Clinical and molecular implications of RGS2 promoter genetic variation in severe asthma

Abstract

Background: Regulator of G protein signaling (RGS) 2 terminates bronchoconstrictive Gαq signaling; murine RGS2 knockout demonstrate airway hyperresponsiveness. While RGS2 promoter variants rs2746071 and rs2746072 associate with a clinical mild asthma phenotype, their impact on human airway smooth muscle (HASM) contractility and asthma severity outcomes is unknown.

Objective: We sought to determine whether reductions in RGS2 expression seen with these 2 RGS2 promoter variants augment HASM contractility and associate with an asthma severity phenotype.

Methods: We transfected HASM with a range of RGS2-specific small interfering RNA (siRNA) concentrations and determined RGS2 protein expression by Western blot analysis and intracellular calcium flux induced by histamine (a Gαq-coupled H1 receptor bronchoconstrictive agonist). We conducted regression-based genotype association analyses of RGS2 variants from 611 patients from the National Heart, Lung, and Blood Institute Severe Asthma Research Program 3.

Results: RGS2-specific siRNA caused dose-dependent increases in histamine-stimulated bronchoconstrictive intracellular calcium signaling (2-way ANOVA, P < .0001) with a concomitant decrease in RGS2 protein expression. RGS2-specific siRNA did not affect Gαq-independent ionomycin-induced intracellular calcium signaling (P = .42). The minor allele frequency of rs2746071 and rs2746072 was 0.46 and 0.28 among African American/non-Hispanic Black patients and was 0.28 and 0.27 among non-Hispanic White patients, among whom these single nucleotide polymorphisms were in stronger linkage disequilibrium (r² = 0.97). Among non-Hispanic White patients, risk allele homozygotes for rs2746072 and rs2746071 each had nearly 2-fold greater asthma exacerbation rates relative to alternative genotypes with wild-type alleles (P_additive = 2.86 × 10^-5/P_recessive = 5.22 × 10^-6 and P_additive = 3.46 × 10^-6/P_recessive = 6.74 × 10^-7, respectively) at baseline, which was confirmed by prospective longitudinal exacerbation data.

Conclusion: RGS2 promoter variation associates with a molecular and clinical phenotype characterized by enhanced bronchoconstrictive stimulation in vitro and higher asthma exacerbations rates in non-Hispanic White patients.

Keywords: G protein–coupled receptors; Regulator of G protein signaling; airway hyperresponsiveness; airway smooth muscle; asthma exacerbations; bronchoconstriction; endotype; genomics; genotype; phenotype.

Figure 1.. Effects of decreased RGS2 on histamine-mediated [Ca²⁺]_i release in human airway smooth muscle cells.

A,B) Decreasing RGS2 by RGS2-specific siRNA increases [Ca²⁺]_i release by histamine (25 μM) in a siRNA dose-dependent manner, while the ionomycin (2 μM) response is unaffected. Results are mean +/− SE of quadruplicate determinations from a single representative experiment, which was performed three times. C,D) RGS2 expression is decrease by ~50% in human airway smooth muscle cells receiving 150 nM RGS2-siRNA. A representative western blot is shown in (C), and the results from 3 experiments is shown in (D). *, P<0.01 vs scrambled control siRNA (siCtrl).

Figure 2.

Linkage disequilibrium plots (r²) for RGS2 variation with minor allele frequency (MAF) >0.05 in (A.) Non-Hispanic Whites (NHW) and (B.) African-American/Black (AA/B) with asthma from SARP3. 20 of 29 in NHW and 44 of 54 variants in AA/B have MAF<0.05. rs2746071 in red, rs2746072 in blue.

Figure 3.

Relationship between variant homozygotes for RGS2 promoter SNPs rs2746071 (GG) and rs2746072 (CC) and annualized exacerbation rates relative to genotypes containing wildtype alleles among Non-Hispanic Whites (NHW) and African-American-Blacks (AA-B) in a cohort enriched for severe asthma (SARP3). We show adjusted p-values for both additive and recessive models that included age, sex and body mass index. Models for NHW are also included the first two principal components of NHW ancestry, while those for AA-B included the first four principal components of AA-B ancestry.