Transforming growth factor-beta signaling pathway in patients with Kawasaki disease

Abstract

Background: Transforming growth factor (TGF)-β is a multifunctional peptide that is important in T-cell activation and cardiovascular remodeling, both of which are important features of Kawasaki disease (KD). We postulated that variation in TGF-β signaling might be important in KD susceptibility and disease outcome.

Methods and results: We investigated genetic variation in 15 genes belonging to the TGF-β pathway in a total of 771 KD subjects of mainly European descent from the United States, the United Kingdom, Australia, and the Netherlands. We analyzed transcript abundance patterns using microarray and reverse transcriptase-polymerase chain reaction for these same genes, and measured TGF-β2 protein levels in plasma. Genetic variants in TGFB2, TGFBR2, and SMAD3 and their haplotypes were consistently and reproducibly associated with KD susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. A SMAD3 haplotype associated with KD susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (A/G, rs4776338) (P=0.000022; odds ratio, 1.50; 95% confidence interval, 1.25 to 1.81). Pathway analysis using all 15 genes further confirmed the importance of the TGF-β pathway in KD pathogenesis. Whole-blood transcript abundance for these genes and TGF-β2 plasma protein levels changed dynamically over the course of the illness.

Conclusions: These studies suggest that genetic variation in the TGF-β pathway influences KD susceptibility, disease outcome, and response to therapy, and that aortic root and coronary artery Z scores can be used for phenotype/genotype analyses. Analysis of transcript abundance and protein levels further support the importance of this pathway in KD pathogenesis.

Figure 1

TGF-β signaling pathway TGF-β is secreted in a latent form and activation is mediated by several molecules including furin and emilin. Active TGF-β peptides (TGFB1, 2, 3) bind to the Type II receptor (TGFBR2), which recruits and activates Type I receptors such as TGFBR1 or ACVRL1. The activated Type I receptor phosphorylates receptor-specific SMAD molecules: TGFBR1 phosphorylates SMAD2 and 3, and ACVRL1 phosphorylates SMAD1, 5 and 8. These activated SMADs form a larger complex with Smad4 and translocate to the nucleus where they regulate gene transcription. Accessory receptors such as ENG have a role in the balance of ACVRL1 and TGFBR1 signaling to regulate endothelial cell proliferation. In addition to the classical SMAD signaling pathway, a SMAD-independent pathway using alternative molecules such as MAP3K7 can also mediate TGF-β signaling. TGFB: Transforming growth factor-β, TGFBR: Transforming growth factor-β receptor, EMILIN1: Elastin microfibril interfacer 1, SMAD: SMAD family member, MAP3K7: Mitogen-activated protein kinase kinase kinase 7, ENG: Endoglin, ACVRL1: Activin A receptor type II-like 1, FURIN: Furin

Figure 2

Cohorts and phenotypes analyzed in genetic association studies

Figure 3

Significant SNP locations for TGFB2 (A), TGFBR2 (B) ans SMAD3 (C) Arrows show the location of significant SNPs genotyped in this study. Gene structure and the location of SNPs are shown: boxes= exons and 3′ and 5′ untranslated regions;. Underlined text highlights SNPs with nominal p<0.01. Letter code above SNP rs# refers to type of analysis and cohort: CC: susceptibility in Cohort 1 (Supplemental Table 5), TDT: susceptibility in Cohort 2 (Table 2), CAA3 and CAA4: association with CAA in Cohort 3 and Cohort 4, respectively (Supplemental Table 6), CAZ: association with CA Z-worst (Supplemental Table 7), AoR; association with AoR Z-worst (Supplemental Table 8), IVIG; association with IVIG treatment response (Supplemental Table 9). LD maps are shown for SNPs of interest.

Figure 3

Significant SNP locations for TGFB2 (A), TGFBR2 (B) ans SMAD3 (C) Arrows show the location of significant SNPs genotyped in this study. Gene structure and the location of SNPs are shown: boxes= exons and 3′ and 5′ untranslated regions;. Underlined text highlights SNPs with nominal p<0.01. Letter code above SNP rs# refers to type of analysis and cohort: CC: susceptibility in Cohort 1 (Supplemental Table 5), TDT: susceptibility in Cohort 2 (Table 2), CAA3 and CAA4: association with CAA in Cohort 3 and Cohort 4, respectively (Supplemental Table 6), CAZ: association with CA Z-worst (Supplemental Table 7), AoR; association with AoR Z-worst (Supplemental Table 8), IVIG; association with IVIG treatment response (Supplemental Table 9). LD maps are shown for SNPs of interest.

Figure 3

Significant SNP locations for TGFB2 (A), TGFBR2 (B) ans SMAD3 (C) Arrows show the location of significant SNPs genotyped in this study. Gene structure and the location of SNPs are shown: boxes= exons and 3′ and 5′ untranslated regions;. Underlined text highlights SNPs with nominal p<0.01. Letter code above SNP rs# refers to type of analysis and cohort: CC: susceptibility in Cohort 1 (Supplemental Table 5), TDT: susceptibility in Cohort 2 (Table 2), CAA3 and CAA4: association with CAA in Cohort 3 and Cohort 4, respectively (Supplemental Table 6), CAZ: association with CA Z-worst (Supplemental Table 7), AoR; association with AoR Z-worst (Supplemental Table 8), IVIG; association with IVIG treatment response (Supplemental Table 9). LD maps are shown for SNPs of interest.

Figure 4

(A-D) RT-PCR analysis of transcript abundance in whole blood from KD patients during the acute and convalescent phase and association between genotype and gene expression. Relative transcript abundance levels were normalized by TAF1B for ACVRL1 (A), SMAD1 (B), SMAD3 (C) and TGFBR3 (D). Solid line: subjects with normal coronary arteries, Dashed line: subjects with coronary artery aneurysms. p value by paired t-test. (E-G) Association between acute and convalescent gene expression levels and ACVRL1 rs11169953 (risk allele= G, GG: n=7, GA: n=5, AA: n=2) (E), SMAD1 rs6537355 (risk allele=A, AA: n=9, AG: n=5, no subject had GG genotype). (F), SMAD3 rs4776338 (risk allele= G, AA: n=6, AG: n=5, GG: n=3) (G). p value by Kruskal-Wallis test (E, G) and Mann Whitney test (F).

Figure 5

(A) Plasma levels of TGF-β2 in KD patients during the acute and convalescent phase. Solid line: subjects with normal coronary arteries, Dashed line: subjects with coronary artery aneurysms. p value by paired t-test. (B) Association between acute and convalescent plasma levels of TGF-β2 and rs12029576 in TGF-β2 (risk allele= C, AA: n=7, AC: n=6, no subject had CC genotype). p value by Mann Whitney test.