Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome

Abstract

Ethnic diversity has been long considered as one of the factors explaining why the severe forms of dengue are more prevalent in Southeast Asia than anywhere else. Here we take advantage of the admixed profile of Southeast Asians to perform coupled association-admixture analyses in Thai cohorts. For dengue shock syndrome (DSS), the significant haplotypes are located in genes coding for phospholipase C members (PLCB4 added to previously reported PLCE1), related to inflammation of blood vessels. For dengue fever (DF), we found evidence of significant association with CHST10, AHRR, PPP2R5E and GRIP1 genes, which participate in the xenobiotic metabolism signaling pathway. We conducted functional analyses for PPP2R5E, revealing by immunofluorescence imaging that the coded protein co-localizes with both DENV1 and DENV2 NS5 proteins. Interestingly, only DENV2-NS5 migrated to the nucleus, and a deletion of the predicted top-linking motif in NS5 abolished the nuclear transfer. These observations support the existence of differences between serotypes in their cellular dynamics, which may contribute to differential infection outcome risk. The contribution of the identified genes to the genetic risk render Southeast and Northeast Asian populations more susceptible to both phenotypes, while African populations are best protected against DSS and intermediately protected against DF, and Europeans the best protected against DF but the most susceptible against DSS.

Fig 1. Global ancestry inferred through RFMix when using three parental ancestries (South, Northeast and Southeast Asian) for the global dataset.

Each vertical line represents an individual, and the three colours represent the proportion of the three parental populations in each genome (light orange for South Asian, dark orange for Southeast Asian and blue for Northeast Asian).

Fig 2

Manhattan plots of BMIX analysis in Vietnamese DSS vs Control (A), Thai DSS vs Control (B) and Thai DF vs Control (C) for Northeast and Southeast Asian ancestries. The red line represents the significance threshold. The protein coding genes with significantly associated SNPs are identified.

Fig 3. Worldwide (from the 1000 Genomes database) and Thai dengue cohorts (control, DF and DSS) frequencies for significantly associated haplotypes in the various genes.

A- PLCB4; B- PLCE1; C- MICB; D- CHST10; E- AHRR; F- GRIP1; G- PPP2R5E. The protective and causative haplotypes are highlighted.

Fig 4

Genetic risk for the various worldwide regions by considering an additive model of protective and causative haplotypes/SNPs for DSS (A) and DF (B). Median (middle line), mean (little square), 95% confidence interval (whiskers) and extreme values (crosses) are indicated.

Fig 5

Significantly altered gene expression for PPP2R5E (A), GRIP1 (B) and AHR (C) in Thai dengue cohort along the course of disease from a transcriptome dataset for whole blood[17]. Significant p-values are indicated. D) Scheme of the xenobiotic metabolism signalling pathway (based on Ingenuity database information), highlighting the three nuclear transcription factors: the constitutive active receptor (CAR); the pregnane X receptor (PXR); and the aryl hydrocarbon receptor (AHR).

Fig 6. Confocal imaging of PPP2R5E and NS5 from DENV1 and DENV2.

A. The main link motifs in NS5 proteins from DENV1 and DENV2. B. Subcellular localization of PPP2R5E in Huh7 control cells. C. Subcellular localization of PPP2R5E in Huh7 control and after 24h, 48h and 72h of transfection with DENV2-NS5 protein. D. Subcellular localization of mutated PPP2R5E in Huh7 control and after 24h, 48h and 72h of transfection with DENV2-NS5 protein. E. Subcellular localization of PPP2R5E in Huh7 control and after 24h, 48h and 72h of transfection with DENV1-NS5 protein. Green immunofluorescence indicates PPP2R5E, red indicates NS5, blue flags nucleus. Yellow signals indicate co-localization of NS5 and PPP2R5E, and was obtained by overlapping the two panels. Original magnification, ×630.