OSBPL10, RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans

RESEARCH ARTICLE OSBPL10,RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans Beatriz Sierra 1☯ *, Petr Triska 2,3,4☯ , Pedro Soares 3 , Gissel Garcia 1 , Ana B. Perez 1 , Eglys Aguirre 1 , Marisa Oliveira 2,3,4,5,6 , Bruno Cavadas 2,3 , Be ´atrice Regnault 5 , Mayling Alvarez 1 , Didye Ruiz 1 , David C. Samuels 7 , Anavaj Sakuntabhai 6 , Luisa Pereira 2,3,8 *, Maria G. Guzman 1 1Virology Department, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Pedro Kourı´Institute of Tropical Medicine (IPK),Havana, Cuba, 2i3S - Instituto de Investigac¸ão e Inovac¸ão em Sau ´de, Universidade do Porto, Porto, Portugal, 3Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal, 4Instituto de Ciências Biome ´dicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal, 5Eukaryote Genotyping Platform, Genopole Pasteur Institute, Paris, France, 7Vanderbilt Genetics Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, United States of America, 6Functional Genetics of Infectious Diseases Unit, Pasteur Institute, Paris, France, 8Faculdade de Medicina da Universidade do Porto (FMUP), Porto, Portugal ☯These authors contributed equally to this work. *[email protected] (BS); [email protected] (LP) Abstract Ethnic groups can display differential genetic susceptibility to infectious diseases. The arthropod-born viral dengue disease is one such disease, with empirical and limited genetic evidence showing that African ancestry may be protective against the haemorrhagic phenotype. Global ancestry analysis based on high-throughput genotyping in admixed populations can be used to test this hypothesis, while admixture mapping can map candidate protective genes. A Cuban dengue fever cohort was genotyped using a 2.5 million SNP chip. Global ancestry was ascertained through ADMIXTURE and used in a fine-matched corrected association study, while local ancestry was inferred by the RFMix algorithm. The expression of candidate genes was evaluated by RT-PCR in a Cuban dengue patient cohort and gene set enrichment analysis was performed in a Thai dengue transcriptome. OSBPL10 and RXRA candidate genes were identified, with most significant SNPs placed in inferred weak enhancers, promoters and lncRNAs. OSBPL10 had significantly lower expression in Africans than Europeans, while for RXRA several SNPs may differentially regulate its transcription between Africans and Europeans. Their expression was confirmed to change through dengue disease progression in Cuban patients and to vary with disease severity in a Thai transcriptome dataset. These genes interact in the LXR/ RXR activation pathway that integrates lipid metabolism and immune functions, being a key player in dengue virus entrance into cells, its replication therein and in cytokine production. Knockdown of OSBPL10 expression in THP-1 cells by two shRNAs followed by DENV2 infection tests led to a significant reduction in DENV replication, being a direct functional proof that the lower OSBPL10 expression profile in Africans protects this ancestry against dengue disease. PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 1 / 21 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Sierra B, Triska P, Soares P, Garcia G, Perez AB, Aguirre E, et al. (2017) OSBPL10,RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans. PLoS Pathog 13(2): e1006220. doi:10.1371/journal.ppat.1006220 Editor: Richard J. Kuhn, Purdue University, UNITED STATES Received: December 6, 2016 Accepted: February 5, 2017 Published: February 27, 2017 Copyright: ©2017 Sierra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data has been deposited at the European Genome-Phenome Archive under the accession EGAS00001002276. Funding: The research leading to these results has received funding from the European Commission Seventh Framework Programme [https://ec. europa.eu/research/fp7/index_en.cfm] for the DENFREE project under Grant Agreement no. 282378. PT had a PhD grant from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (http://ec. Author summary Dengue is a concern of worldwide health authorities given the increase on virus and vector dispersions. So far only one traditional GWAS survey has been performed in Vietnamese children. This disease is also epidemic in tropical and subtropical regions of the Americas, where most populations descend from a dynamic admixture between African, European and Native American backgrounds. Empirical evidence claimed that African descent was protective against dengue haemorrhagic phenotype in the Cuban population, and this study is the first to apply admixture mapping to identify candidate genes that confer African protection. We also present evidence that two candidate genes, OSBPL10 and RXRA, are differentially expressed along dengue disease progression in Cuban patients and in a Thai dengue transcriptome dataset, and directly show that knockdown of OSBPL10 gene expression leads to a significant reduction in DENV2 replication. A very important overall result of our work is that it provides a unifying framework for many genes that have been said to be protective in dengue. Our evidence places the LXR/RXR activation pathway at the center of natural dengue protection, and supports pursuing therapeutic techniques involving synthetic ligands of nuclear receptor genes or kinases inhibitors that interact with proteins involved in lipid metabolism. Introduction Dengue is an emerging arthropod-born viral disease caused by the infection with any of the four dengue viruses (DENV-1 to 4). The virus is transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes. Morbidity and mortality associated with severe dengue infection render this disease a major increasing public health problem throughout tropical and subtropical regions. Dengue illness is also attracting awareness in Europe and in the United States as climate change and globalisation enlarge the geographic dispersion of the vector and the viruses [1]. A dengue infection can evolve from a subclinical infection, a relatively mild, selflimited infection known as dengue fever (DF), to the severe disease called dengue haemorrhagic fever (DHF), which may evolve to a life-threatening hypovolemic shock (dengue shock syndrome, DSS [2]). But only a small proportion of antibody-positive individuals develops DHF/DSS, while the vast majority suffers an asymptomatic infection or the mild disease. This differential susceptibility to disease severity indicates that besides immune factors, the host genetics may influence the infection outcome, acting in a complex interplay with viral and environmental factors. Diverse single nucleotide polymorphisms (SNPs) in genes such as HLA-I,HLA-II,TNF-α,IL-10,TGF-β1,FcγRIIa,VDR,CD209and OAS have been associated with symptomatic dengue or considered protective against the disease, in Asian and Latin American populations [3]. Also, MICA and MICB genes have been associated with susceptibility to dengue in Cuba [4], partially overlapping previous results reported in the only genomewide association study (GWAS) performed so far in Vietnamese children, and showing significant association of MICB and PLCE1 genes with DSS [5]. Evidence supporting the impact of human genetic factors on infection outcome also comes from differences between ethnic groups in developing severe DHF/DSS symptoms [6]. As early as in 1906, it was reported that Cuban dark-skinned individuals showed a remarkable resistance against dengue disease compared with light-skinned individuals [7]. This early observation was confirmed during the 1981 Cuban DHF/DSS epidemic of DENV-2 when ethnicity was recognized for the first time as a possible host risk factor, and confirmed afterwards Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 2 / 21 europa.eu/research/mariecurieactions/) under REA grant agreement no. 290344 (EUROTAST) and MO a PhD grant from FCT, The Portuguese Foundation for Science and Technology (https://www.fct.pt/fct. phtml.en) with reference SFRH/BD/95626/2013. I3S is financed by FEDER funds through COMPETE 2020, Portugal 2020, and by Portuguese funds through FCT/Ministe ´rio da Ciência, Tecnologia e Inovac¸ão (POCI-01-0145-FEDER-007274). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. in several other dengue Cuban outbreaks [8]. The low occurrence of dengue disease in Haitians [9] and in African populations [10] adds further support for this ancestry influence. A genetic characterization of 30 ancestry informative markers conducted in the Colombian population [11] confirmed the protective effect of African ancestry against severe dengue outcomes (odds ratios, ORs in 0.963–0.971 interval). Till recently, population structure as occurs in admixed populations was a major confounding factor, requiring strategies for correction of the association p-values [12]. But admixed populations are a great advantage in cases of differential ancestry-conferred susceptibility/ resistance to a disease through the use of admixture mapping [13]. The rationale of admixture mapping is that the ancestry blocks will be distributed at random across the genome, reflecting the admixture proportions of the parental ancestries, except in candidate gene locations where statistically significantly different proportions for the ancestry with higher disease levels will be observed in cases versus controls. It has been shown that this test is statistically more powerful than traditional GWAS [14]: around 250 samples can provide a 60% power to detect a twofold risk due to ancestry, compared to the thousands of samples required in GWAS. In fact, because of the recentness of admixture, the typical ancestry blocks are significantly larger than haplotype blocks, thus lowering the multiple testing burden. This strategy has been successfully applied in African-Americans and Latin-Americans, in association with various diseases, such as asthma [15] and type 2 diabetes [16]. Cuba is advantageous for studies of ancestry-conferred DHF susceptibility/protection. The current Cuban population is mainly derived from the mix of two well-defined ancestral populations: European colonizers, who began to arrive in 1492 from the Iberian Peninsula, followed by other countries; and enslaved Africans, arriving in the 16 th century, mainly from West Africa. The contribution of the first aboriginal Cuban inhabitants, almost totally exterminated during the Spanish conquest, is almost negligible [17]. The Western and Eastern sides of the island show differences in historic settlement, with European descendants concentrating in the capital [17]. DENV experience has also been different among them. After an absence of 40 years, DENV1 was reported in 1977 and transmitted to nearly one-half of the Cuban population. Four years later, DENV2 (Asian origin) infected approximately 25% of the population, and a large DHF/DSS epidemic occurred. In 1997, another Asian DENV2 virus entered the country, producing a local epidemic in the municipality of Santiago de Cuba. In 2001, DENV3 (Asian genotype), was detected in Havana city [18]. In 2006, the circulation of DENV4 was reported in Havana, while DENV3 affected Guantanamo [19]. We conducted a GWAS of 2.5 million SNPs in 274 Cubans, including patients (DF and DHF) of the 2006 dengue epidemic, from Havana (west) and Guantanamo (east) cities, and in geographically matched asymptomatic individuals and population controls. The high level of admixture in Cuba enabled us to apply the first admixture mapping, thus facilitating the identification of candidate markers ethnically associated with dengue infection. Firstly, we conducted a global admixture analysis which allowed us to confirm the statistically significant decrease of African ancestry in the DHF cohort and to fine tune the association analysis in the admixed Cubans (cases and controls were paired to not differ more than 2% in African ancestry), whose statistical burden is 2.6x10 -8 . Secondly, we performed a local ancestry assignment along chromosomes which identified the precise regions (and genes) where the African ancestry was significantly higher in asymptomatic compared with DHF (above 3SD threshold in the difference in African ancestry between controls-cases). The fine-tuned association analysis can identify a few SNPs (located in a small chromosomal region) that confer reasonable individual risk/protection; the local ancestry assignment can identify ancestry-related blocks (medium to big chromosomal regions, depending on the time since admixture) containing SNPs conferring low-medium risk/protection, which would escape the association test. We also performed Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 3 / 21 a functional test consisting in knocking down the expression of one candidate gene by shRNA, followed by DENV infection assays. Results Global ancestry influence in dengue infection outcome The genome-wide Cuban screening confirmed that all individuals in this study have a mixed ancestral composition. The main ancestry backgrounds (Fig 1A for K = 4) derive from Africa (represented by the blue colour) and Europe (red), but the range follows the entire spectrum of admixture, from nearly 0% African and 90% European to the inverse ratio, while the remaining 10% are from Native American (yellow) and East Asian (orange) influences. Comparing the population control groups from Havana and Guantanamo (HC and GC) as references for the two geographical regions, the average proportions of the African component are statistically different (25.2% and 35.3%, respectively; two-tailed Wilcoxon rank-sum test p = 1.43x10 -3 ), identical to published values [20]. The Native American component was 6.5% in Havana and 13.5% in Guantanamo, values statistically significantly different (p = 1x10 -6 ); while the East Asian component was of 1.9% and 0.7% respectively (p = 0.224). A finer description of Cuban ancestry is presented in Supplementary section 1.2 (Figs A-C in S1 Text). This includes confirmation that, despite the statistical differences in the African/European components between Havana and Guantanamo, the sub-structures within those two components in the two cities are identical, not favouring differential migration events into the two parts of the island (Figs D-E in S1 Text). Focusing on the global ancestry among the Cuban cohorts (Fig 1B), the average African ancestry is significantly lower in DHF (22.9%) when compared with DF, controls and especially asymptomatic groups (30.6%, p-value = 0.025; 30.0%, p-value = 0.041; 34.7%, p-value = 0.013, respectively). These results confirmed that African ancestry is protective against DHF phenotype in Cuba, and the odds ratios are very similar to the ones reported in Colombia [11] (Table 1). Nevertheless, the evidence of ancestry influence in dengue was not so straightforward when the samples were divided according to the city of origin: in Havana, the African ancestry is even more significantly lower in DHF (10.3%) compared with DF, controls and especially asymptomatic (24.4%, p-value = 0.009; 25.2%, p-value = 0.015; 33.0%, p-value = 0.002, respectively) and OR even more protective (Table 1); while in Guantanamo, the African averages are statistically identical between all groups (35.3% in controls, 38.1% asymptomatic, 36.4% DHF and 36.0% DF). This complex relation was confirmed by an iterative model (Fig F in S1 Text). Our data show that there is an African protection conferred against DHF in Cuba, but other currently unknown confounding factors render it a complex relation, even in such a geographical restricted scenario as Cuba. Fine-matched corrected population structure followed by association evaluation The fine-matched corrected association tests (Tables C-E in S1 Text) identified the lowest pvalues (10 −6 –10 −7 ) in the DHF comparison (HCG–haemorrhagic comparison group) for six highly linked (Fig 2A; Table I and Figs N-P in S1 Text) SNPs extending for 8,370 bps in chromosome 3, in a region containing the OSBPL10 (oxysterol binding protein-like 10) gene (Fig 2B). These p-values are close to the significant cutoff of 10 −8 . OSBPL10 protein is involved in lipid transport and steroid metabolism. Curiously, the most frequent haplotypes in African and European populations consist totally of the alternative alleles for all six SNPs, and attain Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 4 / 21 Fig 1. The global ancestry in Cuba and its influence on susceptibility to dengue. (A) ADMIXTURE results for four ancestral populations (blue component represents the European ancestry, red the African, yellow the Native American and Orange the East Asian). HC: Havana controls, HA: Havana individuals with asymptomatic infection, HH: Havana DHF cases, HF: Havana DF cases, GC: Guantanamo controls, GA: Guantanamo individuals with asymptomatic infection, GH: Guantanamo DHF cases, GF: Guantanamo DF cases. (B) Box plots for the African ancestry in the Cuban groups: controls; individuals with asymptomatic infection; DF (dengue fever); DHF (dengue haemorrhagic fever). The boxes represent the interquartile range and the whiskers are the 5% and 95% quartiles. The significant p-values for the two-tailed Wilcoxon rank-sum test between pairs of groups are displayed; non-significant ones are not displayed. doi:10.1371/journal.ppat.1006220.g001 Table 1. Odds ratios of the African ancestry influence in DHF phenotype when compared to asymptomatic subjects, in Cuba in general, only Havana city and in Colombia. Odds ratio 1% African ancestry 50% African ancestry 100% African ancestry Cuba 0.979 0.396 0.151 Havana 0.920 0.045 0.012 Colombia*0.962 0.204 0.042 *From [11]. doi:10.1371/journal.ppat.1006220.t001 Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 5 / 21 Fig 2. The relevant region on chromosome 3 containing the OSBPL10 gene (A) Manhattan plot for the association analysis in the 54 fine-matched population structure corrected Cuban pairs of asymptomatic/control versus DHF subjects. (B) The region on chromosome 3, with the haplotype defined by the six significantly associated SNPs indicated by the red box. Genes on the forward sense are indicated in blue; genes on the reverse sense are indicated in light brown. (C) Worldwide frequency of the African (blue), European (red) and other (grey) OSBPL10 haplotypes for populations of the 1000 Genomes project, and also for asymptomatic/control and DHF in Cuba. (D) mRNA expression for homozygous genotypes for African and European OSBPL10 haplotypes in the 1000 Genomes project transcriptome information. doi:10.1371/journal.ppat.1006220.g002 Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 6 / 21 frequencies higher than 50% in each continent (Fig 2C). The odds ratio calculated in DHF is 0.25 [95% CI 0.13–0.47] for the African haplotype (Table 2). By using the 1000 Genomes transcriptome [21], we confirmed that the homozygous individuals for the OBSPL10 African haplotype have a significantly reduced (by half) expression (mean value = 0.145) when compared with the homozygous for the European haplotype (mean value = 0.257; p<0.001; Fig 2D). This indicates that the haplotype affects mRNA expression. We further ascertained that these non-coding SNPs are not located in the inferred promoter, but they can be related with weaker enhancer regions and are recognised by many transcription factors, including STAT and RXRA(Fig Q in S1 Text). Besides the haplotype region, the segment immediately 5’ to it (within the gene) has some SNPs with significant pvalues in the association test that are also regulatory regions in several cell types (Table O and Fig R in S1 Text). Although there is no current good database to check eQTLs in African populations, we used GTEx despite its highly European-biased dataset (84.3%). The six identified OSBPL10 SNPs are not identified in GTEx as eQTLs, but surveying the eQTLs located on that chromosomal region, there is one with frequency differences between African and European populations (rs7642435, at position 32036787; 1000 Genomes frequency in Africa–A = 0.092, G = 0.908; 1000 Genomes frequency in Europe–A = 0.723, G = 0.277), which presents alternative homozygous genotypes associated with our African/European haplotypes: “African” haplotype with rs7642435_GG and “European” haplotype with rs7642435_AA. As can be confirmed in Fig S in S1 Text, rs7642435_GG has a significant reduction of OSBPL10 expression compared with rs7642435_AA, and this SNP is predicted to have an eQTL effect (posterior probability>0.9) in several tissues: artery (aorta, coronary and tibial), brain (nucleus accumbens and caudate), liver, adrenal gland, pancreas, esophagus (muscularis and gastroesophageal junction), stomach, nerve and cells-transformed fibroblasts. The remaining eQTLs in the region are almost fixed in both African and European populations. Possibly, our haplotype association is identifying the rs7642435 eQTL, although more information on African eQTLs is needed in order to ascertain other possible eQTLs in the OSBPL10 region. This extended OSBPL10 segment (haplotype and immediate 5’ region) has probably been under positive selection in the African Yoruban population, as inferred in the Haplotter tool based on HapMap dataset (iHS measure; Fig T in S1 Text). The positive selection of OSBPL10 gene was confirmed in Cuban HCG when applying XP-EHH measure (Fig M and Tables M-N in S1 Text). Therefore, it seems that the OSBPL10 haplotype and its 5’ region, although nonTable 2. Odds ratios of the African OSBPL10 haplotype and RXRA alleles in DHF when compared with asymptomatic/control and tests (identified by Y) where statistical significant evidence was detected for each gene. Gene Haplotype SNP Position Haplotype allele Odds ratio OR 95% confidence interval A B C D E F OSBPL10 African haplotype rs4600849 rs11129475 rs6419811 rs11718700 rs975406 rs7639637 32027672 32030544 32031135 32033248 32035587 32036042 CTGCCC 0.25 [0.13–0.47] Y Y Y Y Y RXRA rs12339163 137205188 G 0.36 [0.17–0.77] Y Y Y rs62576287 137214888 C 0.10 [0.01–0.83] rs3118593 137426334 A 0.44 [0.25–0.77] rs4262378 137515156 G 0.41 [0.24–0.72] rs4424343 137515158 A 0.43 [0.24–0.76] A—Association; B—Admixture mapping; C—Positive selection (XP-EHH and iHS); D—Expression changes in Cuban patients RT-PCR; E—Expression changes in Thai transcriptome dataset; F—Expression changes between African and European haplotypes/genotypes in 1000 Genomes transcriptome. doi:10.1371/journal.ppat.1006220.t002 Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 7 / 21 coding, bear several expression regulatory SNPs with large frequency differences between Africans and Europeans, probably due to selection events. Several other SNPs located on genes (mostly one or two SNPs per gene) reached association p-values of 10 −5 . By checking which SNPs are more frequent in Africa (information from the 1000 Genomes database) and which genes would have differential gene expression between dengue patients and control/convalescent subjects (in the whole blood transcriptome obtained in a Thai dengue dataset [22] and using a linear discriminant analysis effect size method, LEfSe [23], for high-dimensional class comparisons), besides OSBPL10, a few kinases or kinaserelated genes are amongst the most significant genes (Fig G in S1 Text): CAMK1D(calcium/ calmodulin-dependent protein kinase ID), MAPKAPK5 (mitogen-activated protein kinaseactivated protein kinase 5), PIK3AP1 (phosphoinositide-3-kinase adaptor protein 1), SNRK (sucrose nonfermenting related kinase), GNA14 (guanine nucleotide binding protein (G protein), alpha 14) and DAB-1 (dab, reelin signal transducer, homolog 1 (Drosophila)). The non-African set (Fig H in S1 Text) has more variable and generalized functions, dealing with hemostasis (DOCK10),phospholipid binding (PLEKHM1L), protein serine/threonine kinase activity and ribosomal protein S6 kinase activity (RPS6KA2), protein homodimerization activity and HMG box domain binding (OLIG2), sequence-specific DNA binding transcription factor activity and chromatin binding (TSHZ3). Fine-matched corrected population structure followed by admixture mapping The admixture mapping led to the identification of locally enriched African regions across the genome in the asymptomatic/controls (Figs I-K and Tables F-H in S1 Text). One of these regions is located on chromosome 9 (detected at HCG and FCG/fever comparisons), containing the RXRA(retinoid X receptor alpha), COL5A1(collagen type V alpha 1) and FCN2(ficolin (collagen/fibrinogen domain containing lectin) 2) genes. We confirmed from the expression data on Thai dengue patients [22] that only RXRAhas statistically significant altered expression (Fig L in S1 Text), supporting several lines of evidence linking retinoid receptors with infectious diseases and dengue [24]. Detailed description of other significant regions is presented in supplementary material sections 1.5 and 1.6 (Tables K-L in S1 Text). We checked the SNPs with significant association p-values in the RXRA-COL5A1region in the Cuban HCG (Fig 3; Table J in S1 Text), and verified that two locations have higher significant p-values and OR between 0.103–0.436 (Table 2). The most significant p-values are for three intergenic SNPs (rs4262378, rs4424343 and rs3118593) located in inferred enhancers (Table P in S1 Text). The other location is immediately before and in the beginning of RXRA, and contains SNPs rs12339163 and rs62576287 (the latter only exists in Africa with a 9% frequency) placed in poised and weak promoters and weak enhancers. From these various significant SNPs, only rs3118593 is within a promoter and a lncRNA (RP11-473E2.4;Fig 3), and interestingly, according to the GTEx portal, the patterns of expression for RXRAand RP11473E24genes in the various human tissues are totally opposite, with RXRAbeing mainly expressed in liver, muscle, skin and whole blood while almost not expressed in brain and testis, and the other way around for RP11-473E24(Figs U-W in S1 Text). The opposite expression pattern between the RXRAand RP11-473E24genes raises the possibility that the lncRNA silences the expression of RXRA. When checking the RXRAmRNA expression for the typical African and European genotypes in those SNPs (based on the 1000 Genomes transcriptome from lymphoblastoid cell lines), no statistical differences were observed (data not presented). We further performed association tests for the whole RXRA-COL5A1region in the 1000 Genomes transcriptome comparing RXRAhighand low-expression groups within Africans Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 8 / 21 and Europeans. The African test shows that there are 37 significant (at the 1% level) SNPs surrounding RXRAgene, including rs3118593, that are differently frequent between the highwith low-expression groups, and these can be regions for expression regulation of the gene (Fig 3; Table Q in S1 Text). While in Europe, the number of significantly differentiated SNPs is much lower, only six (Fig 3; Table R in S1 Text). A reasonable hypothesis would be that the advantage in Africans is related with a faster control of RXRAexpression. OSBPL10 and RXRA expression in dengue patients and focused enrichment analysis The genetic evidence collected in this work, summed up in Table 2, for the involvement of OSBPL10 and RXRAgenes in the protection against DHF, led us to check their expression in Cuban patients throughout the infection process (Fig 4). The mRNA expression of RXRAwas significantly higher during convalescence (day 30) compared to day 3 (p = 0.027), and also higher than at day 7 after fever onset (not significant). There were no significant differences between days 3 and 7 after fever onset. These results are comparable with the ones for the Thai dataset, where RXRAexpression is significantly decreased in DF and DHF cohorts when Fig 3. The RXRA-COL5A1 region with the most significant SNPs highlighted. Map obtained with the LocusZoom tool, using recombination rate information from the Yoruba reference population. The symbols above the rule represent significant SNPs, for the Cuban data (the pink triangles), the African comparison between individuals having low (n = 6; lower than 10 RPKM) and high (n = 8; higher than 20 RPKM) RXRA expression (green circles), the same for European individuals (n = 42 and n = 39, respectively; blue squares). doi:10.1371/journal.ppat.1006220.g003 Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 9 / 21 Gene expression analysis in patients with dengue illness A group of 20 patients with suspected dengue acute illness (clinical diagnosis was made by an experienced physician in dengue illness) were recruited at the Salvador Allende Hospital, Havana in 2014. Dengue infection was confirmed as described above. General signs and symptoms as well as warning signs (clinical fluid accumulation, mucosal bleeding, restlessness, severe abdominal pain, persistent vomiting as well as thrombocytopenia<100 platelets x 10 9 /L, and 20% or higher increase of haemo-concentration indicated by haematocrit or HTC) were recorded daily from recruitment to discharge. Three serial peripheral mononuclear cell (PBMC) samples were collected during hospitalization, at days 3, 7 and 30, after symptoms onset. DNase-treated total RNA was isolated from PBMC using the RNeasy Mini kit (Qiagen, Hilden, Germany) and evaluated by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Expression levels of OSBPL10,RXRAand β-actin (housekeeping) genes were determined by RT-PCR (LightCycler 2.0 instrument, Roche), using LightCycler RNA Master SYBR Green I kit (Roche) and designed primers (Table B in S1 Text). Each sample was duplicated and the mean Ct values were normalized to an average Ct value of the housekeeping gene. The assay specificity was evaluated by melting curve analysis. Statistical analysis was performed using the non-parametric Wilcoxon-Mann-Whitney U mean rank test for quantitative variables. mRNA expression in other datasets, potential functional role and positive selection Expression of significant genes and genotypes was checked also in public datasets: 1) 465 RNASeq performed in European and African lymphoblastoid cell lines by the 1000 Genomes project [21], expressed in reads per kilobase of exon per million reads mapped (RPKM) and extracted from ArrayExpress (E-GEUV-1); 2) Thai whole blood transcriptome (GDS5093) [22] from nine healthy controls, 28 samples collected between days 2 and 9 after onset of symptoms (acute illness) from secondarily infected patients (18 DF and 10 DHF), and 19 samples collected at convalescence, four weeks or later after discharge. GSEA software [52] was used for gene set enrichment analysis of the LXR/RXR activation pathway in the Thai dengue dataset [22] by considering three sets of genes: lipid metabolism, LXR/RXR activation and NF-kB activation. Haplotter database (http://haplotter.uchicago.edu/) was used to explore recent positive selection through iHS measure (detects selective sweeps at 50–80% frequencies). By using the selscan package [53], another measure of positive selection (XP-EHH—detects selective sweeps above 80% frequency) was applied to the Cuban HCG. The significant SNPs were ascertained for location in regulatory regions by using EPDNew [54] and HaploReg [55] tools. EDPNew comprises organism-specific (we obviously used H. sapiens) transcription start site (TSS) collections automatically assembled from carefully selected mass sequences for genome annotation data. HaploReg annotates non-coding diversity by using LD information from the 1000 Genomes Project that allows one to visualize linked SNPs and small indels along with their predicted chromatin state, conservation across mammals and their effect on regulatory motifs. Cell culture, construction of plasmids and generation of OSBPL10 stable knockdown and mock cell lines Two OSBPL10 short hairpin RNAs (shRNA; TRCN0000147511 and TRCN0000149806 from Sigma Aldrich, St. Louis, MO USA) were cloned in the mammalian expression vector pcDNA3. Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 16 / 21 These shRNAs were previously reported to induce 80–90% reduction of the OSBPL10 protein [56]. The plasmids and the control/mock plasmid were transformed into E.coli competent cells and the positive recombinant colonies were selected and amplified. The extracted recombinant plasmids were digested, subjected to DNA sequencing and purified by QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany), following the recommended procedure. THP-1 cells obtained from American Type Culture Collection (ATCC, Rockville, MD USA) were cultured in complete grow medium RPMI1640 supplemented with 10% fetal bovine serum (FBS), L-glutamine and 0.1 mM MEM non-essential amino acids solution (Thermo Fisher Scientific, Waltham, MA USA) at 37˚C with 5%CO 2 . Transfection of the two shRNA and mock plasmids into THP-1 cells was done by using Lipofectamine LTX Reagent (Thermo Fisher Scientific, Waltham, MA USA) and the recombinant cells (THP1sh1/ OSBPL10, THP1sh2/OSBPL10, THP1/mock) were selected for Neomicin resistance. Dengue virus preparation and infection of THP-1 cells C6/36 cell lines from A.albopictus were grown to confluence, infected at a multiplicity of infection of 0.1 particle forming units (PFU) per cell with the dengue strain DENV-2 A15 Cuba 1981 and cultured in minimum essential medium (MEM) supplemented with 2% fetal calf serum. When a cytopathic effect higher than 50% was noted, culture supernatant was clarified by centrifugation at 10,000 rpm for 30 min at 4 ˚C. BHK21, clone 15 cell line was used for virus titration as previously described [57]. DENV-2 A15 Cuba titer was of 2x10 6 PFU/ml. Absence of lipopolysaccharide contamination in the viral preparations was confirmed by the Limulus Amebocyte Lysate test (BioWhittaker Inc., Walkersville, MD USA). THP-1 cells were infected with dengue virus at a multiplicity of infection (MOI) of 0.1. The virus inoculum at a 70–90% confluence was incubated in serum-free medium at 37˚C for 1h. The unadsorbed viruses were removed by washing the cells three times with plain medium. The dengue-virus-infected and non-infected cells were replenished with fresh complete medium and incubated for another 72 hours. OSBPL10 mRNA expression analysis and dengue viral load measurement by qRT-PCR DNase-treated total RNA was isolated from all THP-1 cell variants by means of RNeasy Mini kit (Qiagen, Hilden, Germany) and evaluated by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The cDNA was synthesized from mRNA with poly(dT) primers and Superscript II reverse transcriptase (Life Technologies, Rockville, MD, USA) and quantified by real-time PCR analysis using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA). Samples were analyzed in triplicates for the expression of OSBPL10, as well as for the housekeeping gene β-actin. And each experience was repeated three times (nine values for each condition). Specific expression was calculated in relation to that of β-actin, by using the delta/delta Ct method as recommended by ABI. RNA from 140 μl of OSBPL10 silenced cell line control THP-1 supernatant or cell culture lysate was extracted, using the QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) and amplified with TaqMan Assay. Standard curves were obtained with titrated DENV-2 supernatants serially diluted from 10 6 to 10 PFU/mL. The standard curve obtained by serial dilution of titrated DENV-2 supernatants had a -3.4 slope, the amplification efficiency was of 92.2% and the detection limit was estimated at 10 PFU equivalents/mL. We performed quantitative RT-PCR analyses of viral mRNA isolated from cellular extracts (intracellular) and culture supernatants (extracellular). Intracellular RNA levels indicate viral replication, whereas extracellular viral RNA is a measure of released viral particles in the culture supernatant. Admixture mapping in Cuban individuals infected by dengue PLOS Pathogens | DOI:10.1371/journal.ppat.1006220 February 27, 2017 17 / 21 Supporting information S1 Text. Supporting information containing 27 figures and 19 tables. (DOCX) Acknowledgments We dedicate this paper to the memory of Professor Gustavo Kourı ´. We thank all the Cuban individuals from Havana and Guantanamo cities who agreed to participate in this study. The authors express thanks to Dr Torreblanca from the Centre of Hygiene and Epidemiology of Guantanamo City, to Dr. Raiza Martinez from the Salvador Allende Hospital, and to Dr. Rosa Martinez and technicians Barbara Marrero and Jose Rodriguez from the Pedro Kourı ´Tropical Medicine Institute, for their support in the samples collection. The authors also thank the technician Laure Leme ´e, from the Eukaryote Genotyping Platform Institut Pasteur, for her helpful support with the genotyping. Thanks also to Dr. Daniel Limonta for his helpful reviewing of this paper. 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