Association of Polymorphisms in IL1β -511C>T,IL1RN 86 bp VNTR,and IL6 -174G>C Genes with Clinical Dengue Signs and Symptoms in Brazilian Dengue Patients

Abstract

Dengue is an important infectious disease that has high morbidity and mortality rates in most tropical and subtropical areas of the world. The diversity of the clinical manifestations involved in the outcome of dengue virus infection is affected by the relationship between serotype/genotype of the virus, host immune status, host genetic background, and environmental factors. Polymorphisms in interleukin (IL) genes have been associated with risk of developing symptomatic dengue. This study aimed to determine the association of the single-nucleotide polymorphisms of IL1β -511C>T, IL1RN 86 bp VNTR, and IL6 -174G>C genes with the clinical features of 198 individuals admitted to the São José Infectious Diseases Hospital with suspected dengue infection. Dengue was confirmed in 118 of the patients. The control group consisted of 80 other individuals who had symptoms similar to dengue, but negative for that. A higher frequency of increased hematocrit (p = 0.009), leukopenia (p = 0.000007), neutropenia (p = 0.0004), lymphocytosis (p = 0.00001), monocytosis (p = 0.004), atypical lymphocytes (p = 0.03), and thrombocytopenia (p = 0.0000009) was observed in the dengue patients. Among the polymorphisms studied, only IL1β (-511C>T) was associated with dizziness, (p = 0.01), suggesting that IL1β may be related to hypotensive episodes and increased vascular permeability. These results pointed out the importance of the IL1β (-511C>T) polymorphism in the development of clinical symptoms of dengue symptomology.

Introduction

Dengue is a disease caused by the arbovirus known as dengue virus (DENV), and it is one of the most serious infectious diseases due to the high morbidity and mortality rates in most tropical and subtropical areas of the world (21).The wide spectrum of dengue symptoms ranges from an undifferentiated fever to sudden onset of fever, severe headache, dizziness, myalgia and arthralgia, leukopenia, thrombocytopenia, and even bleeding (22). Host immune response resulting from DENV infection has been shown to be the major contributor in disease pathogenesis, and the outcome of DENV infection is affected by the relationship between serotype/genotype of the virus, host immune status, host genetic background, and environmental factors. Some polymorphisms in IL genes have been associated with dengue disease (15) such as IL1β, IL1RN, IL6, and IL10 (2,11,13,15). Therefore, the diversity of clinical features associated with this disease could also be due to the presence of these polymorphisms. Thereby, this study was undertaken to evaluate if the clinical laboratory signs unconfirmed dengue patients could be associated with IL1β [-511C>T (rs16944)], IL1RN [86bp VNTR (rs315952)], and IL6 [-174 G>C (rs1800795)] polymorphisms comparing with symptomatic dengue patients, but negative for dengue infection by laboratory tests.

Patients and Methods

Subjects

This study included 198 patients with suspected dengue infection who were admitted to the São José Infectious Diseases Hospital, Fortaleza, Ceará, Brazil, a public referral hospital for infectious diseases, during August 2011–2013. Patients who had dengue symptoms had a complete blood count and differential count and underwent serological and molecular analysis in the Public Health Laboratory of the State of Ceará–LACEN/CE, which receives all dengue suspected cases in the state. The present study was approved by the Research Ethics Committee of São José Infectious Diseases Hospital. All patients or their parent/guardian signed an informed consent form.

Dengue diagnosis laboratory tests and clinical parameters

Patients suspected of dengue included those who had clinical signs and symptoms described by WHO (22), such as fever, headache, retro-ocular pain, languidness, exanthema, petechiae, myalgia, arthralgia, asthenia, anorexia, dizziness or poor appetite, nausea, vomiting, cough, dyspnea, chest pain, looseness, mucosal bleeding (vaginal bleeding, gum bleeding), epistaxis, hematuria, and some medications used in these patients for susceptibility to the disease.

The peripheral blood samples of all patients suspected of dengue were subjected to dengue tests. The determination of dengue serotypes was first performed by the viral isolation technique, identifying the serotypes by indirect immunofluorescence. In cases of negative isolation, sera of patients were tested by reverse transcriptase-PCR (RT-PCR) (8). All samples were tested for NS1 and IgM assays to determine which of these suspected patients had been exposed to DENV.

The final dengue diagnosis was based on clinical (anamnesis taken by the physician) and laboratory parameters according to WHO clinical classification (22). DENV was confirmed in 118 of the patients studied, among which 110 were classified as dengue without warning signs and eight as dengue with warning signs. These patients were designated the dengue group (DG) and included 64 females and 54 males. The control group consisted of 80 other individuals who had symptoms similar to dengue, but negative for DENV according to the laboratory tests. These patients were designated the non-DG group (NDG), which consisted of 43 females and 37 males.

Genotype analysis

Genomic DNA was extracted from 5 mL of blood, collected at the same time as samples for routine hospital blood tests, using the salting-out method by Maniatis et al. with some modifications (10). DNA quality was determined by 1% agarose gel electrophoresis, and the amount was determined using the NanoDrop 3300 fluorospectrometer.

The sequences of the polymerase chain reaction (PCR) primers were as follows: 5′-TGG CAT TGA TCT GGT TCA TC-3′ (sense) and 5′-GTT TAG GAA TCT TCC CACTT-3′ (antisense) for IL1β (-511 C>T); 5′-CTC AGC AAC ACT CCTAT-3′(sense) and 5′-TCC TGG TCT GCA GGT AA-3′(antisense) for IL1RN 86 bp VNTR; and 5′-TTG TCA AGA CAT GCC AAA GTG-3′ (sense) and 5′-TCA GAC ATC TCC AGT CCT ATA-3′ (antisense) for IL6 (-174 G>C).

The genetic polymorphisms of IL1β (-511 C>T) and IL6 (-174 G>C) were evaluated by PCR-RFLP and IL1RN (86 bp VNTR) polymorphism by PCR according to published protocols (4,19). All PCRs were performed using Master Mix (1×) (QIAGEN) with 25 μg DNA. PCR products of IL1β (-511 C>T) and IL6 (-174 G>C) were visualized in 1% agarose gel electrophoresis with ethidium bromide staining, and PCR products were digested using FastDigest (Fermentas–Thermo) AvaI restriction enzyme for IL-1β (-511C>T) and SfaNI restriction enzyme for IL-6 (-174G>C). The digestion fragments were resolved by 6% nondenaturing polyacrylamide gel electrophoresis and silver staining. For all polymorphisms, 10% of samples were genotyped again on a random basis to confirm the results.

Statistical analysis

Data were analyzed using the statistical software Epi Info version 7. Significant differences were evaluated using the chi-square test and Fisher's exact test. p < 0.05 was considered significant, and p < 0.01 was considered very significant.

Results

Clinical characteristics

The main laboratory data of all subjects distributed according to the clinical characteristics of dengue are given in Table 1. The median age of DG patients was 28.7 years old, ranging from 2 to 81 years (mean of 28.9 ± 10.0), and the median age of NDG patients was 26.7 years old, ranging from 1 to 83 years (mean of 29.8 ± 10.2). Brown skin color was more frequent in both groups (DG and NDG), followed by white skin. No significant differences were found for age, sex, and ethnicity. Almost all laboratory parameters, except leukocytosis and decreased hemoglobin level, were statistically more frequent in the DG, especially thrombocytopenia. No significant differences were found for other clinical parameters (data not shown).

Table 1.

Clinical Characteristics by Laboratory Determination in Dengue and Nondengue Groups

Laboratory determination	NDG 80 (%)	DG 118 (%)	p
Atypical lymphocytes	2 (2.5)	13 (11.0)	0.03 ±
Increased hematocrit (Ht)	4 (5.0)	23 (19.5)	0.009 ±
Decreased hemoglobin (Hb)	0 (0.0)	2 (1.6)	0.24
Leukocytosis	0 (0.0)	4 (3.3)	0.10
Lymphocytosis	0 (0.0)	30 (25.4)	0.00001 ±
Leukopenia	0 (0.0)	45 (56.2)	0.000007 ±
Monocytosis	5 (6.2)	29 (24.5)	0.004 ±
Neutropenia	4 (5.0)	34 (28.8)	0.0004 ±
Thrombocytopenia	3 (3.7)	61 (51.6)	0.0000009 ±

± p < 0.05–statistically significant.

DG, dengue group; NDG, nondengue group; SG, symptomatic group.

Clinical manifestations of dengue and genotyping of SNPs in symptomatic individuals

The genotypic and allelic frequencies for all the single-nucleotide polymorphisms (SNPs) studied are presented in Table 2. In both DG and NDG, the genotype distributions were within Hardy–Weinberg equilibrium. Table 3 displays the frequencies determined for each symptom in DG patients considering IL1β (-511C>T), IL1RN (86 bp VNTR), and IL6 (-174G>C). Association analysis considering the three SNPs was studied and clinical symptoms showed that only IL1β (-511C>T) was statistically associated with clinical manifestation (Table 3). Among the 25 symptoms described, 22 symptoms had a higher prevalence in T genotype carriers (C/T and T/T) with a significant difference observed for dizziness [OR = OR = 8.18 (1.04–64.16); p = 0.01].

Table 2.

Genotypic and Allelic Frequencies of IL1β (-511C>T), IL1RN (86 bp VNTR), and IL6 (-174G>C) SNPs of DG and NDG

	Genotypic frequency			HWE reported	Allelic frequency
IL1β	CC	CT	TT	p	C	T
NDG	31 (0.49)	23 (0.37)	9 (0.14)	0.25	85 (0.67)	41 (0.33)
DG	23 (0.23)	56 (0.57)	20 (0.2)	0.23	102 (0.52)	96 (0.48)
IL1RN	LL	L^*2	^2^2		L	^*2
NDG	39 (0.56)	27 (0.39)	4 (0.06)	1	105 (0.75)	35 (0.25)
DG	55 (0.62)	30 (0.34)	4 (0.04)	1	140 (0.79)	38 (0.21)
IL6	GG	GC	CC		G	C
NDG	31 (0.47)	31 (0.47)	4 (0.06)	0.39	93 (0.7)	39 (0.3)
DG	34 (0.4)	53 (0.54)	12 (0.12)	0.29	121 (0.61)	77 (0.39)

p < 0.05, Hardy–Weinberg equilibrium, exact test.

SNP, single-nucleotide polymorphism.

Table 3.

Association of SNPs IL1β (-511C>T), IL1RN (86 bp VNTR), and IL6 (-174G>C) with Clinical Manifestations of Dengue and Medication Used in the Acute Phase of the Disease

Gene/SNPs	IL1β/(-511 C>T)					IL1RN/(86 bp VNTR)					IL6/(-174G>C)
Genotypes (n)	CC (n = 28)	%	CT+TT (n = 65)	%	p	LL (n = 54)	%	L^2 + ^2^*2 (n = 35)	%	p	GG (n = 10)	%	GC+CC (n = 80)	%	p
Clinical manifestations (symptoms) constitutional
Headache	15	53.6	64	98.5	0.09	45	83.3	28	80.0	0.89	9	90.0	71	88.8	0.92
Retro-orbital pain	18	64.3	59	90.8	0.32	47	87.0	34	97.1	0.72	9	90.0	79	98.8	0.88
Fever	11	39.3	49	75.4	0.10	35	64.8	21	60.0	0.82	5	50.0	56	70.0	0.73
Languidness	10	66.7	33	50.8	0.40	25	46.3	17	48.6	0.90	4	40.0	39	48.8	0.93
Gastrointestinal
Vomiting	9	32.1	35	53.8	0.23	22	40.7	20	57.1	0.36	3	30.0	43	53.8	0.62
Nausea	11	39.3	43	66.2	0.56	30	55.6	22	62.9	0.71	3	30.0	53	66.3	0.82
Diarrhea	5	17.9	16	24.6	0.56	13	24.1	7	20.0	0.86	2	20.0	19	23.8	0.23
Anorexia	0	0.0	1	1.5	0.51	1	1.9	0	0.0	0.42	0	0.0	1	1.3	0.76
Abdominal pain	1	5.6	3	4.6	0.82	1	1.9	2	5.7	0.34	1	10.0	4	5.0	0.67
Musculoskeletal
Myalgia	12	42.9	46	70.8	0.20	34	63.0	21	60.0	0.89	6	0.6	53	66.3	0.62
Arthralgia	13	46.4	37	56.9	0.60	33	61.1	17	48.6	0.53	3	30.0	49	61.3	0.46
Dermatological
Exanthema	10	35.7	23	35.4	0.98	19	35.2	12	34.3	0.95	4	40.0	30	37.5	0.89
Petechiae	3	10.7	14	21.5	0.29	10	18.5	6	17.1	0.89	0	0.0	18	22.5	0.49
Neurological and behavioral
Dizziness	1	3.6	19	29.2	0.01^a,b	2	3.7	4	11.4	0.06	2	20.0	15	18.8	0.11
Fainting	1	3.6	2	3.1	0.90	0	0.0	1	2.9	0.34	1	0.0	2	2.5	0.76
Hemorrhagic manifestations
Mucosal bleeding	2	7.1	4	6.2	0.86	2	3.7	3	8.6	0.34	1	10.0	5	6.3	0.45
Metrorrhagia	0	0.0	0	0.0	0.19	0	0.0	0	0.0	0.35	0	0.0	0	0.0	0.76
Hematuria	1	3.5	0	0.0	0.13	0	0.0	1	2.9	0.21	0	0.0	1	1.3	0.76
Epistaxis	0	0.0	1	1.5	0.51	0	0.0	1	2.9	0.21	0	0.0	2	2.5	0.76
Respiratory
Cough	2	7.1	1	1.5	0.17	0	1.8	1	2.9	0.21	0	0.0	1	1.3	0.76
Dyspnea	0	0.0	0	0.0	0.19	0	0.0	0	0	0.21	0	0.0	0	0.0	0.76
Chest pain	0	0.0	0	0.0	0.19	0	0.0	0	0	0.21	0	0.0	0	0.0	0.76
Medications
Dipyrone	8	28.6	19	29.2	0.96	15	27.8	11	31.4	0.78	2	20.0	27	33.8	0.56
Paracetamol	6	21.4	35	53.8	0.05	25	46.3	12	34.3	0.46	5	50.0	37	46.3	0.67
Others	0	0.0	4	14.3	0.19	2	3.7	1	2.9	0.83	1	10.0	6	7.5	0.79

p < 0.05; Fisher's exact test.

p = 0.01; OR = 8.18 (1.04–64.16).

Discussion

Initial symptoms are common in all dengue manifestations and other viral infections, making laboratory tests, along with a guided clinical examination, necessary to confirm a diagnosis, especially in endemic areas (5). As expected, our data were in agreement with the literature, showing a significantly higher frequency of increased hematocrit, leukopenia, neutropenia, lymphocytosis, monocytosis, atypical lymphocytes, and thrombocytopenia (p < 0.05) in the SG and corroborating other studies involving DENV infection (1,13). Some studies have reported a higher prevalence of leukopenia and also thrombocytopenia in nonsevere cases of dengue (18,20). Evidence suggests that both symptoms occur because of the destruction or growth inhibition of myeloid progenitor cells, which may cause peripheral destruction of platelets or only their reduced production, resulting from the destruction of bone marrow megakaryocytes by the virus (17). However, mechanisms underlying thrombocytopenia in DENV infection are not yet fully understood. Several other hypotheses have been suggested in the elucidation of mechanisms involving the immune response against the virus (11). The virus may directly or indirectly inhibit the production of progenitor cells in bone marrow or reduce reproductive capacity of hematopoietic cells. In addition, DENV interferes with the platelet counts, destroying cells by apoptosis or lysis through the complement system or by the involvement of antiplatelet antibodies (7,9). The lack of statistical significance for hemorrhagic manifestations, particularly mucosal bleeding, was probably due to the small number of patients with warning signs, in whom these symptoms are more common.

Nowadays, there is a consensus that dengue fever is associated with an imbalance of the inflammatory process (1,16). Since polymorphisms in the IL genes have been found to have an influence on the disease (12), it is reasonable to infer that such polymorphisms can be linked to clinical symptoms (1,13,14). There are no studies in the literature associating any of the polymorphisms analyzed (IL1β -511C>T, IL1RN 86 bp VNTR, and IL6 -174G>C) with clinical findings of dengue patients. In this study, only IL1β (-511C>T) showed a statistically significant difference in the DG between the CT/TT genotypes and dizziness. Therefore, an association between IL1β genotypes was explored in confirmed dengue patients, and dizziness was found to be statistically associated with patients carrying the T allele (p = 0.01). This association suggests that IL1β may be related to hypotensive episodes, which could lead to dizziness, and increased vascular permeability in patients infected with dengue, triggering thrombocytopenia and other severe conditions (6).

In conclusion, this study showed that the polymorphic allele of IL1β -511C>T is important in the development of dengue symptoms such as dizziness. This result reveals a new potential marker for studies of susceptibility to dengue and is essential to better understand the dynamics of DENV immunopathogenesis.

Footnotes

Acknowledgments

The authors thank the Public Health Laboratory of the State of Ceará for immunoserological and viral genetic analyses of subjects in this study. The authors are very thankful to the Federal University of Ceará–UFC/Brazil and the Northeast Biotechnology Network–RENORBIO/Brazil for providing most of the scientific training. Dr. A. Leyva helped with English editing of the article. Funding sources: The principal author is grateful for the financial support from the Foundation for Research Support of the State of Piauí–FAPEPI, provided throughout much of his doctorate work.

Author Disclosure Statement

No competing financial interests exist.

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