Research ArticleHEALTH AND MEDICINE

The ferroportin Q248H mutation protects from anemia, but not malaria or bacteremia

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Science Advances  04 Sep 2019:
Vol. 5, no. 9, eaaw0109
DOI: 10.1126/sciadv.aaw0109
  • Fig. 1 Collections with biological samples and phenotype and genetic data available in humans to test associations between anemia, iron, malaria, and bacteremia traits and the FPN Q248H mutation.

    (A) Proportion of total individuals with samples and data available for iron-related traits, and the Q248H mutation are shown in a heatmap of green gradient, representing the proportion of individuals with biological samples available, stratified by population with longitude and latitude represented on a map of the African continent. (B) Individuals with genetic data available from the 1000 Genomes Phase 3 Project with sequence data available stratified by population with longitude and latitude represented on a map of the African continent. MCV, mean cell volume; sTFR, soluble transferrin receptor; ZPP, zinc protoporphyrin; CRP, C-reactive protein; erythrocyte* includes samples available for ex vivo Plasmodium growth assay.

  • Fig. 2 FPN Q248H mutation is associated with protection from anemia, red cell hemolysis, and iron deficiency.

    The Q248H mutation renders FPN partially resistant to hepcidin degradation (4), increasing iron export through enterocytes, erythrocytes, and macrophages. The effects of Q248H on (A) anemia, (B) hemoglobin, (C) MCHC, (D) haptoglobin, (E) iron deficiency, (F) transferrin saturation (TSAT), (G) hepcidin, (H) ferritin, (I) soluble transferrin receptors (sTfR), (J) ZPP, and (K) inflammation indicate reduced hemolysis, limited effect on iron status, and no association with inflammation. Plotted values are geometric means except anemia, iron deficiency, and inflammation, which are percentages. All P values were two tailed and derived from regression models adjusted for age, sex, and cohort. GIT, gastrointestinal tract; DMT1, divalent metal transporter 1.

  • Fig. 3 Effect of FPN Q248H variant on parasite P. falciparum growth rates and parasite density.

    (A) Ex vivo parasite growth rates in fresh erythrocytes from Gambian children (6 to 29 months) and mid-pregnant women stratified by carriage of Q248H FPN mutation (Het, heterozygous; Hom, homozygous). Parasite growth rates are geometric means from log-transformed data from triplicate assays. See the Materials and Methods for details of the assays and the clinical trials from which the samples were derived. A total of 229 genotyped children contributed data on up to three visits each (days 0, 49, and 84 of iron supplementation) and 380 genotyped pregnant women contributed data on up to four visits each (days 0, 14, 49, and 84). P values were calculated by analysis of variance (ANOVA) with visit day as a covariate to allow for supplementation effect. There was no interaction with hemoglobin or anemia or with plasma hepcidin (as a continuous variable or as above or below the thresholds of 5.5 ng/ml in children and 2.5 ng/ml in pregnant women). In the combined analysis of women and children, there was no interaction with study, confirming that merging of the datasets was legitimate. (B) P. falciparum parasite densities in hospitalized Kenyan children with severe malaria stratified by carriage of Q248H. Parasite densities are geometric means from log-transformed data. N, severe malaria cases. P values were derived from regression models adjusted for age, sex, and cohort. *Q248H heterozygotes and homozygotes combined (n = 3 homozygotes).

  • Fig. 4 Frequencies of adenine allele of rs11568350 encoding the Q248H mutation in African populations compared with the composite maximal estimate of P. falciparum transmission in 2- to 10-year-olds between the years 1900 and 1959.

    Regional median estimates of transmission are provided across malaria endemic regions of the natural extent of sub-Saharan Africa including the Island of Madagascar as obtained from Snow et al. (14). Population-specific frequencies of the variant encoding the Q248H allele are shown as pie charts for each of the included populations with the more common ancestral allele colored in black and the rarer adenine allele colored. For illustration purposes, colors are assigned on a scale matched to the P. falciparum parasite prevalence scale using a frequency of 10% as equivalent to a prevalence of 1 (for example, green represents 5% allele frequency). If there were a correlation between the allele frequencies and malaria prevalence, one would expect to see similar colors in the pie chart slices and map contours. The allele frequencies and estimated maximal estimates of transmission per population are plotted against each other in fig. S4.

  • Table 1 The FPN Q248H mutation is associated with protection from anemia, hemolysis, and iron deficiency in community-based children.

    Analyses were adjusted for age, sex, and country. CRP, C-reactive protein; MCV, mean corpuscular volume; sTfR, soluble transferrin receptor; TSAT, transferrin saturation; ZPP, zinc protoporphyrin.

    Q248H*WT
    CategoryAll, nQ248H, nn (%)WT, nn (%)OR (95% CI)P
    Anemia2666237111 (46.8)24291305 (53.7)0.75 (0.57 to 0.98)0.037
    Iron deficiency306532598 (30.2)2740933 (34.1)0.77 (0.60 to 0.99)0.046
    Iron deficiency anemia§236320835 (16.8)2155435 (20.2)0.77 (0.52 to 1.14)0.19
    Inflammation314933466 (19.8)2815587 (20.9)0.94 (0.70 to 1.25)0.65
    BiomarkersAll, nQ248H, nGeometric mean (SD)WT, nGeometric mean (SD)Estimate
    (95% CI)
    P
    Hemoglobin, g/dl266623710.81 (1.15)242910.59 (1.16)0.21 (0.01 to 0.40)0.036
    MCHC, g/dl153713133.10 (1.04)140632.87 (1.04)0.27 (0.05 to 0.50)0.017
    MCV, fL232420572.98 (1.12)211972.76 (1.12)0.16 (−0.87 to 1.19)0.76
    Haptoglobin, μmol/liter58750133.22 (1.98)537109.32 (1.87)0.20 (0.02 to 0.38)0.030
    Ferritin, μg/liter306532520.80 (2.53)274020.03 (2.84)0.08 (−0.03 to 0.20)0.16
    Hepcidin, μg/liter30803198.31 (3.48)27617.33 (3.87)0.14 (−0.02 to 0.30)0.089
    sTfR, mg/liter309132511.98 (1.66)276613.30 (1.67)−0.07 (−0.16 to 0.01)0.088
    Iron, μmol/liter459455.45 (1.91)4144.83 (2.35)0.14 (−0.10 to 0.37)0.25
    Transferrin, g/liter23802582.62 (1.28)21222.70 (1.27)−0.06 (−0.14 to 0.02)0.14
    TSAT, %430407.89 (2.13)3906.95 (2.62)0.09 (−0.18 to 0.36)0.52
    ZPP, μmol/mol heme6846697.43 (1.80)618114.63 (1.77)−0.17 (−0.31 to −0.03)0.018
    CRP, mg/liter24032631.08 (5.07)21401.35 (5.44)−0.19 (−0.41 to 0.02)0.079
    ACT, g/liter737680.47 (1.34)6690.44 (1.30)0.05 (−0.01 to 0.12)0.12

    *Heterozygotes and homozygotes.

    †Anemia was defined as hemoglobin <11 g/dl.

    ‡Iron deficiency was defined as plasma ferritin <12 or <30 μg/liter in the presence of inflammation in children <5 years or <15 μg/liter in children ≥5 years.

    §Iron deficiency anemia was defined as iron deficiency with anemia.

    ║Inflammation was defined as C-reactive protein >5 mg/liter or ACT >0.6 g/liter in The Gambia.

    • Table 2 Little evidence that FPN Q248H provides protection against severe malaria or invasive bacterial infection.

      All P values reflect two-tailed tests. NTS, nontyphoidal Salmonella; RR, relative risk.

      OutcomeAllQ248H*WTRR/OR
      (95% CI)
      P
      Malaria susceptibility
      Mild malaria260/2,550 (10.2%)25/232 (10.8%)235/2,318 (10.1%)1.06 (0.68–1.64)0.79
      Severe malaria5,489/11,982 (45.8%)658/1519 (43.3%)4,831/10,463(46.1%)0.91 (0.81–1.01)0.08
        (The Gambia§)2,419/4,910 (49.3%)225/479 (47.0%)2,194/4,431 (49.5%)0.88 (0.73–1.07)0.21
        (Malawi§)1,023/2,345 (43.6%)174/419 (41.5%)849/1,926 (44.1%)0.89 (0.72–1.10)0.28
        (Kenya§)1,446/2,924 (49.5%)191/405 (47.2%)1,255/2,519 (49.8%)0.98 (0.79–1.22)0.87
        (Ghana)601/1,803 (33.3%)68/216 (31.5%)533/1,587 (33.6%)0.83 (0.59–1.17)0.29
      Cerebral malaria1,948/7,971 (24.4%)249/1043 (23.9%)1,699/6,928 (24.5%)0.90 (0.77–1.06)0.21
        (The Gambia§)758/3,676 (20.6%)60/356 (16.9%)698/3,320 (21.0%)0.73 (0.55–0.99)0.04
        (Malawi§)644/2,057 (31.3%)116/373 (31.1%)528/1,684 (31.4%)0.95 (0.75–1.22)0.71
        (Kenya§)546/2,238 (24.4%)73/314 (23.2%)473/1,924 (24.6%)1.00 (0.75–1.34)0.99
      Severe malarial anemia735/7,971 (9.2%)82/1043 (7.9%)653/6,928 (9.4%)0.90 (0.71–1.16)0.42
        (The Gambia§)428/3,676 (11.6%)43/356 (12.1%)385/3,320 (11.6%)0.96 (0.68–1.35)0.82
        (Malawi§)91/2,057 (4.4%)12/373 (3.2%)79/1,684 (4.7%)0.69 (0.37–1.29)0.24
        (Kenya§)216/2,238 (9.7%)27/314 (8.6%)189/1,924 (9.8%)0.93 (0.60–1.43)0.74
      Malaria-related death677/4,669 (14.5%)86/571 (15.1%)591/4,098 (14.4%)1.00 (0.78–1.29)0.99
        (The Gambia§)309/2,335 (13.2%)27/219 (12.3%)282/2,116 (13.3%)0.91 (0.59–1.39)0.65
        (Malawi§)200/1,018 (19.6%)37/173 (21.4%)163/845 (19.3%)1.14 (0.76–1.71)0.51
        (Kenya§)168/1,316 (12.8%)22/179 (12.3%)146/1,137 (12.8%)0.94 (0.58–1.52)0.80
      Bacteremia1,536/4,213 (36.5%)223/607 (36.7%)1,313/3,606 (36.4%)1.04 (0.87–1.24)0.68
        Streptococcus pneumoniae426/4,213 (10.1%)57/607 (9.4%)369/3,606 (10.2%)0.93 (0.69–1.26)0.65
        NTS180/4,213 (4.3%)31/607 (5.1%)149/3,606 (4.1%)1.25 (0.83–1.87)0.28
        Escherichia coli151/4,213 (3.6%)26/607 (4.3%)125/3,606 (3.5%)1.27 (0.82–1.97)0.29
        Haemophilus influenzae128/4,213 (3.0%)12/607 (2.0%)116/3,606 (3.2%)0.64 (0.35–1.18)0.15
        Staphylococcus aureus175/4,213 (4.2%)20/607 (3.3%)155/3,606 (4.3%)0.80 (0.50–1.30)0.37

      *Heterozygotes and homozygotes.

      †Mild malaria was defined as P. falciparum positive slide measured in community-based cohorts in Uganda, The Gambia, Burkina Faso, and Kenya. Severe malaria was defined as positive for P. falciparum parasites and clinical features of severe malaria (8), including diagnosis of cerebral malaria, severe malarial anemia, and other clinical symptoms.

      ‡Relative risk, CI, and P value were computed by fixed-effect meta-analysis of estimates from the three case-control cohorts and (where applicable) the Ghanaian trios.

      §Association analysis estimated by logistic regression adjusted for the first five principal components. For severe malaria and malaria-related death, results reflect binomial logistic regression of the phenotype compared with controls. For severe malaria subphenotypes, results reflect multinomial logistic regression of cerebral malaria, severe malarial anemia, and other severe malaria cases compared with controls. A dominant mode of effect is assumed.

      ║Counts reflect numbers of probands (affected children) and parents in 608 Ghanaian trios. Relative risk, CI, and P value are computed using a transmission disequilibrium test. A dominant mode of effect is assumed.

      ¶Bacteremia was defined as positive blood culture from hospitalized admission in Kilifi County Hospital in Kenya. Association analysis for all-cause bacteremia was estimated by logistic regression adjusted for sex and the first two principal components of genome-wide genotyping data to account for population structure. Pathogen-specific P values and odds ratios were derived by multinomial logistic regression adjusted for sex and population structure.

      Supplementary Materials

      • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw0109/DC1

        Fig. S1. A meta-analysis of studies investigating the relationship between the FPN Q248H mutation and hemoglobin levels.

        Fig. S2. Meta-analysis of associations of the FPN Q248H mutation with anemia, hemoglobin, and iron status across the study populations.

        Fig. S3. Forest plots of the effect of the FPN Q248H mutation on hemoglobin and measures of iron status.

        Fig. S4. Correlation between P. falciparum prevalence/rate and the derived adenine allele encoding the Q248H mutation.

        Table S1. Summary of studies examining the relationship between the FPN Q248H mutation and hemoglobin, ferritin, and C-reactive protein.

        Table S2. Characteristics of participants by study cohort and FPN Q248H mutation.

        Table S3. Estimates of the effect of Q248H heterozygotes and homozygotes on iron status and anemia.

        Table S4. Estimates of the effect of Q248H heterozygotes and homozygotes on severe malaria and bacteremia status.

        Table S5. Deviation from Hardy-Weinberg equilibrium for the variant causing the FPN Q248H mutation.

        Table S6. Rare alleles present in populations included in the 1000 Genomes Phase 3.

        Appendix A

        References (3640)

      • Supplementary Materials

        This PDF file includes:

        • Fig. S1. A meta-analysis of studies investigating the relationship between the FPN Q248H mutation and hemoglobin levels.
        • Fig. S2. Meta-analysis of associations of the FPN Q248H mutation with anemia, hemoglobin, and iron status across the study populations.
        • Fig. S3. Forest plots of the effect of the FPN Q248H mutation on hemoglobin and measures of iron status.
        • Fig. S4. Correlation between P. falciparum prevalence/rate and the derived adenine allele encoding the Q248H mutation.
        • Table S1. Summary of studies examining the relationship between the FPN Q248H mutation and hemoglobin, ferritin, and C-reactive protein.
        • Table S2. Characteristics of participants by study cohort and FPN Q248H mutation.
        • Table S3. Estimates of the effect of Q248H heterozygotes and homozygotes on iron status and anemia.
        • Table S4. Estimates of the effect of Q248H heterozygotes and homozygotes on severe malaria and bacteremia status.
        • Table S5. Deviation from Hardy-Weinberg equilibrium for the variant causing the FPN Q248H mutation.
        • Table S6. Rare alleles present in populations included in the 1000 Genomes Phase 3.
        • Appendix A
        • References (3640)

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