DC-SIGN (CD209) Promoter -336 A/G Polymorphism Is Not Associated with Dengue Fever at Burkina Faso, West Africa ()
1. Introduction
Dengue fever is widespread in the tropics and subtropics regions. It is the first public health problem caused by arboviruses. Around 40% - 50% or 3.9 billion people in 128 countries are exposed to the Dengue Virus (DENV). Each year, there are 390 million cases of dengue fever with 96 million presenting symptoms and more than 3000 deaths in the world [1] [2]. Recently, outbreaks of the Dengue Fever (DF) epidemic were reported in many European countries, but also in Africa. In Burkina Faso, 1061 probable cases and 15 deaths were reported in 2016.
In August 2019, Burkina Faso once again experienced cases of DF. Some clinical cases were observed in different hospitals in the city of Ouagadougou, the capital and its surroundings. In their study, Ouattara et al. reported a prevalence of 23.5% in 2016 and 13.3% in 2017 [3]. In 2016, the serotypes DENV-1, DENV-2, DENV-3 and even DENV-4 were incriminated in dengue infections [4].
The onset of the severe form of dengue is due to increased endothelial dysfunction and vascular leakage. It could be explained by an increase in viremia, but also by the phenomenon of antigenic sin linked to the genetics of the host [5]. As the vaccine or effective antiviral therapy is not yet available to everyone to prophylactically or therapeutically treat DENV infection, the incidence of dengue is increasing globally around the world, especially in endemic areas [6].
Dendritic Cell-Specific Intercellular adhesive molecule-3-Grabbing Non-integrin (DC-SIGN), or Cluster of Differentiation CD209 is a C-type lectin receptor found on the surface of macrophages. CD209 plays a major role in the pathogenesis of HIV [7], HCV [8], Ebola [9] and dengue [10]. DC-SIGN also mediates the migration of DCs on the endothelium by interacting with ICAM-2. The binding of ligands to DC-SIGN induces internalization from the cell surface and is targeted to late endosomal or lysosomal compartments for processing and subsequent major histocompatibility complex Class II presentation to T cells. DC-SIGN has an intracellular domain that enables the activation of signal transduction pathways that affects the maturation of DCs. Moreover, ligand binding by DC-SIGN also leads to toll-like receptor signaling and subsequent cytokine responses [11]. The gene coding for DC-SIGN, CD209, is located in chromosome 19p 13.3 and has six exons. Complex alternative splicing events in DC-SIGN pre-mRNA generate an array of transcripts that codes for five major groups of isoforms of DC-SIGN. These isoforms include membrane-associated, soluble and truncated forms of DC-SIGN [12]. The gene has several Single Nucleotide Polymorphisms (SNPs) that affect the expression of DC-SIGN. SNPs at positions −871 (rs735239), −336 (rs4804803) and −139 (rs2287886) in the promoter region of the CD209 gene have been investigated for their association with susceptibility to infectious diseases. However, in Burkina Faso, there are yet no studies in the literature showing the influence of the interaction of CD209 receptors on the development of dengue fever. Therefore, the aim of this study is to characterize the polymorphisms of the DC-SIGN (−336 A/G, rs4804803) gene and their association with the immunopathogenicity of dengue fever in Ouagadougou.
2. Materials and Methods
2.1. Ethics Statement and Subjects of Study
A total of three hundred forty-one subjects, patients of all ages, including children and blood donors from all professions and social categories. The blood samples were taken in the laboratories of the Saint Camille Hospital in Ouagadougou (HOSCO), the National Blood Transfusion Center (CNTS) and the Pietro-Annigoni Biomolecular Research Center (CERBA). This was a case-control analytical study realized from June to December 2018.
The study was approved by the ethics committee for health research of the Ministry of Health of Burkina Faso under number: 2018-5-052 of May 16, 2018 and a written informed consent was obtained from the study participants before blood collection. All the participants were not related to each other and were living in or around Ouagadougou.
All patients seen for consultations during the sample collection period and presenting at least two signs suggestive of dengue fever were included. In addition, voluntary blood donors received during the collection period were also included with no known history of dengue. Patients with already know another symptomatic pathology were not included in this study.
2.2. Dengue Virus Diagnostic
Serological markers for DENV were detected using Dengue Duo Comb Test Kits (Abon Biopharm Guangzhou, Co., Ltd. China). The AgNS1, IgM and IgG were detected directly from blood samples obtained by taking venous blood from the bend of the elbow. The results were read between 15 and 20 minutes.
2.3. Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) Diagnostic
We used a rapid diagnostic test “Determines HIV1/2” and SD-Bioline using immune-chromatography as a principle. We used a cobas e 411 automatic analyzers for the determination of AgHbs and Anti-HCV from plasma containing stored in EDTA tube. For HCV, the kit consisted of a vial of microparticles lined with streptavidin “M”, a vial of R1 containing biotinylated HCV antigen, and R2 consisting of ruthenylated HCV antigen.
2.4. Genotyping of CD209 (−336 A/G, rs4804803)
Genotyping of CD209 rs4804803 SNP Genomic DNA was isolated from EDTA-anticoagulated blood sample using the commercial kit called “QIAamp® DNA Mini Kit” according to the manufacturer’s protocol. DNA purity and concentration were determined using a Biodrop (Isogen Life Science, NV/S.A, Temse, Belgium). Approximatively 100 ng/μl of genomic DNA was used to amplify DC-SIGN gene using the rT-PCR method as previously described [13]. Genotyping for the CD209 variant (−336 A/G, rs4804803) was carried out using Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, 59-USA). The primer sequences were 3’-GGACAGTGCTTCCAGGAACT-5’-(forward) and 5’-TGTGTTACACCCCCTCCACTAG-3’ (reverse). The TaqMan minor groove binder probe sequences were 5’-TACCTGCCTACCCTTG-3’- and 5’CTGCCCACC CTTG-3’. The probes were labeled with the TaqMan fluorescent dyes VIC and FAM, respectively. The PCR was conducted in total volume of 15 µL using the following amplification protocol: denaturation at 95˚C for 10 min, followed by 40 cycles of denaturation at 94˚C for 20 s, 60˚C annealing and extension for one minute [13]. After the PCR, the genotype of each sample was determined by measuring the allele-specific fluorescence in the ABI Prism 7500 Sequence Detection System, using SDS 1.1 software for allele discrimination (both applied biosystems).
2.5. Statistical Analysis
Genotype frequency distributions were tested for their confirmation to Hardy-Weinberg equilibrium using the Chi-square test. Comparison of allele and genotype frequencies between different study groups were carried out using the Chi-square test or Fisher’s exact test as appropriate. SPSS program was used to calculate the P-value with Yate’s correction and Odds Ratio (OR) with 95% interval Confidence Limits (CIs). Haplotype frequencies were inferred and compared between the study groups.
3. Results
3.1. Study Population
The study population consisted of 341 subjects with 208 patients presenting clinical signs of dengue fever which were confirmed by diagnostic and 133 Healthy Controls. The percentage of men was 61.88% (211/341) and 38.12% (130/341) for women. Among 208 dengue virus patients, women were the less represented (39.90%). The youngest was 4 years old and the majority had an age between 20 to 40 years. A highest frequency of dengue fever (77.42%) was noted in patients with age between 20 to 40 years. 1.03% of the study population was positive for HIV and concerning hepatitis, 06.15% were carriers of HBV and 1.94% HCV (Table 1).
3.2. Frequencies of CD209 Genotype in Dengue Cases and Healthy Controls
In this study of polymorphism of DC-SIGN gene, genotype distribution was in Hardy-Weinberg equilibrium in both patients and controls (P-value > 0.05). Genotypes AG were more frequent than genotypes AA and GG in patients and controls. The genotype frequencies of CD209 gene are presented in Table 2.
3.3. Allele Frequencies of CD209 Gene Polymorphisms in Dengue and Healthy Controls
Table 3 presented frequencies of the DC-SIGN allele at −336 loci in 180 Dengue Fever (DF) patients and 128 controls. The frequency of allele A were higher to allele G, however statistical analyses showed that there is no significant difference between genotypes AG, GG and AA in patients and controls.
4. Discussion
This study investigated the role of host genetics on dengue susceptibility by determining polymorphisms of the gene encoding DC-SIGN among a Burkinabè population representative of the dynamic one. On the serological level, the study population presents a sex ratio (1.62) that is unfavorable to women, which does
![]()
Table 1. Serogical characteristic of the study population.
![]()
Table 2. Distribution of the CD 209 - 336 A/G (rs4804803) genotypes frequency in patients with dengue infection and controls the study population.
![]()
Table 3. Association between polymorphisms and risk of dengue.
not contradict the CNTS statistics [14] [15]. With a maximum rate of DENV infection in the 20 - 40 age group, our data are not seen as a contradiction because it is the most active part of the population and therefore the most exposed to DENV. Also the serological analysis linked to HIV, HBV and HCV presents us with rates relatively close to those national with a rate of co-infection of HBV which peaks at 6.15% [16].
Although no DC-SIGN −336 (rs4804803) genotype is significantly associated with the development of dengue, we are able to note that the frequency of the heterozygote is lower in dengue cases than in controls (45.00%/52.34%) which is not the case in the Thai population where it is the GG and AG genotype frequencies which are low and associated with an increased risk of dengue [17] [18]. While another study had, it showed that it is rather homozygous AA that would be associated with protection against the development of dengue On the other hand, in the same scope as the present study; a Brazilian study proposed that polymorphisms in the CD209 gene were not associated with dengue fever [13]. The difference in results between different studies could be due environment interactions, ethnicity-specific effects, and differences in circulating genotypes of different serotypes of DENV.
Dendritic Cells (DCs) are known to be the most powerful antigen presenting cells so far. It could not only initiate primary immune response, but down-regulate immune reaction as well [15]. DCs play an important role in maintaining immune homeostasis for their distinguished immune regulatory capability. Also, DCs are initial factors in auto-immune diseases, and play a key role in immune escape of pathogens and tumors. It is known that immune regulatory capability of DCs is closely related to pattern recognition and immune regulation of the receptors on DC surface [5] [15]. DC-SIGN recognizes several pathogens, such virus like as HIV, Dengue, West Nile virus [10] [13] [19] [20] [21] [22], tuberculosis [19]. Thus in the case of Ebola virus infection, a highly contagious and deadly virus, DC-SIGN on the surface of dendritic cells is able to function as a trans-receptor, binding to Ebola virus pseudo typed lentiviral particles and transmitting infection to susceptible cells [9]. Dendritic cells play a major role in HIV pathogenesis. Epithelial dendritic cells seem be one of the first infected cells after sexual transmission and transfer of the virus to CD4 lymphocytes, simultaneously activating these cells to produce high levels of HIV replication [7].
In the present study, no association was found between the CD209 and DF gene polymorphisms. It was suggested earlier that in DF, DENV might use receptors other than DC-SIGN, the receptors that are known to promote antibody-dependent enhancement [13]. The present study also has a medium sample size, in particular, for DF cases and may have limited power in detecting minors the associations revealed for DF also explore the association between these polymorphisms and DHF cases could also provide valuable information in the knowledge of dengue pathogenicity. To our knowledge, this is the first study to report an association between CD209 gene variants and dengue fever in a Burkinabè population. Further studies with larger numbers of samples, especially DHF cases, are needed in the population.
In another study concerning dengue in the Taiwanese population, the authors show that the SNP rs4804803 in the CD209 promoter contributed to the susceptibility to dengue infection and complication of DHF. This SNP with the AG genotype affects the expression of cell surface DC-SIGN related to the immune system increase and less viral replication [13]. Tassaneetrithep and al thinks that DC-SIGN can be considered as a new target for the design of therapies that block dengue infection [10].
DC-SIGN polymorphisms and dengue susceptibility have been described in numerous studies, with a positive association in the Taiwanese population [12], and those from other countries [17] [20]. This does not turn out to be the case in the Burkina population. Indeed, we dare to think that the genetic inheritance of the host could be an explanation for this divergence. However, in the present study, the −336A allele had a frequency of 55.83% whereas this SNP with the AG genotype affects the expression of DC-SIGN at the cell surface related to immune augmentation and replication less viral [12].
5. Conclusion
This related no significant association with dengue for the variant of −336A/G in the DC-SIGN gene in an Ouagadougou population. However, our results offered the SNP frequencies in a West African population (Ouagadougou), which might be useful for the study of ethnic groups.
Acknowledgements
We would like to thank the World Academy of Sciences and the Swedish International Development Cooperation Agency (Sida) for funding this research through grant No. 17-403 RG/BIO/AF/AC_I-FR3240297757. We are also grateful to the staff of Saint Camille Hospital, the Biomolecular Research Center Pietro Annigoni and the National Blood Sanguine Transfusion center for their collaboration.