A genome-wide meta-analysis yields 46 new loci associating with biomarkers of iron homeostasis.

Iron is essential for many biological functions and iron deficiency and overload have major health implications. We performed a meta-analysis of three genome-wide association studies from Iceland, the UK and Denmark of blood levels of ferritin (N = 246,139), total iron binding capacity (N = 135,430), iron (N = 163,511) and transferrin saturation (N = 131,471). We found 62 independent sequence variants associating with iron homeostasis parameters at 56 loci, including 46 novel loci. Variants at DUOX2, F5, SLC11A2 and TMPRSS6 associate with iron deficiency anemia, while variants at TF, HFE, TFR2 and TMPRSS6 associate with iron overload. A HBS1L-MYB intergenic region variant associates both with increased risk of iron overload and reduced risk of iron deficiency anemia. The DUOX2 missense variant is present in 14% of the population, associates with all iron homeostasis biomarkers, and increases the risk of iron deficiency anemia by 29%. The associations implicate proteins contributing to the main physiological processes involved in iron homeostasis: iron sensing and storage, inflammation, absorption of iron from the gut, iron recycling, erythropoiesis and bleeding/menstruation.

Frequent blood donation and offspring scholastic attainment: an assessment of long-term consequences of prenatal iron deficiency.

BACKGROUND: Due to physiological demands, children and premenopausal women are at risk of developing iron deficiency. In premenopausal women, the risk may be further increased by repeated whole blood donations. Short-term consequences of iron deficiency in infancy include impaired cognitive development and lower IQ scores. This prompts concern that maternal iron deficiency before or during pregnancy may have long-term consequences for the offspring, for example, by affecting scholastic attainment. The aim of this study was to evaluate if prepregnancy donation intensity is associated with offspring scholastic attainment measured as grade averages in standardized national written examinations in Denmark. STUDY DESIGN AND METHODS: By using the Danish personal identification number as key, we obtained information on donation intensity before pregnancy, school grade, year of graduation, age of the students, students' sex, and parental length of education and income from various nationwide registers. Linear regression analyses were performed, with grade average as outcome and maternal donation status as explanatory variable (nondonor, n = 177,078; low-frequency donor, n = 4995 [one to five donations in the 3 years before pregnancy]; high-frequency donor, n = 414 [six or more donations in the 3 years before pregnancy), and further adjusted for the covariates listed above. RESULTS: Adjusted normalized (mean, 0; standard deviation [SD], 1) test scores were statistically significantly higher for children of active female donors compared with children of nondonors (SD, 0.104; 95% confidence interval, 0.079-0.129). We observed no differences in scholastic attainment between children of low-frequency donors and high-frequency donors. CONCLUSION: Prepregnancy donation intensity, as a proxy of iron stores, is not associated with subsequent offspring scholastic attainment.

Genetic factors influencing hemoglobin levels in 15,567 blood donors: results from the Danish Blood Donor Study.

BACKGROUND: Blood donors have an increased risk of low hemoglobin (Hb) levels due to iron deficiency. Therefore, knowledge of genetic variants associated with low Hb could facilitate individualized donation intervals. We have previously reported three specific single-nucleotide polymorphisms that were associated with ferritin levels in blood donors. In this study, we investigated the effect of these single-nucleotide polymorphisms on Hb levels in 15,567 Danish blood donors. STUDY DESIGN AND METHODS: We studied 15,567 participants in the Danish Blood Donor Study. The examined genes and single-nucleotide polymorphisms were 1) TMPRSS6, involved in regulation of hepcidin: rs855791; 2) HFE, associated with hemochromatosis: rs1800562 and rs1799945; 3) BTBD9, associated with restless leg syndrome: rs9357271; and 4) TF, encoding transferrin: rs2280673 and rs1830084. Associations with Hb levels and risk of Hb deferral were assessed in multivariable linear and logistic regression models. RESULTS: The HFE,rs1800562 G-allele and the HFE rs1799945 C-allele were associated with lower Hb levels in men and women, and with an increased risk of Hb below 7.8 mmol/L (12.5 g/dL) in women. Only the rs1799945 C-allele increased the risk of Hb below 8.4 mmol/L (13.5 g/dL) in men. In TMPRSS6, the rs855791 T-allele was associated with lower Hb levels in both men and women, and with an increased risk of low Hb among women. CONCLUSION: With this study we demonstrate that HFE and TMPRSS6 are associated with Hb levels and risk of Hb below the limit of deferral. Thus, genetic testing may be useful in a future assay for personalized donation intervals.

Frequent blood donation and offspring birth weight-a next-generation association?

BACKGROUND: The prevalence of iron depletion is high among premenopausal women who donate blood frequently. Studies in nondonor populations indicate that iron deficiency anemia is associated with an increased risk of low birth weight. This prompts concerns that iron deficiency induced by frequent blood donation might impair subsequent fetal development. STUDY DESIGN AND METHODS: The aim of this study was to assess whether prepregnancy donation intensity affects the birth weight of singletons born at term (gestational week 38 or later) to nulliparous female donors in Denmark. We identified 293,897 first live singleton births to Danish women between 1997 and 2012 with complete information on gestational age, birth weight, child sex, parental age, maternal smoking status during pregnancy, and parental education length and annual income. Linear regression analysis was applied, with birth weight as outcome, number of donations within the 3 years before pregnancy as the explanatory variable, and confounding variables as described. RESULTS: Birth weight among children of low-intensity donors (n = 22,120) was 12.6 g (95% confidence interval, 6.7-18.6) higher than nondonors (n = 268,253) after controlling for the above-mentioned factors. The higher birth weight among low-intensity donors can be explained by the healthy donor effect. In fully adjusted analyses, birth weight among children of high-intensity donors (n = 3,524) was 20.2 g (95% confidence interval, 5.1-35.3 g) lower compared with low-intensity donors. This reduced birth weight among high-intensity donors compared to low-intensity donors may reflect blood donation-induced iron deficiency. CONCLUSIONS: Our results show that high prepregnancy donation intensity is inversely associated with birth weight of singletons born at term to nulliparous women.

Restless legs syndrome is associated with major comorbidities in a population of Danish blood donors.

BACKGROUND: Restless Legs Syndrome (RLS) is characterized by uncomfortable nocturnal sensations in the legs making sedentary activities and sleep difficult, and is thus linked with psychosocial distress. Due to the symptomatology and neurobiology of RLS (disrupting brain iron and dopamine) it is likely that RLS associates with poorer health-related quality of life (HRQL) and depressive disorder. The objective of this study was to investigate the RLS-HRQL and the RLS-depressive disorder links in a generally healthy population that is not biased by medications. METHODS: Complete data, including the Cambridge-Hopkins RLS questionnaire, the 12-item short-form standardized health survey (SF-12), the Major Depression Inventory (MDI), body mass index, smoking status, alcohol consumption, and education were available for 24,707 participants enrolled in the Danish Blood Donor Study from May 1, 2015 to February 1, 2017. Information on quality of sleep was available for all RLS cases. T-tests and multivariable logistic regression models were applied to examine the associations of RLS and MDI scores, and the physical and mental component scores (PCS and MCS) of SF-12, respectively. Analyses were conducted separately for men and women. RESULTS: RLS associated with poorer MCS and poorer PCS. Moreover, Participants with RLS were more likely to classify with depressive disorder. Poor quality of sleep was associated with depressive disorder and poorer MCS among RLS cases, and with poorer PCS in female RLS cases. CONCLUSION: Thus, we demonstrated that RLS is associated with a significantly lower HRQL and a higher prevalence of depressive disorder among otherwise healthy individuals.

Calcium in drinking water: effect on iron stores in Danish blood donors-results from the Danish Blood Donor Study.

BACKGROUND: Studies confirm that calcium inhibits iron absorption. Danish tap water comes from groundwater, which contains varying amounts of calcium depending on the subsoil. We investigated the association of calcium in drinking water with iron levels in Danish blood donors. STUDY DESIGN AND METHODS: We used data on Danish blood donors including dietary and lifestyle habits, blood donation history, and physiologic characteristics including measures of ferritin levels along with information on area of residence from The Danish Blood Donor Study. Data on calcium levels in groundwater ("water hardness") were obtained through the Geological Survey of Denmark and Greenland. We performed multiple linear and logistic regression analyses to evaluate the effect of water hardness on ferritin levels and risk of having iron deficiency (defined as ferritin levels <15 ng/mL), stratified by sex. RESULTS: There was a significant negative association between water hardness and ferritin levels in both men and women. Risk of iron deficiency was correspondingly increased in both men (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.14-2.12) and women (OR, 1.20; 95% CI, 1.03-1.40) with increasing water hardness. In analyses restricted to individuals who received supplemental iron tablets no significant association between groundwater hardness and ferritin levels was observed. CONCLUSIONS: As measured by ferritin levels, residential drinking water calcium content is associated with blood donors- iron levels and risk of iron deficiency. However, effect sizes are small.

Reduced ferritin levels in individuals with non-O blood group: results from the Danish Blood Donor Study.

BACKGROUND: Genomewide association studies have reported alleles in the ABO locus to be associated with ferritin levels. These studies warrant the investigation of a possible association between the ABO blood group and ferritin levels. We aimed to explore if ABO blood group is associated with iron stores expressed as ferritin levels. STUDY DESIGN AND METHODS: Ferritin levels were measured at least once for 30,595 Danish Blood Donor Study participants. Linear regression analyses were performed with the ABO blood group as explanatory variable and adjusted for age, number of donations 3 years before the ferritin measurement, and time since latest donation. In addition, a subanalysis was performed on 15,280 individuals in which further adjustments for body mass index, smoking status, and C-reactive protein levels were possible. Furthermore, logistic regression analyses were performed to determine if ABO blood group was associated with a ferritin level of less than 15 ng/mL. RESULTS: Non-O blood group donors had lower ferritin levels than blood group O donors, regardless of sex. Accordingly, risk of ferritin level of less than 15 ng/mL was increased for individuals with non-O blood group compared with O blood group. In subanalyses similar associations were observed, albeit in women the association between blood group and risk of a ferritin level below 15 ng/mL was no longer significant. ABO blood group was not associated with red blood cell indices such as mean cell volume and mean cell hemoglobin content. CONCLUSION: Donors with non-O blood group have lower ferritin levels than donors with other blood groups.

Prevalence of restless legs syndrome and associated factors in an otherwise healthy population: results from the Danish Blood Donor Study.

OBJECTIVE: Restless legs syndrome (RLS) is a neurological sensorimotor disorder characterized by uncomfortable sensations in the legs. RLS often occurs as a comorbid condition. Besides an increased risk of iron deficiency, blood donors are considered to be generally healthy. Blood donors are therefore an ideal population for studying factors associated with RLS occurrence, herein the role of iron. It is suggested that RLS is linked to sex, age, low socioeconomic status, unhealthy lifestyle, and iron deficiency. The objective of this study is therefore to estimate the RLS prevalence and identify associated biological, sociodemographic, economic, and lifestyle factors in a population of blood donors. METHODS: A total of 13,448 blood donors enrolled in the Danish Blood Donor Study from May 2015 to May 2016. RLS cases were identified using the validated Cambridge-Hopkins RLS-questionnaire. Logistic regression models were applied to assess the relationship between RLS and data on socially related factors collected using questionnaires and population registers. RESULTS: In this study, 7.2% women and 4.5% men were classified with RLS. RLS was associated with: female sex, high age, smoking, frequent alcohol consumption, and low education. RLS-related symptoms were associated with obesity, parity and donation intensity three years prior to inclusion among women. RLS was not related to: reduced plasma ferritin, employment status, and income. CONCLUSIONS: RLS is a frequent disorder in otherwise healthy individuals. The associations discovered in this study can be utilized in preventing or reducing RLS symptoms.

Deferral for low hemoglobin is not associated with increased risk of infection in Danish blood donors.

BACKGROUND: Low hemoglobin (Hb) is associated with poor general health and adverse outcomes in a wide range of diseases. However, a link between Hb levels and the risk of infection among healthy individuals has yet to be investigated. STUDY DESIGN AND METHODS: Using data from the Scandinavian Donations and Transfusions database, 497,390 donors were followed after 5,458,499 donations in health registers. With 1,339,362 person-years of follow-up, Andersen-Gill Cox regression was used to study the association of Hb levels below deferral thresholds, very low Hb levels (in the lowest 0.1 percentile), and declining Hb levels with the risk of infection as measured by hospital or outpatient contact for infection and filling of prescription for antimicrobials, respectively, within 3 months of donation. Analyses were stratified by sex, menopausal status, and frequency of donation. RESULTS: Hb levels below deferral thresholds were not associated with a risk of hospital contact for infection among premenopausal women (hazard ratio [HR], 1.04; 95% confidence interval [CI], 0.95-1.14), postmenopausal women (HR, 0.77; 95% CI, 0.54-1.11), or men (HR, 0.97; 95% CI, 0.81-1.16), nor was there any association with hospital contact for very low Hb levels or patterns of declining Hb levels. However, subthreshold Hb levels were associated with a reduced risk of antimicrobial prescriptions among premenopausal women (HR, 0.92; 95% CI, 0.91-0.93), postmenopausal women (HR, 0.93; 95% CI, 0.89-0.97), and men (HR, 0.91; 95% CI, 0.88-0.94). CONCLUSIONS: Neither Hb levels below deferral thresholds nor very low or declining Hb levels were associated with an increased risk of infection. This is reassuring, because blood donation can lead to lower Hb levels.

Genetic factors influencing ferritin levels in 14,126 blood donors: results from the Danish Blood Donor Study.

BACKGROUND: Many biologic functions depend on sufficient iron levels, and iron deficiency is especially common among blood donors. Genetic variants associated with iron levels have been identified, but the impact of genetic variation on iron levels among blood donors remains unclear. STUDY DESIGN AND METHODS: The effect of six single-nucleotide polymorphisms (SNPs) on ferritin levels in 14,126 blood donors were investigated in four genes: in Human Hemochromatosis Protein gene (HFE; rs1800562 and rs179945); in Transmembrane Protease gene, Serine 6 (TMPRSS6-regulating hepcidin; rs855791); in BTB domain containing protein gene (BTBD9-associated with restless legs syndrome; rs9357271); and in the Transferrin gene (TF; rs2280673 and rs1830084). Multiple linear and logistic regression analyses were used to evaluate the effect of each SNP on ferritin levels and the risk of iron deficiency (ferritin < 15 ng/mL). RESULTS: In HFE, the G-allele of rs1800562 was associated with lower iron stores in both sexes. This was also true for the C-allele of rs179945, but in men only. Also, the T-allele of TMPRSS6 rs855791 was negatively associated with iron stores in men. Homozygocity for C in rs1799945 was associated with iron deficiency in women. Results for all other genetic variants were insignificant. CONCLUSION: Genetic variants associated with hemochromatosis may protect donors against depleted iron stores. In addition, we showed that presence of the T-allele at rs855791 in TMPRSS6 was associated with lower iron stores in men.

No association between iron status and self-reported health-related quality of life in 16,375 Danish blood donors: results from the Danish Blood Donor Study.

BACKGROUND: Health-related quality of life (HRQL) represents people's subjective assessment of their mental and physical well-being. HRQL is highly predictive of future health. The effect of iron deficiency without anemia induced by blood donation on HRQL is presently unknown. The aim was to explore the relationship between iron status and self-reported mental component score (MCS; SF-12) and physical component score (PCS; SF-12) in Danish blood donors. STUDY DESIGN AND METHODS: Complete relevant data, including the 12-item short-form health survey (SF-12), plasma ferritin levels, age, body mass index, smoking status, C-reactive protein levels, number of donations in the previous 3 years, and PCS and MCS, were available for 8692 men and 7683 women enrolled from March 1, 2010, to December 31, 2010. Multivariable linear and logistic (cutoff at the 10th percentile) regression analyses were used to assess the relationship between iron deficiency (ferritin < 15 ng/mL) and MCS and PCS, respectively. Analyses were performed separately for men and women. RESULTS: There was no significant relationship between iron deficiency and self-reported mental or physical health. CONCLUSION: This study found no association between iron stores and self-reported HRQL among Danish blood donors.

Predictors of hemoglobin in Danish blood donors: results from the Danish Blood Donor Study.

BACKGROUND: It is well known that blood donors are at increased risk of iron deficiency and subsequent development of iron deficiency anemia. We aimed to investigate the effect of factors influencing hemoglobin (Hb) levels. STUDY DESIGN AND METHODS: Initiated in 2010, the Danish Blood Donor Study is a population-based study and biobank. We performed multivariable linear regression analysis to assess the effects of donation activity, physiologic and lifestyle factors, and diet on Hb levels among 15,197 donors. We also performed multivariable logistic regression to evaluate the effects of these factors on the risk of having low Hb (defined as Hb below the 10th percentile among men and women, respectively) and of a decrease in Hb greater than 0.5 mmol/L (0.8 g/dL) between successive donations. All analyses were performed stratified for sex and smoking status. We also tested a previously used model for the prediction of Hb. RESULTS: The strongest predictors of Hb and risk of low Hb were low ferritin (<15 ng/mL) and current use of iron supplementation (yes/no). No dietary factors were found to be consistently significant in multivariable models predicting Hb levels, risk of having low Hb, or risk of a decrease in Hb greater than 0.5 mmol/L. We found similar effects to previous studies of factors in the predictive model, with little additional effect of including smoking status and ferritin. CONCLUSIONS: As ferritin was the strongest predictor of Hb, this study supports the implementation of regular ferritin measurement as a method of risk assessment among blood donors.

No association between rs6897932 in the gene encoding interleukin-7 receptor α and low-grade inflammation or self-reported health - results from the Danish Blood Donor Study.

The T-allele in the single nucleotide polymorphism rs6897932 in the gene encoding the IL-7 receptor α (IL7RA) is associated with reduced risk of autoimmune diseases including multiple sclerosis and also affects the course of HIV infection. Low-grade inflammation (LGI) and self-reported, health-related quality of life (HRQL) are often associated with chronic diseases and widely used in assessing and monitoring health status. The aim of the present study was to evaluate whether the T-allele in rs6897932 is associated with reduced risk of LGI (hsCRP 3-10 mg/L), history of infectious mononucleosis (IM), and HRQL in healthy individuals. A total of 17, 293 healthy Danish individuals from the Danish Blood Donor Study were included in the analyses. We tested rs6897932 as a predictor of LGI, self-reported IM, and HRQL in univariable and multivariable models stratified by sex. No associations between rs6897932 and LGI, self-reported IM or HRQL were found in men or women. This suggests that rs6897932 is not associated with general inflammation, and the reported associations between the T-allele in rs6897932 with several autoimmune diseases may be mediated through effects on a restricted part of the immune system.

Combined oral contraception and obesity are strong predictors of low-grade inflammation in healthy individuals: results from the Danish Blood Donor Study (DBDS).

BACKGROUND: C-reactive protein (CRP) is a well-established marker of inflammation. The level of CRP is affected by several lifestyle factors. A slightly increased CRP level, also known as low-grade inflammation (LGI), is associated with increased risk of several diseases, especially cardiovascular disease. The aim of this study was to identify predictors of increased CRP levels in healthy individuals. We therefore assessed CRP in a large cohort of blood donors. METHODS: We measured plasma CRP levels in 15,684 participants from the Danish Blood Donor Study. CRP was measured by a commercial assay. Furthermore, all participants completed a standard questionnaire on smoking status, alcohol consumption, physical activity, diet, and various body measurements. Female participants also reported the use of contraception, childbirth, and menopausal status. The relationship between LGI (defined here as a plasma CRP level between 3 mg/L and 10 mg/L) and predictors was explored by multivariable logistic regression analysis. Results were presented as odds ratios (OR) with 95% confidence intervals (CI). RESULTS: We found LGI in a total of 1,561 (10.0%) participants. LGI was more frequent in women using combined oral contraception (OC) (29.9%) than in men (6.1%) and women not using OC (7.9%). Among premenopausal women, OC was the strongest predictor of LGI (odds ratio = 8.98, p<0.001). Additionally, body mass index (BMI) and waist circumference were positively associated with LGI. CONCLUSION: High BMI and abdominal obesity strongly predicted LGI among healthy individuals. However, the most striking finding was the high prevalence of LGI among premenopausal women who used combined oral contraception. Although the significance of CRP as a marker of inflammation is well known, the role of CRP in pathogenesis is still uncertain. The impact of oral contraception on CRP levels should nevertheless be considered when CRP is used in risk assessment.