the EFP, it shows no clear differences between the phases of the menstrual cycle (clinicaltrials.gov: NCT04458662).

Investigating the Molecular Mechanisms of Renal Hepcidin Induction and Protection upon Hemoglobin-Induced Acute Kidney Injury.

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCDcl1) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis (p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCDcl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM (p < 0.001), NQO1 (p < 0.001), and TXNRD1 (p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers (IL-6, TNFα) but not Hamp1. However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.

Transferrin Saturation/Hepcidin Ratio Discriminates TMPRSS6-Related Iron Refractory Iron Deficiency Anemia from Patients with Multi-Causal Iron Deficiency Anemia.

Pathogenic TMPRSS6 variants impairing matriptase-2 function result in inappropriately high hepcidin levels relative to body iron status, leading to iron refractory iron deficiency anemia (IRIDA). As diagnosing IRIDA can be challenging due to its genotypical and phenotypical heterogeneity, we assessed the transferrin saturation (TSAT)/hepcidin ratio to distinguish IRIDA from multi-causal iron deficiency anemia (IDA). We included 20 IRIDA patients from a registry for rare inherited iron disorders and then enrolled 39 controls with IDA due to other causes. Plasma hepcidin-25 levels were measured by standardized isotope dilution mass spectrometry. IDA controls had not received iron therapy in the last 3 months and C-reactive protein levels were <10.0 mg/L. IRIDA patients had significantly lower TSAT/hepcidin ratios compared to IDA controls, median 0.6%/nM (interquartile range, IQR, 0.4-1.1%/nM) and 16.7%/nM (IQR, 12.0-24.0%/nM), respectively. The area under the curve for the TSAT/hepcidin ratio was 1.000 with 100% sensitivity and specificity (95% confidence intervals 84-100% and 91-100%, respectively) at an optimal cut-off point of 5.6%/nM. The TSAT/hepcidin ratio shows excellent performance in discriminating IRIDA from TMPRSS6-unrelated IDA early in the diagnostic work-up of IDA provided that recent iron therapy and moderate-to-severe inflammation are absent. These observations warrant further exploration in a broader IDA population.

Novel concepts in red blood cell clearance.

PURPOSE OF REVIEW: Red blood cell (RBC) clearance has been studied for decades in many different pathologies, which has revealed different routes of RBC degradation, depending on the situation. This review summarizes the latest mechanistic insights on RBC clearance in different contexts; during homeostatic removal, immune-mediated destruction, and systemic inflammation. RECENT FINDINGS: Besides the recognition of a variety of potential 'eat me' signals on RBCs, recent evidence suggests that normal RBC degradation is driven by the increase of the adhesive properties of RBCs, mediating the retention in the spleen and leading to RBC hemolysis. Furthermore, immune-mediated degradation of RBCs seems to be fine-tuned by the balance between the density of the antigens expressed on RBCs and the presence of 'don't eat me' signals. Moreover, besides RBC clearance by macrophages, neutrophils seem to play a much more prominent role in immune-mediated RBC removal than anticipated. Lastly, RBC clearance during systemic inflammation appears to be driven by a combination of extreme macrophage activity in response to proinflammatory cytokines as well as direct damage of RBC by the inflammation or inflammatory agent. SUMMARY: Recent studies on RBC clearance have expanded our knowledge on their destruction in different contexts.

Controversies in optimal anemia management: conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Conference.

In chronic kidney disease, anemia and disordered iron homeostasis are prevalent and associated with significant adverse consequences. In 2012, Kidney Disease: Improving Global Outcomes (KDIGO) issued an anemia guideline for managing the diagnosis, evaluation, and treatment of anemia in chronic kidney disease. Since then, new data have accrued from basic research, epidemiological studies, and randomized trials that warrant a re-examination of previous recommendations. Therefore, in 2019, KDIGO decided to convene 2 Controversies Conferences to review the latest evidence, explore new and ongoing controversies, assess change implications for the current KDIGO anemia guideline, and propose a research agenda. The first conference, described here, focused mainly on iron-related issues, including the contribution of disordered iron homeostasis to the anemia of chronic kidney disease, diagnostic challenges, available and emerging iron therapies, treatment targets, and patient outcomes. The second conference will discuss issues more specifically related to erythropoiesis-stimulating agents, including epoetins, and hypoxia-inducible factor-prolyl hydroxylase inhibitors. Here we provide a concise overview of the consensus points and controversies resulting from the first conference and prioritize key questions that need to be answered by future research.

Absorption of nonheme iron during gastric acid suppression in patients with hereditary hemochromatosis and healthy controls.

Phlebotomies are performed in hereditary hemochromatosis (HH) to maintain normal iron concentrations. Proton-pump inhibitors (PPIs) can reduce the number of phlebotomies in patients with HH. However, in patients without HH, the iron concentrations do not appear to be compromised when using PPIs. Therefore, we aim to explain the differences in iron absorption between patients with and without HH. In 10 p.cysteine282tyrosine (p.C282Y) homozygous HH patients with normalized iron stores and 10 healthy control subjects (HCs), the iron parameters and hepcidin concentrations were determined before ingestion of a pharmacological dose of 50 mg iron [ferric iron (Fe3+)] polymaltose and hourly for 4 h afterward. This was repeated after 7 days of treatment with pantoprazole 40 mg once daily. Serum iron concentrations and transferrin saturation percentages dropped significantly during PPI use in the patients with HH, whereas no changes were observed in the HCs. Hepcidin concentrations were lower in the patients with HH compared with the HCs both before and during PPI use. In both groups, hepcidin levels did not significantly decrease during the treatment. Seven-day PPI use significantly reduces iron absorption in patients with HH but not in HCs. Changes in hepcidin concentrations could not explain these different PPI effects on iron absorption probably due to a small sample size.NEW & NOTEWORTHY This study confirms that lowering gastric acidity by proton pump inhibitors results in a reduction in iron absorption in patients with hemochromatosis and not in healthy control subjects. The presupposition that a decrease in hepcidin concentration in healthy control subjects in response to lowering gastric acidity can explain the difference in iron absorption between these groups could not be confirmed probably because of a small sample size.

Genetic Diagnosis in Hereditary Hemochromatosis: Discovering and Understanding the Biological Relevance of Variants.

BACKGROUND: Hereditary hemochromatosis (HH) is a genetic disease, leading to iron accumulation and possible organ damage. Patients are usually homozygous for p. Cys282Tyr in the homeostatic iron regulator gene but may have mutations in other genes involved in the regulation of iron. Next-generation sequencing is increasingly being utilized for the diagnosis of patients, leading to the discovery of novel genetic variants. The clinical significance of these variants is often unknown. CONTENT: Determining the pathogenicity of such variants of unknown significance is important for diagnostics and genetic counseling. Predictions can be made using in silico computational tools and population data, but additional evidence is required for a conclusive pathogenicity classification. Genetic disease models, such as in vitro models using cellular overexpression, induced pluripotent stem cells or organoids, and in vivo models using mice or zebrafish all have their own challenges and opportunities when used to model HH and other iron disorders. Recent developments in gene-editing technologies are transforming the field of genetic disease modeling. SUMMARY: In summary, this review addresses methods and developments regarding the discovery and classification of genetic variants, from in silico tools to in vitro and in vivo models, and presents them in the context of HH. It also explores recent gene-editing developments and how they can be applied to the discussed models of genetic disease.

Hepcidin response to interval running exercise is not affected by oral contraceptive phase in endurance-trained women.

The use of oral contraceptives (OCs) by female athletes may lead to improved iron status, possibly through the regulation of hepcidin by sex hormones. The present work investigates the response of hepcidin and interleukin-6 (IL-6) to an interval exercise in both phases of the OC cycle. Sixteen endurance-trained OC users (age 25.3 ± 4.7 years; height 162.4 ± 5.7 cm; body mass 56.0 ± 5.7 kg; body fat percentage 24.8 ± 6.0%; peak oxygen consumption [VO2peak ]: 47.4 ± 5.5 mL min-1 kg-1 ) followed an identical interval running protocol during the withdrawal and active pill phases of the OC cycle. This protocol consisted of 8 × 3 minutes bouts at 85% VO2peak speed with 90 seconds recovery intervals. Blood samples were collected pre-exercise, and at 0 hour, 3 hours, and 24 hours post-exercise. Pre-exercise 17ß-estradiol was lower (P = .001) during the active pill than the withdrawal phase (7.91 ± 1.81 vs 29.36 ± 6.45 pg/mL [mean ± SEM]). No differences were seen between the OC phases with respect to hepcidin or IL-6 concentrations, whether taking all time points together or separately. However, within the withdrawal phase, hepcidin concentrations were higher at 3 hours post-exercise (3.33 ± 0.95 nmol/L) than at pre-exercise (1.04 ± 0.20 nmol/L; P = .005) and 0 hour post-exercise (1.41 ± 0.38 nmol/L; P = .045). Within both OC phases, IL-6 was higher at 0 hour post-exercise than at any other time point (P < .05). Similar trends in hepcidin and IL-6 concentrations were seen at the different time points during both OC phases. OC use led to low 17ß-estradiol concentrations during the active pill phase but did not affect hepcidin. This does not, however, rule out estradiol affecting hepcidin levels.

Standardized serum hepcidin values in Dutch children: Set point relative to body iron changes during childhood.

BACKGROUND: Use of serum hepcidin measurements in pediatrics would benefit from standardized age- and sex-specific reference ranges in children, in order to enable the establishment of clinical decision limits that are universally applicable. PROCEDURE: We measured serum hepcidin-25 levels in 266 healthy Dutch children aged 0.3-17 years, using an isotope dilution mass spectrometry assay, standardized with our commutable secondary reference material (RM), assigned by a candidate primary RM. RESULTS: We constructed age- and sex-specific values for serum hepcidin and its ratio with ferritin and transferrin saturation (TSAT). Serum hepcidin levels and hepcidin/ferritin and TSAT/hepcidin ratios were similar for both sexes. Serum hepcidin and hepcidin/ferritin ratio substantially declined after the age of 12 years and TSAT/hepcidin ratio gradually increased with increasing age. Serum hepcidin values for Dutch children <12 years (n = 170) and >12 years (n = 96) were 1.9 nmol/L (median); 0.1-13.1 nmol/L (p2.5-p97.5) and 0.9 nmol/L; 0.0-9.1 nmol/L, respectively. Serum ferritin was the most significant correlate of serum hepcidin in our study population, explaining 15.1% and 7.9% of variance in males and females, respectively. Multivariable linear regression analysis including age, blood sampling time, iron parameters, ALT, CRP, and body mass index as independent variables showed a statistically significant negative association between age as a dichotomous variable (≤12 vs >12 years) and log-transformed serum hepcidin levels in both sexes. CONCLUSIONS: We demonstrate that serum hepcidin relative to indicators of body iron is age dependent in children, suggesting that the set point of serum hepcidin relative to stored and circulating iron changes during childhood.

Optimizing hepcidin measurement with a proficiency test framework and standardization improvement.

Objectives: Hepcidin measurement advances insights in pathophysiology, diagnosis, and treatment of iron disorders, but requires analytically sound and standardized measurement procedures (MPs). Recent development of a two-level secondary reference material (sRM) for hepcidin assays allows worldwide standardization. However, no proficiency testing (PT) schemes to ensure external quality assurance (EQA) exist and the absence of a high calibrator in the sRM set precludes optimal standardization. Methods: We developed a pilot PT together with the Dutch EQA organization Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek (SKML) that included 16 international hepcidin MPs. The design included 12 human serum samples that allowed us to evaluate accuracy, linearity, precision and standardization potential. We manufactured, value-assigned, and validated a high-level calibrator in a similar manner to the existing low- and middle-level sRM. Results: The pilot PT confirmed logistical feasibility of an annual scheme. Most MPs demonstrated linearity (R2>0.99) and precision (duplicate CV>12.2%), although the need for EQA was shown by large variability in accuracy. The high-level calibrator proved effective, reducing the inter-assay CV from 42.0% (unstandardized) to 14.0%, compared to 17.6% with the two-leveled set. The calibrator passed international homogeneity criteria and was assigned a value of 9.07±0.24 nmol/L. Conclusions: We established a framework for future PT to enable laboratory accreditation, which is essential to ensure quality of hepcidin measurement and its use in patient care. Additionally, we showed optimized standardization is possible by extending the current sRM with a third high calibrator, although international implementation of the sRM is a prerequisite for its success.

Iron handling by the human kidney: glomerular filtration and tubular reabsorption both contribute to urinary iron excretion.

In physiological conditions, circulating iron can be filtered by the glomerulus and is almost completely reabsorbed by the tubular epithelium to prevent urinary iron wasting. Increased urinary iron concentrations have been associated with renal injury. However, it is not clear whether increased urinary iron concentrations in patients are the result of increased glomerular iron filtration and/or insufficient tubular iron reabsorption and if these processes contribute to renal injury. We measured plasma and urine iron parameters and urinary tubular injury markers in healthy human subjects ( n = 20), patients with systemic iron overload ( n = 20), and patients with renal tubular dysfunction ( n = 18). Urinary iron excretion parameters were increased in both patients with systemic iron overload and tubular dysfunction, whereas plasma iron parameters were only increased in patients with systemic iron overload. In patients with systemic iron overload, increased urinary iron levels were associated with elevated circulating iron, as indicated by transferrin saturation (TSAT), and increased body iron, as suggested by plasma ferritin concentrations. In patients with tubular dysfunction, enhanced urinary iron and transferrin excretion were associated with distal tubular injury as indicated by increased urinary glutathione S-transferase pi 1-1 (GSTP1-1) excretion. In systemic iron overload, elevated urinary iron and transferrin levels were associated with increased injury to proximal tubules, indicated by increased urinary kidney injury marker 1 (KIM-1) excretion. Our explorative study demonstrates that both glomerular filtration of elevated plasma iron levels and insufficient tubular iron reabsorption could increase urinary iron excretion and cause renal injury.

A rare splice donor mutation in the haptoglobin gene associates with blood lipid levels and coronary artery disease.

Common sequence variants at the haptoglobin gene (HP) have been associated with blood lipid levels. Through whole-genome sequencing of 8,453 Icelanders, we discovered a splice donor founder mutation in HP (NM_001126102.1:c.190 + 1G > C, minor allele frequency = 0.56%). This mutation occurs on the HP1 allele of the common copy number variant in HP and leads to a loss of function of HP1. It associates with lower levels of haptoglobin (P = 2.1 × 10-54), higher levels of non-high density lipoprotein cholesterol (ß = 0.26 mmol/l, P = 2.6 × 10-9) and greater risk of coronary artery disease (odds ratio = 1.30, 95% confidence interval: 1.10-1.54, P = 0.0024). Through haplotype analysis and with RNA sequencing, we provide evidence of a causal relationship between one of the two haptoglobin isoforms, namely Hp1, and lower levels of non-HDL cholesterol. Furthermore, we show that the HP1 allele associates with various other quantitative biological traits.

Variant ASGR1 Associated with a Reduced Risk of Coronary Artery Disease.

BACKGROUND: Several sequence variants are known to have effects on serum levels of non-high-density lipoprotein (HDL) cholesterol that alter the risk of coronary artery disease. METHODS: We sequenced the genomes of 2636 Icelanders and found variants that we then imputed into the genomes of approximately 398,000 Icelanders. We tested for association between these imputed variants and non-HDL cholesterol levels in 119,146 samples. We then performed replication testing in two populations of European descent. We assessed the effects of an implicated loss-of-function variant on the risk of coronary artery disease in 42,524 case patients and 249,414 controls from five European ancestry populations. An augmented set of genomes was screened for additional loss-of-function variants in a target gene. We evaluated the effect of an implicated variant on protein stability. RESULTS: We found a rare noncoding 12-base-pair (bp) deletion (del12) in intron 4 of ASGR1, which encodes a subunit of the asialoglycoprotein receptor, a lectin that plays a role in the homeostasis of circulating glycoproteins. The del12 mutation activates a cryptic splice site, leading to a frameshift mutation and a premature stop codon that renders a truncated protein prone to degradation. Heterozygous carriers of the mutation (1 in 120 persons in our study population) had a lower level of non-HDL cholesterol than noncarriers, a difference of 15.3 mg per deciliter (0.40 mmol per liter) (P=1.0×10(-16)), and a lower risk of coronary artery disease (by 34%; 95% confidence interval, 21 to 45; P=4.0×10(-6)). In a larger set of sequenced samples from Icelanders, we found another loss-of-function ASGR1 variant (p.W158X, carried by 1 in 1850 persons) that was also associated with lower levels of non-HDL cholesterol (P=1.8×10(-3)). CONCLUSIONS: ASGR1 haploinsufficiency was associated with reduced levels of non-HDL cholesterol and a reduced risk of coronary artery disease. (Funded by the National Institutes of Health and others.).

Association of anemia with health-related quality of life and survival: a large population-based cohort study.

Anemia is highly prevalent, especially in older individuals. In selected populations, anemia has been reported to be associated with impaired survival and health-related quality of life. However, data on this impact in the general population are rare. Furthermore, discussions on the optimal definition of anemia have not been conclusive. We investigated these issues using survival data, scores from a health-related quality of life questionnaire (RAND-36), and hemoglobin concentration from 138670 subjects, aged 18-93 years, participating in the Lifelines cohort. Anemia was defined according to World Health Organization criteria and was further subclassified in participants over 60 years old. Anemia was present in 5510 (4.0%) of all 138670 subjects and 516 (2.8%) in the 18667 individuals older than 60 years. Anemia had no impact on overall survival and limited impact on health-related quality of life in individuals less than 60 years old. In contrast, in individuals over 60 years old anemia significantly impaired overall survival and health-related quality of life. The lower health-related quality of life was mainly observed in subscales representing physical functioning. Although consensus on the subclassification of anemia is lacking, our data suggest that particularly anemia of chronic inflammation was associated with worse overall survival and decreased health-related quality of life. Multivariate models confirmed that anemia was an independent risk factor for decreased health-related quality of life in older individuals. Finally, women with a hemoglobin concentration between 12.0-13.0 g/dL (considered anemia in men, but not in women) experienced a significantly lower health-related quality of life. This large, prospective, population-based study indicates that anemia is associated with worse overall survival and health-related quality of life in older individuals, but not in younger individuals. The findings of this study challenge the definition of anemia in women over 60 years old, and suggest that the optimal definition of anemia, in the perspective of health-related quality of life, in women over 60 years old should be altered to a hemoglobin concentration below 13.0 g/dL (8.0 mmol/L), which is comparable to that in men.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Effects of Weekly Iron and Folic Acid Supplements on Malaria Risk in Nulliparous Women in Burkina Faso: A Periconceptional, Double-Blind, Randomized Controlled Noninferiority Trial.

Background: The safety of iron supplementation for young women is uncertain in malaria-endemic settings. Methods: This was a double-blind, randomized controlled noninferiority trial in rural Burkina Faso. Results: A total of 1959 nulliparae were assigned to weekly supplementation (60 mg iron and 2.8 mg folic acid) (n = 980) or 2.8 mg folic acid (n = 979) until first antenatal visit (ANC1), or 18 months if remaining nonpregnant. Three hundred fifteen women attended ANC1, and 916 remained nonpregnant. There was no difference at ANC1 in parasitemia prevalence (iron, 53.4% [95% confidence interval {CI}, 45.7%-61.0%]; control, 55.3% [95% CI, 47.3%-62.9%]; prevalence ratio, 0.97 [95% CI, .79-1.18]; P = .82), anemia (adjusted effect, 0.96 [95% CI, .83-1.10]; P = .52), iron deficiency (adjusted risk ratio [aRR], 0.84 [95% CI, .46-1.54]; P = .58), or plasma iron biomarkers. Outcomes in nonpregnant women were parasitemia (iron, 42.9% [95% CI, 38.3%-47.5%]; control, 39.2% [95% CI, 34.9%-43.7%]; prevalence ratio, 1.09 [95% CI, .93-1.28]; P = .282); anemia (aRR, 0.90 [95% CI, .78-1.05]; P = .17), and iron deficiency (aRR, 0.99 [95% CI, .77-1.28]; P = .96), with no iron biomarker differences. Conclusions: Weekly iron supplementation did not increase malaria risk, improve iron status, or reduce anemia in young, mostly adolescent menstruating women, nor in early pregnancy. World Health Organization Guidelines for universal supplementation for young nulliparous women may need reassessment. Clinical Trials Registration: NCT01210040.