A novel functional variant in the stem cell growth factor promoter protects against severe malarial anemia

Abstract

Plasmodium falciparum malaria is a leading global cause of infectious disease burden. In areas in which P. falciparum transmission is holoendemic, such as western Kenya, severe malarial anemia (SMA) results in high rates of pediatric morbidity and mortality. Although the pathophysiological basis of SMA is multifactorial, we recently discovered that suppression of unexplored hematopoietic growth factors that promote erythroid and myeloid colony development, such as stem cell growth factor (SCGF) (C-type lectin domain family member 11A [CLEC11A]), was associated with enhanced development of SMA and reduced erythropoietic responses. To extend these investigations, the relationships between a novel SCGF promoter variant (-539C/T, rs7246355), SMA (hemoglobin [Hb] < 6.0 g/dl), and reduced erythropoietic responses (reticulocyte production index [RPI], <2.0) were investigated with Kenyan children (n = 486) with falciparum malaria from western Kenya. Circulating SCGF was positively correlated with hemoglobin levels (r = 0.251; P = 0.022) and the reticulocyte production index (RPI) (r = 0.268; P = 0.025). Children with SMA also had lower SCGF levels than those in the non-SMA group (P = 0.005). Multivariate logistic regression analyses controlling for covariates demonstrated that individuals with the homologous T allele were protected against SMA (odds ratio, 0.57; 95% confidence interval [95% CI] 0.34 to 0.94; P = 0.027) relative to CC (wild-type) carriers. Carriers of the TT genotype also had higher SCGF levels in circulation (P = 0.018) and in peripheral blood mononuclear cell culture supernatants (P = 0.041), as well as an elevated RPI (P = 0.005) relative to individuals with the CC genotype. The results presented here demonstrate that homozygous T at -539 in the SCGF promoter is associated with elevated SCGF production, enhanced erythropoiesis, and protection against the development of SMA in children with falciparum malaria.

FIG. 1.

Relationship between SCGF and anemia. Plasma was obtained from children with acute malaria and SCGF concentrations were determined by ELISA. (A) Correlation between SCGF concentrations and Hb levels in parasitemic children (n = 83) determined by Spearman's correlation coefficient. (B) Association between SCGF levels and anemia severity in parasitemic children with SMA (n = 25) and non-SMA (n = 58). Boxes represent the interquartile range, the line through the box is the median, and whiskers show 10th and 90th percentiles. Statistical significance was determined by the Mann-Whitney U test.

FIG. 2.

Functional association between SCGF −539C/T promoter variants and SCGF. (A) Circulating SCGF levels were measured using ELISA. Data are shown for the CC (n = 17), CT (n = 33), and TT (n = 33) genotypes. Data are presented as box plots, where the box represents the interquartile range, the line through the box is the median, and whiskers show the 10th and 90th percentiles. Cross-group comparisons were determined by Kruskal-Wallis tests followed by Mann-Whitney U tests for pairwise comparison. The presence of the TT genotype was associated with significantly higher circulating SCGF levels relative to individuals with CC genotype (P = 0.018). (B) PBMCs were isolated from peripheral blood (<3.0 ml) and cultured (1 × 10⁶ cells/ml) in serum-containing media. Supernatants were obtained at 48 h for SCGF determination by ELISA. Children were stratified according to genotypes: CC (n = 11), CT (n = 13), and TT (n = 13). Data are presented as box plots, where the box represents the interquartile range, the line through the box is the median, and whiskers show the 10th and 90th percentiles. Statistical significance was determined by Mann-Whitney U test. The presence of the TT genotype was associated with significantly higher culture supernatant SCGF levels relative to individuals with the CC genotype (P = 0.041).

FIG. 3.

Association between SCGF variants and erythropoiesis. Plasma was obtained from children with acute malaria, and SCGF concentrations were determined by ELISA. (A) Correlation between SCGF concentrations and reticulocyte production index (RPI) levels in children with anemia (n = 70). Relationships between SCGF and RPI were determined by Spearman's correlation coefficient. (B) Association between SCGF −539C/T promoter variants and the RPI in anemic children. Data are shown for the CC (n = 14), CT (n = 29), and TT (n = 27) genotypes. Boxes represent the interquartile range, the line through the box is the median, and whiskers show 10th and 90th percentiles. Cross-group comparisons were determined by Kruskal-Wallis tests followed by Mann-Whitney U tests for pairwise comparison. The presence of the TT genotype was associated with significantly higher RPI relative to individuals with the CC genotype (P = 0.005).