To evaluate the role of FCN2 polymorphism and ficolin-2 protein concentration in pathophysiological disorders in dengue fever; 278 patients with Dengue patients were collected in the 2017 outbreak at Military Hospital 103, diagnosed and grouped according to the “Guidelines for diagnosis and treatment for Dengue of Vietnam Ministry of Health” in 2011, the polymorphisms in the promoter region and exon 8 were determined by Sanger sequencing method. Serum ficolin-2 protein concentration was determined by ELISA method.
Journal of military pharmaco-medicine no5-2020 POLYMORPHISM OF FCN2 GENE AND SERUM FICOLIN-2 LEVELS IN DENGUE HEMORRHAGIC FEVER PATIENTS Do Quyet1, Ngo Truong Giang1, Nguyen Thanh Huyen2 Hoang Van Tong1, Nguyen Linh Toan1 SUMMARY Objectives: To evaluate the role of FCN2 polymorphism and ficolin-2 protein concentration in pathophysiological disorders in dengue fever Subjects and methods: 278 patients with Dengue patients were collected in the 2017 outbreak at Military Hospital 103, diagnosed and grouped according to the “Guidelines for diagnosis and treatment for Dengue of Vietnam Ministry of Health” in 2011, the polymorphisms in the promoter region and exon were determined by Sanger sequencing method Serum ficolin-2 protein concentration was determined by ELISA method Results: Serum ficolin-2 level of the patient group was significantly higher than the control group (median 2,856 compared to 1,994 ng/mL) and dengue haemorrhagic fever (DHF) group was significantly higher than dengue with warning signs (DWS) (median 3,087 versus 2,181 ng/mL), the difference was statistically significant with p < 0.001 single nucleotide polymorphic points -986G>A, -902C>A, -602G>A, +6031A>G, +6220T>G, +6424G>T were observed and analyzed statistically There were statistically significant differences of rare genotypes between research groups Conclusions: Patients with genotypes or low serum ficolin-2 protein levels will be more susceptible to Dengue * Keywords: Dengue haemorrhagic fever; Ficolin-2; FCN2 gene; Single nucleotide polymorphism INTRODUCTION Dengue is a major public-health problem which is caused by dengue virus (DENV) [1] The Dengue virus belongs to the Flaviviridae family which has four antigenically serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) [3] Secondary infection with one of these serotypes may result in a wide range of clinical symptoms [4] According to the World Health Organization, Dengue occurs in three main clinical forms: Dengue haemorrhagic fever (DHF), dengue with warning signs (DWS) and severe dengue Patients with DHF often experience rapid onset of fever, severe headache, myalgia, arthralgia, gastrointestinal discomfort, positive tourniquet test, minor haemorrhagic manifestations, epistaxis, and gingival bleeding Besides the inclusive symptoms of DHF, DWS manifests with liver pain, mucosal bleeding, Vietnam Military Medical University Military Technical Academy Corresponding author: Hoang Van Tong (hoangvantong@vmmu.edu.vn) Date received: 9/4/2020 Date accepted: 10/7/2020 128 Journal of military pharmaco-medicine no5-2020 urine < 500 mL per 24 hours, increased vomiting, increased hematocrit, rapid reduction of platelet count Severe dengue is characterized by all the symptoms of DWS and hyposphyxia, heavy bleeding, multiple organ failure [5, 6] There are many factors related to dengue pathogenesis, involving components essential of the virus and the host Despite extensive studies, the pathogenesis of dengue is still not fully understood The most important hypotheses that explained for the pathogenesis of dengue involve immunerelated factors, namely: complementary system and activation factors Complements are an important part of innate immunity Complements must be activated before performing their functions There are three pathways to activate the complements system The lectin pathway is the newly discovered one which relates to concentration and function of two lectin proteins, namely: Ficolin and mannosebinding lectin [7] There are several studies about lectin pathway which show the close relationship between concentration, function of ficolin and mannose-binding lectin and some diseases, for example, rheumatic fever, rheumatic heart disease, leprosy and in malaria [8, 9, 10] However, the role of ficolin in dengue pathogenesis has been unclear Ficolins act as pattern recognition receptors that binds to specific pathogenassociated molecular patterns on the agents surface and activate the immune response by initiating the complement lectin pathway In humans, there are three ficolins: ficolin-1 (M-Ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin) which are encoded by the FCN1, FCN2 and FCN3 genes respectively [11] The three proteins have different function and effect on different cells and tissues FCN2 gene includes eight exons located on the chromosome The close relationship between some variants of FCN2 and ficolin-2 serum levels was verified such as positions -986G>A, -602G>A and -4A>G in the promoter region and at +6359C>T, +6424G>T in exon Low ficolin-2 serum levels and FCN2 gene polymorphisms were associated to several infectious diseases such as respiratory infections in children and invasive pneumococcal disease in adults [13] Thus, ficolin-2 serum levels and FCN2 gene polymorphisms may cause a wide spectrum of pathophysiological disorders in dengue In this study, we focus on the role of ficolin-2 in dengue pathogenesis and the relationship between its serum levels, FCN2 gene polymorphisms and clinical, subclinical symptoms in dengue patients and the change of ficolin-2 serum levels during infection Hence, we conducted this study: To evaluate the roles of FCN2 gene polymorphisms and ficolin-2 protein concentration in dengue pathogenesis SUBJECTS AND METHODS Subjects * The study group: 278 patients with dengue infection were enrolled in this study The dengue patients were divided into two different 129 Journal of military pharmaco-medicine no5-2020 groups based on clinical, subclinical symptoms The clinically classified groups include DHF (n = 171) and DWS (n = 107) All patients were confirmed positive for NS1 antigen or/and positive for dengue-IgM, dengue-IgG This study was conducted in 2017 at Military Hospital 103 * The control group: We recruited 200 Vietnamese blood donors All control individuals were confirmed negative for HBsAg, anti-HCV and antiHIV by routine serological procedures Methods * DNA extraction: Genomic DNA was extracted from whole blood samples of the two groups using the commercially available QIAamp Blood mini kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions * FCN2 genotyping: We focus on variants in two regions of FCN2 gene with two fragments: promoter + exon1 and exon7+8 These fragments were sequenced in both groups Primers used to amplify are forward primer 5’-ATT GAA GGA AAA TCC GAT GGG-3’ Reverse primer 5’-GAA GCC ACC AAT CAC GAA G-3’ for promoter + exon1; Forward primer 5’-CCA GCT CCC ATG TCT AAA GG-3’ Reverse primer 5’-TTA CAA ACC GTA GGG CCA AG-3’ for exon7+8 PCR amplifications were carried out in 25 mL volume of reaction mixture containing 1X Master Mix (Promega), 0.25 mM of each primer and 50 ng of genomic DNA The cycling conditions were performed as 130 follows: Denaturation at 95oC for minutes followed by 40 cycles of 94oC for 30-sec denaturation, 56oC for 30-sec annealing, followed by an extension at 72oC for 30 sec and a final extension of 72oC for minutes All the PCR products were purified using ethanol purification method and sequenced by VNTAB company * Ficolin-2 ELISA: The ficolin-2 serum levels were measured in two groups: using the human ficolin-2 ELISA kit (Hycult Biotech, Frontstraat 2a 5405 PB Uden, Netherland) following the manufacturer’s instructions * Statistical analysis: By SPSS 20.0 software and the level of significance was set to p < 0.05 Clinical and individual characteristics data were performed in median values with range for continuous variables Quantitative variables were performed in percentage values Mann Whitney U tests were used to analyze the association between serum ficolin-2 levels and genotypes, haplotype in different clinical groups Chi-square and Fisher exact tests were executed to determine the differences in allele frequencies and genotype distributions in clinical groups Haplotypes were created by using Arlequin v.3.5.1.2 software Genotype or haplotype frequencies were determined by simple gene counting The significance of deviations from Hardy-Weinberg equilibrium was tested using the randompermutation procedure as implemented in the Arlequin v.3.5.1.2 software (http://lgb unige.ch/arlequin) Linkage disequilibrium (LD) analysis was performed using Haploview v 3.2 programs Journal of military pharmaco-medicine no5-2020 RESULTS Characteristics of dengue-infected patients Table 1: Clinical, subclinical characteristics of the study group Baseline characteristics Age (mean, - max) DHF (n = 171) DWS (n = 107) p 31 (14 - 81) 31 (13-68) NS Sex NS Male 82 (47.7) 48 (44.9) Female 89 (52.3) 59 (55.1) Positive 152 (88.9) 93 (86.9) Negative 16 (9.4) 10 (9.3) Undetermined (1.7) (3.7) Positive 28 (16.4) 27 (25.2) Negative 138 (80.7) 75 (70.1) (2.9) (4.7) Positive 30 (17.5) 31 (29.0) Negative 136 (79.6) 71 (66.4) (2.9) (4.7) Immune status NS1 IgM Undetermined IgG Undetermined Bleeding NS NS 0.024 < 0.001 Absence 137 (80.1) 46 (43.0) Presence 34 (19.9) 61 (57.0) GOT (min-max) (U/L) 61.8 (18 - 369.2) 108.3 (20.5 - 2.322) < 0.001 GPT (min-max) (U/L) 39 (8 - 301) 73.8 (8.6 - 992.3) < 0.001 RBC (min-max) (T/L) 4.6 (2.8 - 6.81) 4.75 (2.88 - 7.17) 0.048 WBC (min-max) (G/L) 3.43 (1 - 14.6) 3.38 (1.19 - 17.29) NS PLT (min-max) (G/L) 88.7 (19 - 316) 60 (8 - 1.100) < 0.001 HCT (min-max) (G/L) 0.40 (0.2 - 0.53) 0.407 (0.24 - 0.52) NS Liver injury Plasma leakage In the DHF group, the proportion of patients with bleeding was lower than the DWS group (19.9% compared to 57%, p < 0.001) Plasma levels of GOT and GPT in the DWS group were higher than the DHF group (108.3 and 73.8 U/L compared to 61.8 and 39 U/L, respectively; p < 0.001) The platelet count of the DWS group was lower than that of the DHF group (60 compared to 88.7 G/L; p < 0.001) 131 Journal of military pharmaco-medicine no5-2020 Serum ficolin-2 concentration of the study groups Figure 1: Ficolin-2 protein level in both groups In figure 1A, ficolin-2 levels in the study group was higher than that in the control group (median 2,856 compared to 1,994 ng/mL), the difference was statistically significant p < 0.0001 In figure 1B, the ficolin-2 levels in both DHF and DWS groups was higher than the control group (median 3,087 and 2,181 compared to 1,994 ng/mL, respectively), the difference was statistically significant p < 0.0001 The levels of ficolin2 in DHF group was higher than in DWS group (median 3,087 compared to 2,181 ng/mL), the difference was statistically significant (p < 0.0001) The correlation between allele frequency and FCN2 genotype with the clinical groups Table 2: Relationship between allele frequency, FCN2 genotype and clinical groups Genotype and allele The study group (n,%) The control group p value DHF (1) n = 171 DWS (2) n = 107 Total (3) n = 278 n = 200 (n,%) GG 111 (84.7) 77 (86.5) 188 (85.5) 134 (79.8) Reference GA 18 (13.7) 10 (11.2) 28 (12.7) 31 (18.5) NS AA (1.6) (2.3) (1.8) (1.7) NS G 240 (91.6) 164 (92.1) 404 (91.8) 299 (89) Reference A 22 (8.4) 14 (7.9) 36 (8.2) 37 (11) NS Promoter rs3124952 (-986G/A) 132 OR Journal of military pharmaco-medicine no5-2020 rs3811143 (-902G/T) CC 115 (74.2) 70 (70) 185 (72.5) 138 (79.3) Reference AC 37 (23.9) 29 (29) 66 (25.9) 34 (19.5) NS AA (1.9) (1) (1.6) (1.2) NS C 267 (86.1) 169 (84.5) 436 (85.5) 310 (89.1) Reference A 43 (13.9) 31 (15.5) 74 (14.5) 38 (10.9) NS GG 155 (97.5) 100 (98) 255 (97.7) 163 (93.1) Reference GA (2.5) (2) (2.3) 12 (6.9) NS AA 0 0 NA G 314 (98.7) 202 (99) 516 (98.9) 338 (96.7) Reference A (1.3) (1) (1.1) 12 (3.4) NS rs11103563 (+6031A/G) 103 (66) 65 (66.3) 168 (66.1) 109 (58.6) AA 43 (27.6) 25 (25.5) 68 (26.8) 72 (38.7) Reference AG 10 (6.4) (8.2) 18 (7.1) (2.7) NS GG 249 (79.8) 155 (79.1) 404 (79.5) 290 (78) p2,4 = 0.034 A 63 (20.1) 41 (20.9) 104 (20.5) 82 (22) Reference rs3124953 (-602G/A) Exon G rs7872508 (+6220T/G) 2.4 (1.1 - 5.4) NS 103 (66.9) 64 (65.3) 167 (66.3) 112 (60.2) TT 40 (26) 25 (25.5) 65 (25.8) 69 (37.1) Reference TG 11 (7.1) (9.2) 20 (7.9) (2.7) NS GG 246 (79.9) 153 (78.2) 399 (79.2) 293 (78.8) p2,4 = 0.029 T 62 (20.1) 43 (21.9) 105 (20.8) 79 (21.2) Reference G 2.4 (1.1 - 5.4) NS rs7851696 (+6424G/T) 100 (66.2) 61 (64.2) 161 (65.4) 113 (60.8) GG 40 (26.5) 23 (24.2) 63 (25.6) 69 (37.1) Reference GT 11 (7.3) 11 (11.6) 22 (9) (2.1) NS TT 240 (79.5) 145 (76.3) 385 (78.3) 295 (79.3) p3,4 = 0.037 p2,4 = 0.012 G 62 (20.5) 45 (23.7) 107 (21.7) 77 (20.7) Reference T 111 (84.7) 77 (86.5) 188 (85.5) 134 (79.8) NS 2.2 (1 - 4.5) (1.3 - 7.3) In the promoter region, there was no correlation between genotype and allele at points -986G>A; -902C>A and -602G>A with the clinical form of the study groups On 133 Journal of military pharmaco-medicine no5-2020 exon 8, at point +6031A>G, +6220T>G, there was a relationship between GG genotype and DWS group (p = 0.034, OR = 2.4 and p = 0.029, OR = 2.4) At points +6424G>T, there was a correlation between TT genotype and general disease group and DWS group (p = 0.037, OR = 2.2 and p = 0.012, OR = 3) The relationship between haplotype gene FCN2 and clinical form Table 3: The relationship between haplotype genotype FCN2 and clinical forms The study group The control group (n = 326) p value 221 184 Reference 36 85 68 NS 28 27 55 35 NS ACGATG 19 10 29 25 NS Other haplotype 14 14 NA Haplotype DHF n = 244 DWS n = 160 Total n = 404 GCGATG 140 81 GCGGGT 49 GAGATG # OR In the study population, there were main types of haplotype: GCGATG, GCGGGT, GAGATG, ACGATG There was no correlation between main types of haplotype and the clinical form of the study groups Relationship between genotype, FCN2 haplotype and serum ficolin-2 protein concentration of the study groups Figure 2: Relationship between genotype and serum ficolin-2 concentration in study groups 134 Journal of military pharmaco-medicine no5-2020 There was a clear difference in serum ficolin-2 concentration between the genotypes of polymorphism -902G>T, +6031A>G, +6220T>G, +6424G>T In particular, the rare genotype of polymorphism -902C>A contributes to the increased ficolin-2 level (p = 0.009) In addition, the rare genotype of polymorphism +6031A>G, +6220T>G, +6424G>T contributes to the reduction of ficolin-2 level (p < 0.001) Figure 3: Relationship between haplotype and serum ficolin-2 concentration in both groups There was a statistically significant difference in serum ficolin-2 levels between haplotype types in both groups (p < 0.0001 and p = 0.016) The haplotype GAGATG had the highest serum ficolin-2 concentration, while the haplotype GCGGGT had the lowest serum ficolin-2 concentration DISCUSSION So far, the pathogenesis of dengue hemorrhagic dengue disease is still unclear In this study, we have shown the role of FCN2 polymorphism and serum ficolin-2 concentration in dengue hemorrhagic patients Serum ficolin-2 levels have been shown to have variable amplitudes for different races [5, 9, 12, 13] Ficolin-2 can recognize and bind to specific carbohydrate structures on the surface of pathogens such as GlcNAc After binding to them, MASP complexes are created which activates the complement systems according to the lectin pathway, which can stimulate the body's immune system to limit the multiplication and spread of pathogens 135 Journal of military pharmaco-medicine no5-2020 [14] Therefore, when pathogens are present, the ficolin-2 level will be increased and different serum ficolin-2 level may affect the severity of clinical symptoms Our research results are completely consistent with this hypothesis: the serum ficolin-2 level in the study group is much higher than the control group (median 2,856 compared to 1,994 ng/mL) and it is much higher in the DHF group than in the DWS group (median 3,087 compared to 2,181 ng/mL), the difference was statistically significant with p < 0.001 Previous studies have shown a close association between polymorphisms at 986G>A, 602G>A and -4A>G in the promoter region and +6424G>T in exon with some other pathologies [5, 9] However, there was a difference in our findings on dengue haemorrhage patients, which indicates the close association of some other polymorphisms: +6031A>G; +6220T>G on exon and the risk of dengue haemorrhage with OR was 2.4 and 2.4, respectively In contrast, no correlation between genotypes at points -986G>A; 902C>A and -602G>A and risk of dengue hemorrhagic disease was found Previous studies have shown that three polymorphisms in the promoter region are -986G>A, -602G>A and -4A>G were associated with increased serum Ficolin-2 levels; +6424G>T was associated with decreased serum ficolin-2 level [1] Our study adds a rare genotype of -902C>A that also contributes to an increase in serum ficolin-2 levels In contrast, the rare genotype of +6031A>G, +6220T>G contributes to decrease serum ficolin-2 levels 136 CONCLUSION Through the study of polymorphism of FCN2 gene and serum ficolin-2 levels in dengue patients, we have drawn some conclusions: - Ficolin-2 levels in the study group was higher than the control group (median 2,856 compared to 1,994 ng/mL) and DHF group was much higher than in DWS (median 3,087 compared to 2,181 ng/mL) - Among FCN2 polymorphisms, the rare genotype of polymorphism -902C>A contributes to an increase in the ficolin-2 levels, the rare genotype of the polymorphism +6031A>G, +6220T>G +6424G>T contributes to a decrease in the ficolin-2 levels - In the research population, there were main types of haplotype: GCGATG, GCGGGT, GAGATG, ACGATG REFERENCES Hoang Van Tong, et al Ficolin-2 levels and FCN2 haplotypes influence hepatitis B infection outcome in Vietnamese patients PLoS One 2011; 6(11):e28113 Simmons CP, et al Dengue New England Journal of Medicine 2012; 366(15): 1423-1432 Green S, A Rothman Immunopathological mechanisms in dengue and dengue hemorrhagic fever Current Opinion in Infectious Diseases 2006; 19(5):429-436 Harris E, et al Clinical, epidemiologic, and virologic features of dengue in the 1998 epidemic in Nicaragua The American Journal of Tropical Medicine and Hygiene 2000; 63(1):5-11 Journal of military pharmaco-medicine no5-2020 Kilpatrick DC, T Fujita, M Matsushita P35, an opsonic lectin of the ficolin family, in human blood from neonates, normal adults, and recurrent miscarriage patients Immunology Letters 1999; 67(2):109-112 10 Messias‐Reason I, et al Ficolin (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease Clinical & Experimental Immunology 2009; 157(3):395-399 Organization WH Dengue haemorrhagic fever: Diagnosis, treatment, prevention and control World Health Organization 1997 11 Hummelshoj T, et al Molecular organization of human Ficolin-2 Molecular Immunology 2007; 44(4):401-411 Medzhitov R, CA Janeway Jr Innate immunity: Impact on the adaptive immune response Current Opinion in Immunology 1997; 9(1):4-9 12 Chapman SJ, et al Functional polymorphisms in the FCN2 gene are not associated with invasive pneumococcal disease Molecular Immunology 2007; 44(12):3267-3270 de Messias-Reason I, PG Kremsner, JF Kun Functional haplotypes that produce normal ficolin-2 levels protect against clinical leprosy The Journal of Infectious Diseases 2009; 199(6):801-804 13 Le Y, et al Human l‐ficolin: Plasma levels, sugar specificity, and assignment of its lectin activity to the fibrinogen‐like (FBG) domain FEBS Letters 1998; 425(2):367-370 Faik I, et al Ficolin-2 levels and genetic polymorphisms of FCN2 in malaria Human Immunology 2011; 72(1):74-79 14 Endo Y, M Matsushita, T Fujita Role of ficolin in innate immunity and its molecular basis Immunobiology 2007; 212(4-5):371-379 137 ... clinical, subclinical symptoms in dengue patients and the change of ficolin-2 serum levels during infection Hence, we conducted this study: To evaluate the roles of FCN2 gene polymorphisms and. .. spectrum of pathophysiological disorders in dengue In this study, we focus on the role of ficolin-2 in dengue pathogenesis and the relationship between its serum levels, FCN2 gene polymorphisms and. .. ficolin-2 levels In contrast, the rare genotype of +6031A>G, +6220T>G contributes to decrease serum ficolin-2 levels 136 CONCLUSION Through the study of polymorphism of FCN2 gene and serum ficolin-2