Complex genetic control of susceptibility to malaria: positional cloning of the Char9 locus

Abstract

Mouse strains AcB55 and AcB61 are resistant to malaria by virtue of a mutation in erythrocyte pyruvate kinase (Pklr(I90N)). Linkage analysis in [AcB55 x A/J] F2 mice detected a second locus (Char9; logarithm of odds = 4.74) that regulates the blood-stage replication of Plasmodium chabaudi AS independently of Pklr. We characterized the 77 genes of the Char9 locus for tissue-specific expression, strain-specific alterations in gene expression, and polymorphic variants that are possibly associated with differential susceptibility. We identified Vnn1/Vnn3 as the likely candidates responsible for Char9. Vnn3/Vnn1 map within a conserved haplotype block and show expression levels that are strictly cis-regulated by this haplotype. The absence of Vnn messenger RNA expression and lack of pantetheinase protein activity in tissues are associated with susceptibility to malaria and are linked to a complex rearrangement in the Vnn3 promoter region. The A/J strain also carries a unique nonsense mutation that leads to a truncated protein. Vanin genes code for a pantetheinase involved in the production of cysteamine, a key regulator of host responses to inflammatory stimuli. Administration of cystamine in vivo partially corrects susceptibility to malaria in A/J mice, as measured by reduced blood parasitemia and decreased mortality. These studies suggest that pantetheinase is critical for the host response to malaria.

Figure 1.

The effect of Char9 alleles on peak parasitemia and reticulocytosis. The effect of chromosome 10 (Char9) alleles on peak parasitemia (percentage of infected RBCs; A and B) and reticulocytosis (C and D) is shown in the context of WT or mutant (I90N) alleles at pklr (Char4) in [AcB55 × A/J] F2 mice. Each dot represents a mouse. Alleles at D10Mit189 are represented by the following: a, A/J; b, AcB55; h, heterozygote. Genotypes at Pklr are represented by the following: +/+, WT; +/−, heterozygote; −/−, mutant. Males (A and C) and females (B and D) are shown separately. Horizontal bars indicate the mean peak parasitemia value for each group.

Figure 2.

mRNA expression of positional candidates in the Char9 interval. 28 genes from the Char9 region showing expression in the spleen were examined for strain-specific differences in RNA expression and possible association with malaria susceptibility or resistance. Real-time RT-PCR was performed using a LightCycler (see Materials and methods). The histograms represent the ratio of mRNA expression of each transcript measured in A/J (blue), AcB55 (purple), and AcB61 (yellow) relative to the levels measured in B6 (red). The expression level has been normalized for various control transcripts, including Gapd, actin, and Hprt (representative results with Hprt are shown). Error bars represent the SEM of normalized expression (2^−ΔCt) for three to six replicates. The unique ratios detected for Vnn3 and indicative of a Char9 effect are outlined with a box, and the highly significant difference in expression levels between A/J and AcB55 for Vnn3 is indicated.

Figure 3.

Haplotype structure of the Char9 congenic segment of AcB55. (A) The position of the B6-derived chromosome 10 segment (gray) of AcB55 is shown on the A/J chromosome 10 background (white). Microsatellite markers used in Char9 linkage mapping studies are identified along with their positions on the physical map (in megabases). SNP-based genotypes (spacing of ∼100 kB) for 11 inbred strains (identified on top) were obtained from the WTCTC SNP database. B6 alleles are gray, and the minor alleles are white. Inbred strains were segregated into P. chabaudi–susceptible (S) and –resistant (R) strains (*, DBA/1J is susceptible), and blocks of conserved haplotypes that segregated with susceptibility were noted. The region of the haplotype covering the Char9 congenic segment is outlined, and the minimal interval is indicated. The 11 genes mapping to this minimal interval are shown, with genes expressed in the spleen highlighted in black. (B) The level of mRNA expression of positional candidates Vnn3, Stx7, Ctgf, Enpp1, Crsp3, and Epb4.1l2 was determined in the spleen of 10 inbred mouse strains by quantitative RT-PCR. Strains carrying the A/J-like haplotype in this region are shown in white, and strains with the B6-like haplotype are shown in gray. Expression level is shown as a mean of three replicates and relative to B6 (B6 = 1) after normalization to Hprt. Error bars represent SEM. (C) Inbred strains of mice display two major haplotypes defined by SNPs in the Vnn3 gene that are associated with susceptibility (white) or resistance (gray) to infection with P. chabaudi (P.c.) susceptibility. Int, intronic; sil, silent/synonymous; MS, missense/nonsynonymous; NS, nonsense/premature STOP.

Figure 4.

Effect of the Char9 genomic haplotype on Vanin mRNA expression and pantetheinase activity. Levels of Vanin mRNA and pantetheinase enzyme activity in mouse strains carrying the A/J or B6 haplotype. (A) Northern blot analysis of total liver RNA from A/J, B6, AcB55, and AcB61 using isoform-specific hybridization probes corresponding to the Vnn1 and Vnn3 genes. Ethidium-bromide staining of the agarose gel is shown to demonstrate similar RNA loading on each lane. (B) Levels of pantetheinase activity in the liver (n = 4) of A/J, B6, AcB55, and AcB61 mice as measured by spectrophotometric enzyme assay. Activity is measured in milliunits/milligram of tissue. The limit of detection was 0.1 mU/mg (dashed line). Horizontal bars indicate the mean peak parasitemia value for each group.

Figure 5.

Analysis of the Vnn3 proximal promoter region. Examination of the genomic sequence found upstream of the Vnn3 transcription start site. (A) A schematic representation of the genomic region in the putative proximal promoter of Vnn3. The 2,000 bp that were analyzed for nucleotide sequence integrity are displayed with PCR primers indicated by arrows and SNPs indicated by triangles. BamH1 restriction sites and the localization of the specific probe for Southern blotting are labeled. The major genomic rearrangement distinguishing the B6-like strains from the A/J-like strains is shown in greater detail with repeat regions and deleted regions indicated. (B) All identified polymorphisms in the promoter region are listed with respect to the reference (B6) genomic sequence. The double asterisks represent a deletion at this position. (C) The Southern blot from a BamH1 digest of genomic DNA displaying the size of the genomic rearrangement; A/J-like strains carry ∼340 bp of extra sequence. The arrows indicate the two distinct bands produced by the genomic rearrangement: the smaller band is representative of the B6-like promoters, and the larger band is representative of the A/J-like promoters.

Figure 6.

Administration of cystamine in vivo and the effect on P. chabaudi replication in A/J and B6 mice. The effect of cystamine treatment on P. chabaudi AS infection in A/J and B6 mice. (A) Male and female mice were treated with cystamine (120 mg/kg of body weight) or with PBS for 2 d before infection with 10⁶ parasitized erythrocytes (pRBCs) and continuing daily for 15 d. Boxes and whisker plot shows parasitemia (percent pRBC) levels for 8–10 mice per group at day 7 after infection for control (gray bars) and treated mice (white bars). The median parasitemia is indicated by a horizontal line, whereas the box represents the quartiles, and the whiskers represent the end points of the parasitemia distribution. Error bars represent SEM. Statistical differences between groups are indicated by asterisks: **, P < 0.001; *, P < 0.02. (B) Effect of cystamine treatment on the survival of A/J mice after P. chabaudi AS infection. Dashed lines represent treated mice, whereas solid lines represent control animals. Cont, control; treat, treated; pRBC, parasitized RBC.