Genome-wide association study identifies susceptibility loci for dengue shock syndrome at MICB and PLCE1.

Hypovolemic shock (dengue shock syndrome (DSS)) is the most common life-threatening complication of dengue. We conducted a genome-wide association study of 2,008 pediatric cases treated for DSS and 2,018 controls from Vietnam. Replication of the most significantly associated markers was carried out in an independent Vietnamese sample of 1,737 cases and 2,934 controls. SNPs at two loci showed genome-wide significant association with DSS. We identified a susceptibility locus at MICB (major histocompatibility complex (MHC) class I polypeptide-related sequence B), which was within the broad MHC region on chromosome 6 but outside the class I and class II HLA loci (rs3132468, P(meta) = 4.41 × 10(-11), per-allele odds ratio (OR) = 1.34 (95% confidence interval: 1.23-1.46)). We identified associated variants within PLCE1 (phospholipase C, epsilon 1) on chromosome 10 (rs3765524, P(meta) = 3.08 × 10(-10), per-allele OR = 0.80 (95% confidence interval: 0.75-0.86)). We identify two loci associated with susceptibility to DSS in people with dengue, suggesting possible mechanisms for this severe complication of dengue.

Introduction

Dengue is an acute systemic viral infection caused by one of four serotypes of dengue (DEN) virus and is globally the commonest mosquito-borne infection after malaria [1]. The burden of dengue is growing, with an estimated 100 million infections now occurring annually and with 2.5 billion people living in areas at risk of transmission. A wide spectrum of disease manifestations is seen, ranging from subclinical infection to severe and fatal disease. Severe dengue in children is characterised by an increase in vascular permeability that leads to life-threatening hypovolemic shock (dengue shock syndrome-DSS). This is often accompanied by thrombocytopenia and haemostatic dysfunction, which may result in severe bleeding. Children are at greatest risk of developing DSS but with careful supportive care the case fatality rate is less than 1% [2]. In southern Vietnam, serological studies have estimated the population based exposure to dengue virus infection to reach 85% by the end of childhood (15 years old) [3], while the incidence of DSS is estimated to occur at less than 1% of exposed individuals [2] (see “The use of population controls” in the Methods section). A host genetic basis to susceptibility to severe dengue has been alluded to in epidemiological studies, and various candidate gene studies of modest sample sizes have been performed [4-8].

To estimate the genetic contribution underlying severe dengue, we genotyped 2,118 DNA samples from Vietnamese children with established or incipient DSS and 2,089 cord blood controls in a genome-wide association study (GWAS). After exclusion of samples for discrepancies between clinical and genetically inferred gender, relatedness or for per-sample call rates of less than 95 percent (Supplementary Figure 1), there were 2,008 DSS cases and 2,018 controls available for analysis. The clinical and virological characteristics of the case population are described in Supplementary Table 1. A total of 657,366 SNPs were initially included within the Illumina 660W Beadchip used for genome-wide genotyping. After various stringent QC exclusions (Supplementary Figure 2), a total of 481,342 SNPs were retained for downstream association analysis.

Upon conducting the routine GWAS statistical tests (see Statistical findings in the Methods section), detailed examination of the overall scan results revealed strong evidence of disease association at two distinct loci; (Figure 1) MICB on Chromosome 6 and PLCE1 on Chromosome 10, both represented by SNPs which were close to the formal threshold for genome-wide significance (P = 5.38 × 10−8 for MICB rs3132468 and P = 5.84 × 10−8 for PLCE1 rs3740360) (Table 1). Together with the SNPs at MICB and PLCE1, a total of 85 SNPs exceeded P < 10−4 on single SNP analysis (Supplementary Table 2). We were able to design assays for 72 out of these 85 SNPs using the Sequenom Mass-Array platform. The remaining 13 SNPs in the broad MHC region were refractory to assay design, thus necessitating ABI Taqman assays to be designed for the sentinel SNP at MICB (rs3132468) and rs3134899 (also within MICB; GWAS P = 1.03 × 10−4, OR = 1.31). We then genotyped these 74 SNPs (72 non-MHC SNPs and two SNPs within MICB) in a replication sample of 1,824 DSS cases and 3,019 controls. We applied the same GWAS QC filters for the replication set: five SNPs had poor genotyping clusters and were excluded from analysis (Supplementary Table 2), and 132 samples (87 cases and 85 controls) had per-sample call-rates of less than 95 percent; these were excluded from further analysis. This left 69 SNPs to be analyzed in 1,737 cases and 2,934 controls for the replication stage. In keeping with the GWAS observations, the strongest evidence of association was observed with SNPs at MICB (rs3132468, Prepl = 9.32 × 10−5 and rs3134899, Prepl = 0.0082) and PLCE1 (3 SNPs with Prepl ranging from 5.23 × 10−4 to 1.6 × 10−4, Table 1). Using inverse-variance weights, data from both the GWAS and replication cohorts (N = 3,745 DSS cases and N = 4,952 controls) were combined in formal meta-analysis, and this revealed strong evidence of association with rs3132468 at MICB (P = 4.41 × 10−11; per-allele odds ratio (OR) = 1.34, [1.23 - 1.46]) and 7 SNPs at PLCE1 (4.18 × 10−9 ≤ P ≤ 3.08 × 10−10; 0.75 ≤ OR ≤ 0.87, Table 1). To aid in refining the original signal of association, we performed imputation analysis at regions flanking both loci (Chr. 6: 30 - 32 Mb, and Chr. 10: 95.5 - 96.5 Mb). This did not reveal signals of association over and above that of the directly genotyped SNPs. The associations observed at MICB and PLCE1 were not specific to any Dengue virus serotype on subgroup analysis of viral serotype, nor were they associated with the degree of thrombocytopenia or the degree of clinical shock (data not shown).

Figure 1

Manhattan plot showing directly genotyped SNPs plotted according to chromosomal location (X-axis, with −Log10P-values (Y-axis) derived from the trend test. The lower horizontal dotted line indicates the threshold for bringing SNPs forward to the replication stage (P < 10−4). SNPs surpassing P < 10−8 (upper horizontal dotted line) on combined analysis of both GWAS and replication data are reflected by red dots, and gene names are given for these loci. SNPs in MICB and PLCE1 have significant associations.

Table 1

Association analysis between Dengue shock syndrome and SNP genotypes at MICB and PLCE1.

Gene/Marker(Alleles)	Chromosome(Position)	Stage	MAFCases	MAFControls	OR	P	ORmeta(95% CI)	P meta
MICB/rs3132468	6	GWAS	0.176	0.132	1.41	5.39 × 10−8
(C/T)	31583465	Replication	0.163	0.134	1.27	9.32 × 10−5	1.34 (1.23 -1.46)	4.41 × 10−11
MICB/rs3134899	6	GWAS	0.130	0.102	1.31	1.09 × 10−4
(G/A)	31581265	Replication	0.114	0.096	1.20	0.0082	1.26 (1.14 -1.38)	4.08 × 10−6
PLCE1/rs3765524	10	GWAS	0.249	0.300	0.77	2.68 × 10−7
(T/C)	96048288	Replication	0.265	0.302	0.83	1.60 × 10−4	0.80 (0.75 -0.86)	3.08 × 10−10
PLCE1/rs2274223	10	GWAS	0.250	0.303	0.77	1.19 × 10−7
(G/A)	96056331	Replication	0.267	0.300	0.85	5.23 × 10−4	0.81 (0.75 -0.86)	6.89 × 10−10
PLCE1/rs3740360	10	GWAS	0.219	0.271	0.75	5.84 × 10−8
(C/A)	96015481	Replication	0.242	0.273	0.85	0.0012	0.80 (0.75 -0.86)	1.15 × 10−9
PLCE1/rs12263737	10	GWAS	0.250	0.301	0.77	3.73 × 10−7
(A/G)	96034903	Replication	0.266	0.300	0.84	3.95 × 10−4	0.81 (0.75 -0.87)	1.22 × 10−9
PLCE1/rs11187842	10	GWAS	0.219	0.269	0.76	1.19 × 10−7
(T/C)	96042501	Replication	0.240	0.271	0.85	0.0011	0.80 (0.75 -0.86)	1.78 × 10−9
PLCE1/rs753724	10	GWAS	0.219	0.269	0.76	1.28 × 10−7
(T/G)	96041407	Replication	0.242	0.272	0.85	0.0012	0.81 (0.75 -0.86)	2.27 × 10−9
PLCE1/rs3781264	10	GWAS	0.229	0.278	0.77	3.43 × 10−7
(G/A)	96060365	Replication	0.250	0.280	0.85	0.0011	0.81 (0.76 -0.87)	4.18 × 10−9

MAF cases: Minor allele frequency in DSS cases

MAF controls: Minor allele frequency in the controls

OR: Odds of DSS per-copy of the minor allele

P: P-value using the 1 degree of freedom score-test.

ORmeta : Odds ratio for the combined GWAS and replication cohorts.

Pmeta: P-value for the combined GWAS and replication cohorts.

95% CI: 95% confidence interval for the OR.

Phet: Heterogeneity P-value.

GWAS: Sample size of 2,008 DSS cases and 2,018 cord blood controls.

Replication: Sample size of 1,737 DSS cases and 2,934 cord blood controls.

Found within the broad Major Histo-Compatability (MHC) locus, MICB lies just outside both the type I and type II HLA regions, ~140,000 base-pairs centromeric to the nearest Class I gene (HLA-B) and more than 1 million base-pairs away from the nearest Class II gene (HLA-DR). Apart from the peak signal at rs3132468 which was observed directly within MICB, twelve other SNPs in this region also showed association signals exceeding P < 10−4 on single-SNP analysis. We thus performed conditional analysis to assess the independence of the association observed at MICB rs3132468 from that of the nearby genes. Although the most significant SNP from the GWAS (rs3132468) could account for the majority of the association signal across the locus, we observed residual signals of association (0.0003 < P < 0.05) with SNPs near the vicinity of HLA-B and HLA-C as well as other neighboring genes (Supplementary Figure 3). These residual associations indicate that definitive identification of MICB as a gene associated with DSS could be complicated by its location within the broad MHC region, which is known for its extensive linkage disequilibrium (LD) spanning multiple genes (Supplementary Figure 4). This precludes definitive identification of the causative gene without extensive further fine-mapping and re-sequencing. With regards to PLCE1 on Chromosome 10, association analysis conditioning for the lead SNP (rs3743060, directly genotyped) did not reveal any secondary signals of association (Supplementary Figure 5), which suggests that the lead SNP -or any of its close correlates in complete LD with it and confined within their distinct genomic region (Supplementary Figure 6)- best explains the association signal at the locus. We did not observe any evidence of epistasis between SNPs at MICB and PLCE1 (P = 0.11).

MICB appears to be a promising candidate based on the present strength of the statistical associations observed in the Chromosome 6 hit region. MICB encodes for MHC class I polypeptide-related sequence B, an inducible activating ligand for the NKG2D type II receptor on natural killer (NK) and CD8+ T cells [9-10] Ligation of NKG2D by MICB stimulates anti-viral effector functions in NK cells including cytokine expression and the cytolytic response [11]. We have previously reported that MICB, together with other genes associated with NK cell activation, are highly expressed in the leukocytes of acute dengue patients [12]. We therefore propose the association between the MICB rs3132468 genotype and susceptibility to severe dengue might reflect altered or dysfunctional NK and/or CD8+ T cell activation early in infection that results in a higher viral burden in vivo, a recognized factor in clinical outcome [13-14]. The recent finding that a SNP near the closely related MICA gene (rs2596542) is associated with Hepatitis C virus induced hepatocellular carcinoma is suggestive of a pivotal role for MIC proteins in the pathogenesis of these Flaviviridae infections [15].

Mutations within PLCE1 are associated with nephrotic syndrome [16]. Nephrotic syndrome is a kidney disorder in which dysfunction of the glomeruli basement membrane results in proteinuria and hypoproteinemia that when severe leads to reduced vascular oncotic pressure and edema. These elements of nephrotic syndrome have striking similarities with severe dengue and suggest an important role for PLCE1 in maintaining normal vascular endothelial cell barrier function. In summary, our study identifies genetic variants in MICB and PLCE1 as being associated with severe dengue.