A genome-wide association study identifies novel and functionally related susceptibility Loci for Kawasaki disease

Abstract

Kawasaki disease (KD) is a pediatric vasculitis that damages the coronary arteries in 25% of untreated and approximately 5% of treated children. Epidemiologic data suggest that KD is triggered by unidentified infection(s) in genetically susceptible children. To investigate genetic determinants of KD susceptibility, we performed a genome-wide association study (GWAS) in 119 Caucasian KD cases and 135 matched controls with stringent correction for possible admixture, followed by replication in an independent cohort and subsequent fine-mapping, for a total of 893 KD cases plus population and family controls. Significant associations of 40 SNPs and six haplotypes, identifying 31 genes, were replicated in an independent cohort of 583 predominantly Caucasian KD families, with NAALADL2 (rs17531088, p(combined) = 1.13 x 10(-6)) and ZFHX3 (rs7199343, p(combined) = 2.37 x 10(-6)) most significantly associated. Sixteen associated variants with a minor allele frequency of >0.05 that lay within or close to known genes were fine-mapped with HapMap tagging SNPs in 781 KD cases, including 590 from the discovery and replication stages. Original or tagging SNPs in eight of these genes replicated the original findings, with seven genes having further significant markers in adjacent regions. In four genes (ZFHX3, NAALADL2, PPP1R14C, and TCP1), the neighboring markers were more significantly associated than the originally associated variants. Investigation of functional relationships between the eight fine-mapped genes using Ingenuity Pathway Analysis identified a single functional network (p = 10(-13)) containing five fine-mapped genes-LNX1, CAMK2D, ZFHX3, CSMD1, and TCP1-with functional relationships potentially related to inflammation, apoptosis, and cardiovascular pathology. Pair-wise blood transcript levels were measured during acute and convalescent KD for all fine-mapped genes, revealing a consistent trend of significantly reduced transcript levels prior to treatment. This is one of the first GWAS in an infectious disease. We have identified novel, plausible, and functionally related variants associated with KD susceptibility that may also be relevant to other cardiovascular diseases.

Figure 1. Quantile-Quantile Plot of Allelic Distributions.

Allelic association analysis of expected versus observed P-values of 223,922 SNPs in 107 KD cases and 134 controls. Red dots showing deviations from the line of equality indicate either that the theoretical distribution was incorrect, or that the sample was contaminated with values generated by a true association.

Figure 2. Selection and Verification of SNPs And Haplotypes from the GWAS Analysis.

GWAS SNPs and haplotypes were ranked by P-value and the top 1101 (of 14,065 total associated) SNPs and 35 (of 3,549 non-overlapping total associated) haplotypes were carried through to the follow-up stage. Numbers in parenthesis indicate the number of variants selected from the GWAS by each method of selection that were subsequently replicated in the family-based study.

Figure 3. Linkage Disequilibrium Plot of a Region of NAALADL2 Containing the Most Significantly Associated Variants.

The upper portion shows -log (p-values) of tagging SNPs (r²>0.8) of the polymorphisms, initially identified in our GWAS (rs2861999) and replication study (rs17531088), which are highlighted in blue. D′ values are shown inside each diamond. Red diamonds without a number represent D′ = 1.

Figure 4. Putative Gene Network Derived from Ingenuity Pathway Analysis Software.

Edges are displayed with labels that describe the nature of the relationship between the nodes. The lines between genes represent known interactions, with solid lines representing direct interactions and dashed lines representing indirect interactions. Nodes are displayed using various shapes that represent the functional class of the gene product (see legend). Blue highlighting indicates GWAS associated genes and non-highlighted genes are those identified by IPA. Genes which showed differential transcript levels in acute versus convalescent KD are colored red if the level was significantly higher during acute KD, green if the level was significantly lower and yellow if there was no significant change, with color intensity related to fold change. Genes not colored were not measured.