Polymorphism of DC-SIGN ( CD209 ) Promoter in Association with Clinical Symptoms of Dengue Fever

Abstract

C-type lectin DC-SIGN receptor, encoded by CD209, plays a key role in the infection of dendritic cells by dengue virus (DENV). Because the -336A/G SNP (rs4804803) polymorphism in the promoter of CD209 modulates DC-SIGN expression, we investigated the putative association of this polymorphism with DENV infection and its pathogenesis. A control sample of 72 individuals, rigorously selected through a clinical investigation for absence of past dengue fever (DF) was compared to a sample of 168 patients (156 classical DF; 12 dengue hemorrhagic fever), all residents from Pará, Brazil. However, the prevalence of symptoms showed a trend higher in the AA genotype (Wilcoxon test; Z=2.02; p=0.04). Hence, our findings indicate that the G allele downregulates the spectrum of symptoms during the early acute phase of DENV infection, putatively decreasing the viremia, as suggested in the literature.

Introduction

Dengue fever (DF) is a major arbovirus epidemic afflicting tropical and subtropical regions caused by a flavivirus called dengue virus (DENV), mainly transmitted by Aedes aegypti and A. albopictus mosquitoes. In Brazil, almost 650,000 new cases were recorded in the first months of 2013, with the highest incidence rates occurring in the Brazilian Northern Region (32). Classic DF is self-limited and characterized by a wide spectrum of clinical symptoms associated with a rapid onset of fever in combination with laboratorial findings (19). According to the current World Health Organization (WHO) classification (31), symptomatic DENV infections are grouped into three categories: dengue, dengue with warning signs, and severe dengue.

Together, the large heterogeneity of the disease clinical outcome and ethnic epidemiological differences suggest that a genetic background may influence such variation (21,28). Indeed, many association studies have identified a number of infection-related candidate genes, for example HLA-A and HLA-B loci (27), cell receptors for IgG, vitamin D, mannose binding lectin, ABO blood group (11), human platelet antigens, and DC-SIGN (16).

DENV uses C-type lectin DC-SIGN receptor to infect dendritic cells (DC), encoded by the CD209 gene (located at 19p13.2 and encompassing three exons). At the initial point of infection (during blood feeding) DENV is injected and initially infects Langerhans cells (epidermal dendritic cell) and myeloid dendritic cells. Therefore, variability in the CD209 gene may modulate infection outcome. After this primary viremia, other cells are infected, as the mononuclear lineage and cells from several tissues such as liver, brain, spleen, and muscle (8,18,19). A noteworthy polymorphism of the CD209 gene is the single nucleotide polymorphism (SNP) -336 A/G (rs4804803) located in its promoter region, where the carrying of the G allele is related to a higher expression of DC-SIGN at the DC surface (30). That polymorphism has been extensively reported as a protective factor against many pathogens that use this receptor to infect DC, such as Mycobacterium tuberculosis (3,29,33), Human T-cell lymphotropic virus type 1 (12), and tick-borne encephalitis virus (2). Regarding DENV, few studies have been conducted using -336A/G CD209 SNP (1,24,26,30), and they have shown contradictory results in their association with DENV infection.

Although Wang et al. (30) tried to explain those divergences based on differences in allelic frequencies between Taiwan and Thailand populations and differences in DHF definition criteria, the question remains open, and only further association studies may clarify the real existence of such an association. Moreover, those authors performed functional assays, showing that AG heterozygous displayed an increased production of chemokines compared to AA homozygous, more efficiently controlling viral replication. Hence, the predisposition to DHF conferred by the G allele could be attributed to the exacerbation of the immune response, as already suggested by other studies (4,10). Those latter findings may agree with the protective effect of the G allele against a number of other pathogens, by controlling their replication, probably through chemokine production.

The most common associations investigated were restricted to severe DF, likely DHF. To our knowledge, there is no study associating classical DF pathogenesis with host genetic factors, but a recent study has suggested association of a SNP (other than -336 A/G) in the promoter region of CD209 with thrombocytopenia, but not clearly approaching associations with clinical signs and symptoms (1). CD209 polymorphism was also associated with fever in tuberculosis (33), highlighting an interesting aspect of studying genetic modulation of classical DF symptoms.

In this paper, we aimed to perform a case-control study in a Brazilian population, with a rigorous selection of the control sample through a clinical investigation for past DF symptoms. To our knowledge, a control sample selection criteria has not been used in previous studies. We also investigated for associations with classic DF pathogenesis, detecting a protective effect of the G allele, once their carriers showed a less exuberant spectrum of symptom manifestations.

Materials and Methods

Ethics statement

All individuals included in this research provided written informed consent. In addition, the study protocol met the ethical demands of Brazilian legislation, which follows the Declaration of Helsinki and was approved by the Ethic Committee from the Health Sciences Institute from the Federal University of Pará: registration 135/10.

Sampling strategy

The sample comprised 240 unrelated individuals, resident in Belém (Pará State, Brazil) for at least 5 years, divided into three samples, matched for gender, age, and socioeconomic level: control (72 individuals: 53% female, average age 35.3 years), DF (156 individuals: 49% female; average age 35.9 years), and DHF (12 individuals: 17% female; average age 38 years). The individuals were assigned to each sample group according to the following criteria.

The control recruitment method included a starting selection of individuals who had never reported a conclusive diagnosis of DF confirmed by a physician. They were submitted to a rigorous anamnesis, always performed by the same person, in order to guarantee the homogeneity of any subjective criteria used in this screening. All samples were tested for both IgG and IgM antibodies against DENV antigens (described in the next section). Approximately 89% (64 individuals) of the control sample showed low but detectable IgG levels.

The DF sample was collected during the months of higher transmissibility, when infected patients were searching for a diagnosis of their illness at the Evandro Chagas Institute, a national reference center on arbovirus health surveillance. DF cases were confirmed by positive virus isolation and/or positive viral RNA detection by molecular methods, and/or seroconversion with IgM detection 1 week after the show of any symptoms. Only DENV-2 and DENV-3 were detected (all methods are described in the next section). All DF patients reported DF symptoms during the anamnesis. The symptoms reported were headache, rash, retro-orbital pain, prostration, fever, myalgia, nausea/vomiting, arthralgia, diarrhea, cutaneous congestion, pruritus, petechiae, gingival bleeding, anorexia, epigastralgia, and hiporexia. Patients in this group would also be classified as belonging to the WHO's DF group (31).

DHF samples were identified in the Municipal Health Surveillance Agency Database. Because all confirmed DHF cases are compulsorily registered on this Database, we performed an active search to include those patients in the study. The Brazilian Ministry of Health criteria was used to define DHF (20). These criteria match those currently used by the WHO, according to which DHF would be classified as severe dengue (31). All patients on the database considered as DHF had their infection with DENV confirmed and showed the following clinical and laboratorial features: (i) fever for at least 7 days; (ii) thrombocytopenia (≤100.000/mm³;); (iii) bleeding trends (tourniquet test positive and spontaneous bleeding); and (iv) increased vascular permeability (increasing in hemoconcentration, pleural effusion, ascites, and hypoproteinemia).

Additionally, both DF and control samples were collected at the same time, and were geographically (all samples are from Belém) and ethnically matched (other genetic markers were also genotyped and no differences in allelic frequencies were observed; data not shown).

Sample processing and genotyping

Virus isolation on mosquito cells (9) and/or positive viral RNA detection by reverse transcriptase-polymerase chain reaction (RT-PCR) (17) were conducted in 79% of the DF sample. For all samples, serological tests included immunoenzymatic assay for IgM detection (MAC-ELISA) (14), hemagglutination inhibition for total antibodies detection (5), adapted for microplate assay (25), and enzyme linked immunosorbant assay (ELISA) in order to detect IgG (15).

Genomic DNA was isolated from EDTA-anticoagulated blood samples using a standard phenol-chloroform extraction followed by 70% alcohol precipitation. CD209 (DC-SIGN) genotyping for -336 A/G polymorphism (rs4804803) was performed using Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA; Assay ID C_1999340_10). The real-time-based SNP genotyping was carried out in the ABI Prism 7500 Sequence Detection System, using SDS 1.1 software for allele discrimination, following the PCR conditions suggested by the manufacturer.

Statistical analysis

Genotypic and allelic frequencies were estimated by direct count, and deviations of genotypic frequencies from the Hardy–Weinberg expectations were tested by chi-square. Allelic and genotypic frequencies comparisons between sample groups were also performed by either chi-square or Fisher's exact test, depending on the sample size distribution on the contingency table. The distributions of symptoms frequencies were compared by Wilcoxon's signed-rank test.

Results

The sample of DF was subdivided in two groups: allele G carriers (individuals that presented GG or AG genotypes of -336 A/G SNP), and non-G carriers (individuals presenting only AA genotype, without any copy of G allele). Frequencies of each symptom for both G carriers and non-G carriers groups were compared by Fisher's exact test with correction for multiple tests. A paired Wilcoxon's test across all symptoms was also applied to evaluate any trend for higher prevalence of symptoms among non-G carriers patients compared to the G carriers ones.

Among all sample groups, allelic and genotypic frequencies were similar, displaying no statistical significance when tested (Table 1). Thus, the SNP studied was not associated with DF (control vs. DF pooled with DHF; Fisher's exact test; p=0.89). Genotype proportions were in Hardy–Weinberg equilibrium.

Table 1.

Percentage of CD209 -336A/G SNP Genotypic and Allelic Frequencies in Controls, Dengue Fever (DF), and Dengue Hemorrhagic Fever (DHF) Patients

	Sample groups
Genotypes/alleles	Control (n=72)	DF (n=156)	DHF (n=12)
GG	4	4	0
AG	26	26	33
AA	69	70	67
G	17.4	17	16.6
A	82.6	83	83.4

A total of 16 symptoms were described in the DF sample group. The prevalence of the symptoms showed a trend to be higher in non-G carriers than in G carriers; 11 symptoms had a higher prevalence in non-G carriers (Table 2). Such a trend was tested with a paired Wilcoxon's test, revealing statistical significance (Z=2.02; p=0.04). When comparing G carriers and non-G carriers for each symptom, the only significant differences observed were for headache (Fisher's exact test; p=0.024) and arthralgia (Fisher's exact test; p=0.016), but this significance was lost after correction for multiple tests.

Table 2.

CD209 -336A/G (rs4804803) and Clinical Manifestations of Dengue Fever

		Genotype frequency (%)
Symptom	Frequency (%)	G carriers	G noncarriers	p-Value^*
Fever	100.0	100.0	97.6	1.000
Headache	91.2	80.6	95.2	0.0238^**
Myalgia	77.2	67.7	80.7	0.210
Arthralgia	72.8	54.8	79.5	0.016^**
Prostration	50.9	45.2	53.0	0.530
Nausea/vomiting	55.3	58.1	54.2	0.83
Retro-orbital pain	68.4	74.1	66.3	0.500
Rash	38.6	38.7	38.6	1.000
Pruritus	34.2	32.3	34.9	0.82
Hiporexia	36	35.5	36.2	1.000
Diarrhea	23.7	12.9	27.7	0.130
Epigastralgia	11.4	9.7	12	1.000
Anorexia	16.7	12.9	18.1	0.58
Cutaneous congestion	9.6	9.7	9.6	0.990
Petechiae	3.5	0.0	4.8	0.330
Gingival bleeding	2.6	3.2	2.4	0.580

p-Value was determined by Fisher's exact test. ^**Significance lost after correction for multiple tests.

Discussion

We detected association of the CD209 -336A/G polymorphism with symptom development during DENV infection, although this SNP was not associated with DENV infection. Our results agree with those of Silva et al (26), suggesting that CD209 -336A/G polymorphism is associated neither with protection nor with predisposition to DENV infection. For instance, Sakuntabhai et al. (24) described the allele -336G as protective against DF but not against DHF. On the other hand, Silva et al. (26) and Alagarasu et al. (1) did not find any association of this polymorphism with either DF or DHF, while Wang et al. (30) reported the same allele as predisposing to DHF but not associated with classic DF. Hence, the heterogeneity of the published results could mean that the associations observed were weak enough to be biased by environmental factors and/or sampling strategies. Indeed, no previous case-control studies detected differences >10% in allelic frequencies, which are more susceptible to biases. Regarding DHF, our sample was very small, and although the allelic frequencies were similar to those from DF and control sample groups, the small sample size does not exclude a putative association with disease severity.

Our findings suggest that DF patients carrying the G allele would show fewer symptoms than AA homozygous. Hence, a putative protective effect of the G allele, although not easily detected in case-control studies, could be related to modulation of DENV pathogenesis during the infection.

Some functional aspects of the G allele are remarkable, such as the higher expression of DC-SIGN at the cell surface (30). Moreover, the low viral replication and higher chemokines production in G carriers (30) provide interesting insights into the functional basis for the protective effect of the G allele, in agreement with our results.

In this context, a recent study (7) identified the proportion of inapparent dengue infections in households of febrile dengue cases in a multinational, prospective clinical study involving South-East Asia and Latin America. Interestingly, that study detected only 21% positivity for the NS1 antigen among inapparent infected cases, while classic DF patients showed 79% positivity for the NS1 antigen. In terms of symptoms, considering (i) the strong positive correlation of NS1 production with viremia (6,8), (ii) inapparent infections as a mild manifestation of DENV infection, and (iii) classic DF as a most exuberant outcome, the results of Dussart et al. (7) corroborate ours, at least indirectly. We might infer that high viremia, controlled by CD209 polymorphism, is associated with the exuberant symptom profiles at the early acute phase.

Another interesting point is the quite heterogeneous worldwide distribution of the G allele. This allele shows the highest frequencies among Sub-Saharan African or African-derived populations, ranging from 0.38 to 0.50 (12,33). Among European populations, it ranges from 0.18 to 0.27 (12,23,33), and among East Asians it shows low frequencies, varying from 0.04 to 0.13 (12,13,33). The high frequency of the protective G allele in African populations agrees with the lower prevalence of severe dengue in Africa and in African-derived populations (28).

In agreement with those observations, the findings of Restrepo et al. (22) suggest differences between African descents and admixed Colombian DENV infected patients regarding the expression of chemokines such as TNF-α, IL-6, and IFN-γ. Special TNF-α, which showed higher levels of expression among African descents, was also reported as having higher expression in AG genotype than in AA genotype, correlating with an augmented immune response (30).

Obviously, production of chemokines and control of viral replication are not only regulated by DC-SIGN expression. The expression of DC-SIGN itself does not depend exclusively on the -336 A/G polymorphism. However, evidence suggests a protective role for the allele G, which is apparent even considering a multifactorial context. Additionally, viral genetic factors, such as differences in serotypes circulating, were also excluded as putative bias in our study. Since approximately 77% of the infections were by DENV-3, this homogeneity might not influence our results. Moreover, since there are no reported associations between DC-SIGN genotype and DENV serotypes, the distribution of DENV serotypes in the genotypes is likely random. Finally, the distribution of symptoms across both serotype groups was quite similar and not statistically significant (Wilcoxon's test; data not shown).

In conclusion, we detected a putative protective role of the G allele during DF. That effect is evidenced by modulation of the symptom profile, and the presence of the G allele is associated with a decrease in symptoms. To the best of our knowledge, this is the first report in the literature to associate a CD209 SNP with dengue symptoms. The case-control association has not detected protective or predisposing effects for the G allele. These results match a model where the viremia and chemokine production, modulated by -336A/G polymorphism, influence the spectrum of symptoms during the early acute phase of DENV infection. Further studies, approaching viremia and chemokine profiles associated to CD209 genotypes in larger samples, besides functional approaches, will be essential to understand better the dynamics of the DENV pathogenesis.

Footnotes

Acknowledgments

We are grateful to the patients who collaborated with this study. This work was funded by Brazilian National Research Council—CNPq grant (INCT-FH). L.F.O. was supported by a CNPq fellowship.

Author Disclosure Statement

No competing financial interests exist.

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