Replication and meta-analysis of GWAS identified susceptibility loci in Kawasaki disease confirm the importance of B lymphoid tyrosine kinase (BLK) in disease susceptibility

Abstract

The BLK and CD40 loci have been associated with Kawasaki disease (KD) in two genome-wide association studies (GWAS) conducted in a Taiwanese population of Han Chinese ancestry (Taiwanese) and in Japanese cohorts. Here we build on these findings with replication studies of the BLK and CD40 loci in populations of Korean and European descent. The BLK region was significantly associated with KD susceptibility in both populations. Within the BLK gene the rs2736340-located linkage disequilibrium (LD ) comprising the promoter and first intron was strongly associated with KD, with the combined results of Asian studies including Taiwanese, Japanese, and Korean populations (2,539 KD patients and 7,021 controls) providing very compelling evidence of association (rs2736340, OR = 1.498, 1.354-1.657; P = 4.74×10(-31)). We determined the percentage of B cells present in the peripheral blood mononuclear cell (PBMC) population and the expression of BLK in the peripheral blood leukocytes (leukocytes) of KD patients during the acute and convalescent stages. The percentage of B cells in the PBMC population and the expression of BLK in leukocytes were induced in patients in the acute stage of KD. In B cell lines derived from KD patients, and in purified B cells from KD patients obtained during the acute stage, those with the risk allele of rs2736340 expressed significantly lower levels of BLK. These results suggest that peripheral B cells play a pathogenic role during the acute stage of KD. Decreased BLK expression in peripheral blood B cells may alter B cell function and predispose individuals to KD. These associative data suggest a role for B cells during acute KD. Understanding the functional implications may facilitate the development of B cell-mediated therapy for KD.

Figure 1. Per-cohort analysis of BLK rs2736340.

Boxes indicate the odds ratio for each cohort, and horizontal lines denote the 95% confidence interval for the corresponding odds ratio. Diamonds represent summary odds ratios for the respective meta-analyses. Solid vertical line indicates an odds ratio of 1.0, and dashed vertical line denotes the odds ratio obtained from meta-analysis of all samples.

Figure 2. B cell population in peripheral blood mononuclear cells (PBMC) and BLK expression in peripheral blood leukocytes (PBLs) induced at the acute stage of KD.

(A) PBMCs were stained with anti-CD19 or anti-CD3 monoclonal antibodies, and the percentage of CD19⁺ B cells and CD3⁺ T cells in samples taken from a patient at acute and convalescent stages of KD and from a healthy control were determined by multicolor flow cytometry. (B) Levels of BLK expression were determined by real-time RT-PCR, and levels in KD patients were compared to those in fever controls. Values are expressed as mean ± standard error (SE). RNA was harvested from PBLs from KD patients at different stages of disease development or from age-matched fever controls. KD1, n = 20, before IVIG treatment (within 24 h before IVIG treatment); KD2, n = 12, after IVIG treatment (3–7 days after IVIG treatment); and KD3, n = 10, convalescence stage (3 weeks after IVIG treatment). FC, n = 19, fever controls.

Figure 3. Genotypes of rs2736340 are associated with BLK expression and down stream signaling of B cell receptor in B cell lines established from KD patients and in B cells purified from the acute stage of KD patients.

(A) BLK expression was detected by real-time RT-PCR, and expression from patients with T/T or T/C genotypes was compared to that from patients with C/C genotypes. Values are mean ± SE (C/C, n = 4; T/T, n = 5; and T/C, n = 5). (B) BLK expression and ERK1/2 expression and activation were detected by Western blot. BLK, antibody against BLK; ERK1/2, antibody against p44/p42 ERK1/2; p- ERK1/2, antibody against phospho-p44/p42 ERK1/2; GAPDH, antibody against GAPDH.