Decreased sphingolipid synthesis in children with 17q21 asthma-risk genotypes

Abstract

Risk for childhood asthma is conferred by alleles within the 17q21 locus affecting ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) expression. ORMDL3 inhibits sphingolipid de novo synthesis. Although the effects of 17q21 genotypes on sphingolipid synthesis in human asthma remain unclear, both decreased sphingolipid synthesis and ORMDL3 overexpression are linked to airway hyperreactivity. To characterize the relationship of genetic asthma susceptibility with sphingolipid synthesis, we analyzed asthma-associated 17q21 genotypes (rs7216389, rs8076131, rs4065275, rs12603332, and rs8067378) in both children with asthma and those without asthma, quantified plasma and whole-blood sphingolipids, and assessed sphingolipid de novo synthesis in peripheral blood cells by measuring the incorporation of stable isotope-labeled serine (substrate) into sphinganine and sphinganine-1-phosphate. Whole-blood dihydroceramides and ceramides were decreased in subjects with the 17q21 asthma-risk alleles rs7216389 and rs8076131. Children with nonallergic asthma had lower dihydroceramides, ceramides, and sphingomyelins than did controls. Children with allergic asthma had higher dihydroceramides, ceramides, and sphingomyelins compared with children with nonallergic asthma. Additionally, de novo sphingolipid synthesis was lower in children with asthma compared with controls. These findings connect genetic 17q21 variations that are associated with asthma risk and higher ORMDL3 expression to lower sphingolipid synthesis in humans. Altered sphingolipid synthesis may therefore be a critical factor in asthma pathogenesis and may guide the development of future therapeutics.

Keywords: Asthma; Pulmonology.

Figure 1. Sphingolipid composition in whole blood.

Sphingolipids in control subjects (Co) and subjects with asthma (As), stratified by allergy group. Boxes represent the interquartile ranges with the median, and whiskers indicate the range. Low eosinophil counts (Low-Eos): <300/mL, high eosinophil counts (High-Eos): ≥300/mL in peripheral blood. The means are indicated by a plus sign and were compared by 2-sample t test for normal data; for non-normal data, comparison of the median values is indicated by a pound sign. The corresponding P values are reported if they were less than 0.05. Significant P values following the BH procedure (FDR = 0.05) are indicated by an asterisk if they fell below the BH-critical value of 0.014. Controls with low-eosinophil counts (n = 43), asthma subjects with low-eosinophil counts (n = 16); controls with high-eosinophil counts (n = 14), asthma subjects with high-eosinophil counts (n = 34).

Figure 2. Whole-blood sphingolipids with an acyl-chain length of C16-18 in all subjects.

Examples of sphingolipid masses in combined asthma and control subjects based on genotype at (A) rs7216389 (n = 8 CC; n = 53 CT; n = 51 TT) and (B) rs8076131 (n = 6 GG; n = 51 AG; n = 55 AA). In total, 18 individual sphingolipids were measured for each genotype. Individual values are shown; bars represent the median with a 95% CI, compared by Mann-Whitney U test. Significant P values following adjustment for a FDR of 0.05 are indicated with an asterisk, as they fell below the BH-critical value of 0.0092.

Figure 3. De novo sphingolipid synthesis is decreased in children with asthma.

Newly synthesized sphinganine and sphinganine-1-phosphate in control subjects (n = 7) and subjects with asthma (n = 13) following metabolic labeling of the substrate (C¹³N¹⁵-serine) in PBMCs. Individual values are shown, and the means were compared using a 2-sample t test.

Figure 4. De novo sphingolipid synthesis is decreased in children with 17q21 asthma–risk genotypes.

Newly synthesized sphinganine (Sa) and sphinganine-1-phosphate (Sa1P) in PBMCs of all subjects, incubated with C¹³N¹⁵-serine and stratified by genotype (A) rs7216389 (n = 44 Sa CC/CT; n = 38 TT; n = 41 Sa1P CC/CT; n = 39 TT) and (B) rs8076131 (n = 41 Sa GG/AG; n = 42 TT; n = 39 Sa1P GG/AG; n = 38 AA). Individual data points are shown; bars represent the median and a 95%CI, compared by Mann-Whitney U test. (C) De novo synthesis of sphinganine and sphinganine-1-phosphate by rs7216389 genotype and stratified by group. Bars represent the median with a 95%CI. Comparisons between asthma groups were done with a Kruskal-Wallis H test [H(4) = 6.534, P = 0.08].