Association of Single Nucleotide Polymorphisms in TNFA and IL10 Genes with Disease Severity in Influenza A/H1N1pdm09 Virus Infections: A Study from Western India

Abstract

Susceptibility to severe influenza A/H1N1pdm09 virus is multifactorial. The present study was carried out in 246 patients infected with A/H1N1pdm09 virus to find out whether single nucleotide polymorphisms (SNPs) in the genes coding for proinflammatory and anti-inflammatory cytokines are associated with disease severity. Among the cases, 129 had mild disease, whereas 117 had severe disease. There were 27 fatal cases. TNFA rs1800629, IFNG rs2430561, IL10 rs1800872, IL10 rs1800896, and CCL2 rs1024611 SNPs were genotyped by polymerase chain reaction-based methods. A significantly higher frequency of TNFA rs1800629 “G/A” genotype was observed in severe and fatal cases compared with mild and survived cases, respectively. In a dominant mode, IL10 rs1800896 “G” allele was significantly negatively associated with disease severity. IL10 rs1800896 “C/A” genotype was significantly associated with fatality in influenza A/H1N1pdm09 infections. The results suggest that SNPs in the IL10 and TNFA genes might be associated with disease severity in influenza A/H1N1pdm09-infected patients.

Introduction

The emergence of influenza A/H1N1pdm09 virus in 2009 has globally created havoc in the public health system. Since 2009, India has experienced multiple waves of influenza A/H1N1pdm09 virus (14). Influenza A/H1N1pdm09 virus contributes to 51% of the influenza-associated hospitalizations (5). In India, A/H1N1pdm09 virus has been reported to be associated with severe disease in terms of hospitalization and mortality (13). Interindividual variation in the outcome of influenza virus infections is determined by pathogen and host factors (16). The question why some individuals develop severe disease while majority does not, still remains elusive. Evidence for contribution from a heritable component to death due to influenza has been reported (1).

Among the various host components to be implicated in the pathogenesis of severe A/H1N1pdm09 viral infections, presence of high systemic levels of T helper (Th)1 and Th17 cytokines, also identified as hypercytokinemia, has been reported as a characteristic feature in severe cases of A/H1N1pdm09 infection (4). However, production of chemokines such as monocyte chemoattractant protein-1 and interleukin (IL)8 as well as cytokines, such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL6, IL10, IL12p70, IL15, and various others, represent a host response to A/H1N1pdm09 infection, exacerbated secretion of proinflammatory and immunomodulatory cytokines alters host immune response leading to poor virus control, perpetuates inflammation, and leads to tissue damage and respiratory failure leading to severe disease (2).

Age, gender, and environmental factors have been shown to influence cytokine production (20). Above all, genetic variations contribute to 25–75% in the interindividual variability in cytokine production when the peripheral blood mononuclear cells were stimulated by microbial antigens (11). Host genetic variations by affecting cytokine production can influence susceptibility to infectious diseases and progression to severe disease. Single nucleotide polymorphisms (SNPs) in the TNFA, IFNG, IL10, and CCL2 have been shown to be associated with variations in the levels of the respective cytokines (6,17,21,22). In the present study, functional SNPs in the TNFA, IFNG, IL10, and CCL2 genes were investigated to find out their association with the development of severe disease in A/H1N1pdm09 virus-infected subjects from Western India.

Study Subjects and Methods

Study subjects

The study was approved by the Institutional Ethics Committees of ICMR-National Institute of Virology (NIV) and the collaborating hospitals. Based on the line list of laboratory-confirmed influenza A/H1N1pdm09 virus patients available from 2009 with ICMR-NIV, patients were contacted and requested to participate in the study. Influenza A/H1N1pdm09 virus infection in these patients was confirmed by a real-time reverse transcriptase polymerase chain reaction. A written informed consent was obtained from all the study subjects or their legal guardians before sample collection. Blood sample (5 mL) from the consenting patients was collected in an Ethylenediaminetetraacetic acid (EDTA) vaccutainer. Blood samples were also prospectively collected from patients who were admitted to the collaborating hospitals with confirmed influenza A/H1N1pdm09 virus infection.

DNA was extracted from the blood using a commercial DNA extraction kit (QIAamp Blood DNA Midi Kit; Qiagen) according to the instructions of the manufacturer. Based on the data from case history sheets, study subjects were classified into those with mild disease or severe disease based on the presence of severe acute respiratory illness (SARI). As per the World Health Organization definition, patients with influenza-like illness and shortness of breath or difficulty in breathing and requiring hospital admission are considered as SARI patients and were considered as severe cases.

Genotyping of SNP in TNFA, IFNG, IL10, and CCL2 genes

Genotyping of TNFA rs1800629, and IL10 rs1800896 SNPs was performed using the polymerase chain reaction with sequence specific primers (PCR-SSP)-based method as described earlier (8,23). IFNG rs2430561, IL10 rs1800872, and CCL2 rs1024611 polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based methods as described earlier (6,18,25).

Statistical analysis

Statistical comparisons were made between mild and severe disease categories as well as between survived and fatal cases. Survived cases include all the mild cases and those who had favourable outcome among severe cases. Mean age was compared between the study groups using Student's t-test. Distribution of gender between the study groups was compared using Fisher's exact test. Allele and genotype frequencies were calculated by direct counting. Chi-square test was used to find out whether the genotype frequency distributions confirmed to Hardy–Weinberg equilibrium. Allele and genotype frequencies were compared between the study groups using Fisher's exact test.

For allele frequencies, p-values with Yate's correction, and odds ratio (OR) with 95% confidence limits (CI) were calculated using Statcalc program, Epi info version 7 (CDC, Atlanta, GA). p-Values with OR adjusted for gender and age were calculated by logistic regression under codominant, dominant, recessive, and overdominant models using the online SNP stats program for genotypic associations (19). Akaike information criterion (AIC) and Bayesian information criterion (BIC) provided by the software were used to find out the best fitting model of association. The model, which had the lowest AIC and BIC values, was considered as the best fitting model. Haplotype frequencies were predicted and compared between the study groups using the online SNPstats program. p-Values less than 0.05 were considered significant.

Results

Demographic and disease severity information of the study subjects

A total of 246 patients were recruited in the study. Among the 246 patients, based on the presence or absence of SARI, 117 patients were grouped as those having severe disease, whereas the remaining 129 patients were considered as having mild disease. There were 27 fatal cases, whereas the outcome was favourable in the remaining patients (n = 219).

Mean age was significantly higher in severe cases compared with the mild cases (p = 5 × 10⁻⁹). Fatal cases had a significantly higher mean age compared with the survived cases (p = 0.021). Proportion of males to females was not different between the mild and severe cases (p = 0.201). Fatal cases had a higher proportion of males compared with survived cases, although it was not statistically significant (p = 0.104) (Table 1).

Table 1.

Demographic Characteristics of Influenza A/H1N1pdm09 Virus-Infected Patients with Different Grades of Disease Severity

Demographic characteristics	Mild cases (n = 129)	Severe cases (n = 117)	p	Survived cases (n = 219	Fatal cases (n = 27)	p
Age	33.3 ± 18.0	46.1 ± 14.8	5.02 × 10⁻⁹	38.5 ± 17.9	46.8 ± 14.3	0.021
Male:female ratio	1:1.3	1:0.92	0.201	1:1.2	1:0.59	0.104

Allele frequencies in influenza A/H1N1pdm09 virus-infected positive patients with different degrees of disease severity

Minor allele frequencies of different SNPs in A/H1N1pdm09 patients are provided in Table 2. A higher frequency of “A” allele of TNFA rs1800629 was observed among the severe cases and fatal cases compared with the mild as well as the survived cases, respectively. However, the difference was significant for the comparison between the mild and severe cases (OR with 95% CI 2.76 [1.18–6.43]; p = 0.025).

Table 2.

Percent Allele Frequencies of Cytokine Gene Polymorphisms in Influenza A/H1N1pdm09 Virus-Infected Patients with Different Grades of Disease Severity

SNP	Alleles	Percent minor allele frequency n (%) ^a	Mild vs. severe cases		Survived vs. fatal cases
SNP	Alleles	Percent minor allele frequency n (%) ^a	Odds ratio with 95% confidence intervals	p	Odds ratio with 95% confidence intervals	p
TNF rs1800629	G > A	A
Mild cases		8 (3.2)	—	—	—	—
Severe cases		19 (8.1)	2.76 (1.18–6.43)	0.025	—	—
Survived cases		21 (4.8)	—	—	—	—
Fatal cases		6 (11.1)	—	—	2.48 (0.96–6.45)	0.108
IFNG rs2430561	A > T	T
Mild cases		99 (38.4)	—	—	—	—
Severe cases		88 (37.6)	0.97 (0.67–1.39)	0.935	—	—
Survived cases		164 (37.4)	—	—	—	—
Fatal cases		23 (42.6)	—	—	1.24 (0.70–2.20)	0.557
IL10 rs1800896	A > G	G			—	—
Mild cases		71 (27.5)	—	—	—	—
Severe cases		49 (20.9)	0.70 (0.46–1.06)	0.111	—	—
Survived cases		110 (25.1)	—	—	—	—
Fatal cases		10 (18.5)	—	—	0.68 (0.33–1.39)	0.370
IL10 rs1800872	C > A	A
Mild cases		113 (43.7)	—	—	—	—
Severe cases		108 (46.1)	1.08 (0.76–1.55)	0.721	—	—
Survived cases		199 (45.4)	—	—	—	—
Fatal cases		22 (40.7)	—	—	0.82 (0.46–1.45)	0.591
CCL2 rs1024611	A > G	G
Mild cases		97 (37.6)	—	—	—	—
Severe cases		82 (35.0)	0.90 (0.62–1.29)	0.621	—	—
Survived cases		161 (36.7)	—	—	—	—
Fatal cases		18 (33.3)	—	—	0.86 (0.47–1.56)	0.731

Bold values represent significant values.

n = 129 for mild cases; 117 for severe cases; 219 for survived cases; and 27 for fatal cases for all SNPs, except for IL10 rs1800871, where n = 128 for mild cases.

n represents allelic counts and the numbers in parentheses represent percentages.

IL, interleukin; SNP, single nucleotide polymorphism.

A trend toward lower frequency of the “G” allele of IL10 rs1800896 was observed among the severe cases and fatal cases compared with the mild cases and survived cases, respectively, although the difference was not significant.

The allele frequencies of IFNG rs2430561, IL10 rs1800872, and CCL2 rs1024611 were not different between the study groups (Table 2).

Genotype frequencies in influenza A/H1N1pdm09 virus-infected patients with different degrees of disease severity

Percentage genotype frequencies of different SNPs in A/H1N1pdm09-positive patients are provided in Table 3. The genotype frequencies of different SNPs conformed to Hardy–Weinberg equilibrium in the overall study group (p > 0.05).

Table 3.

Percentage Genotype Frequencies of Cytokine Gene Polymorphisms in Influenza A/H1N1pdm09 Virus-Infected Patients with Different Grades of Disease Severity

Genotypes	Mild cases (n = 129), n (%)	Severe cases (n = 117), n (%)	Mild vs. severe cases		Survived cases (n = 219), n (%)	Fatal cases (n = 27), n (%)	Survived vs. fatal cases
Genotypes	Mild cases (n = 129), n (%)	Severe cases (n = 117), n (%)	Odds ratio with 95% confidence intervals ^a	p	Survived cases (n = 219), n (%)	Fatal cases (n = 27), n (%)	Odds ratio with 95% confidence intervals ^a	p
TNF rs1800629
G/G	121 (93.8)	98 (83.8)	1.00	—	198 (90.4)	21 (77.8)	1.00
G/A	8 (6.2)	19 (16.2)	3.20 (1.27–8.05)	0.0099	21 (9.6)	6 (22.2)	2.90 (1.02–8.26)	0.06
IFNG rs2430561
A/A	48 (37.2)	39 (33.3)	1.00		80 (36.5)	7 (25.9)	1.00
A/T	63 (48.8)	68 (58.1)	1.27 (0.71–2.29)	0.37	114 (52.0)	17 (63.0)	1.71 (0.66–4.40)	0.51
T/T	18 (13.9)	10 (8.6)	0.70 (0.28–1.77)		25 (11.4)	3 (11.1)	1.64 (0.38–7.00)
IL10 rs1800896
A/A	70 (54.3)	76 (65%)	1.00		128 (58.5)	18 (66.7)	1.00
A/G	47 (36.4)	33 (28.2)	0.56 (0.32–0.98)	0.039	72 (32.9)	8 (29.6)	0.76 (0.31–1.87)	0.43
G/G	12 (9.3)	8 (6.8)			19 (8.7)	1 (3.7)	0.31 (0.04–2.55)
IL10 rs1800872
C/C	41 (32.0)	36 (30.8)	1.00		70 (32.1)	7 (25.9)	1.00
C/A	61 (47.7)	54 (46.1)	1.07 (0.57–2.00)	0.66	97 (44.5)	18 (66.7)	2.50 (1.06–5.89)	0.032
A/A	26 (20.3)	27 (23.1)	1.40 (0.66–2.98)		51 (23.4)	2 (7.4)
CCL2 rs1024611
A/A	46 (35.7)	48 (41.0)	1.00		84 (38.4)	10 (37.0)	1.00
A/G	56 (47.9)	56 (47.9)	0.88 (0.49–1.56)	0.88	109 (49.8)	16 (59.3)	1.31 (0.55–3.09)	0.35
G/G	14 (10.8)	13 (11.1)	1.04 (0.41–2.62)		26 (11.9)	1 (3.7)	0.36 (0.04–3.02)

Bold values represent significant values.

n = 129 for mild cases; 117 for severe cases; 219 for survived cases; and 27 for fatal cases for all SNPs, except for IL10 rs1800871, where in n = 128 for mild cases.

p-Values and odds ratio were usually based on codominant models, except for IL10 rs1800896 in the comparison between mild and severe cases, wherein they were based on dominant model (A/A vs. A/G+G/G) and for IL10 rs1800871 in the comparison between survived and fatal cases, wherein they were based on overdominant model (C/C+A/A vs. C/A).

When the genotype frequencies were compared between the mild and severe cases, G/A genotype of TNFA rs1800629 was significantly higher among the severe cases (OR with 95% CI 3.20 [1.27–8.05]; p = 0.0099). In a dominant mode (A/A vs. G/A+G/G), the G allele of IL10 rs1800896 was significantly negatively associated with disease severity (OR with 95% CI 0.56 [0.32–0.98]; p = 0.039). The genotype frequencies of other SNPs were not different between the mild and severe cases (p > 0.05).

Comparison of the genotype frequencies between the fatal and survived cases revealed a higher frequency of G/A genotype of TNFA rs1800629 in fatal cases (OR with 95% CI 2.90 [1.02–8.26]; p = 0.06). In an overdominant mode (C/C+A/A vs. C/A), the C/A genotype of IL10 rs1800872 was significantly associated with fatal cases (OR with 95% CI 2.50 [1.06–5.89]; p = 0.032).). The genotype frequencies of other SNPs were not different between the fatal and survived cases (p > 0.05).

IL10 haplotypes in influenza A/H1N1pdm09 virus-infected patients with different degrees of disease severity

Investigation of IL10 rs1800896-rs1800872 haplotypes revealed higher frequency of A-C haplotype among the severe cases as well as the fatal cases compared with the mild and survived cases, respectively. The increased frequency among the fatal cases compared with the survived cases was significant (OR with 95% CI 2.71 [1.17–6.28]; p = 0.021). In contrast, the frequency of the G-C haplotype was lower among the severe as well as fatal cases compared with the mild cases and survived cases and the decreased frequency in severe cases compared with mild cases was significant (OR with 95% CI 0.60 [0.36–0.99]; p = 0.045). The frequency of the rare haplotype G-A was significantly higher in the fatal cases compared with the survived cases (OR 31.75 96% CI [2.67–378.23]; p = 0.0067) (Table 4).

Table 4.

Percent-Predicted Haplotype Frequencies of IL10 Single Nucleotide Polymorphisms in Influenza A/H1N1pdm09-Infected Patients with Different Grades of Disease Severity

Haplotypes IL10 rs1800896-rs1800872	Mild cases (n = 129), % (n) ^a	Severe cases (n = 117), % (n) ^a	Mild vs. severe cases		Survived cases (n = 219), % (n) ^a	Fatal cases (n = 27), % (n) ^a	Survived vs. fatal cases
Haplotypes IL10 rs1800896-rs1800872	Mild cases (n = 129), % (n) ^a	Severe cases (n = 117), % (n) ^a	Odds ratio with 95% confidence intervals	p^b	Survived cases (n = 219), % (n) ^a	Fatal cases (n = 27), % (n) ^a	Odds ratio with 95% confidence intervals	p^c
A-A	43.3 (112)	44.3 (104)	1.00	—	44.5 (195)	29.2 (16)	1.00	—
A-C	29.2 (75)	34.8 (81)	1.12 (0.72–1.76)	0.61	30.3 (133)	52.3 (28)	2.71 (1.17–6.28)	0.021
G-C	26.5 (68)	19.1 (45)	0.60 (0.36–0.99)	0.045	23.9 (105)	7.0 (4)	0.23 (0.03–1.82)	0.17
G-A	0.98 (3)	1.9 (4)	2.09 (0.31–14.14)	0.45	1.2 (5)	11.5 (6)	31.75 (2.67–378.23)	0.0067

Bold values represent significant values.

Numbers in the parentheses are absolute number of haplotypes, which were back calculated from the estimated relative frequency of haplotypes. Since each subject will have two haplotypes, the sum of the haplotype numbers will be twice the number of subjects in each group.

Global haplotype association p-value 0.13.

Global haplotype association p-value 0.02.

Discussion

Severe disease in influenza A/H1N1pdm09 virus-infected patients has been linked to the association between viral replication and its rate of clearance and inflammatory cytokine response (2,10). Since inflammatory cytokine response is influenced by SNPs in the genes coding for them, the present study investigated the association between SNPs in the genes coding for inflammatory cytokines and severe disease in influenza A/H1N1pdm09 virus-infected patients. In the present study, SNPs in the genes coding for two proinflammatory cytokines: TNF-α and IFN-γ, a proinflammatory chemokine: CCL-2, and an anti-inflammatory cytokine: IL10, were investigated.

The results revealed that “A” allele and G/A genotype of TNFA rs1800629 was associated with susceptibility to disease severity in influenza A/H1N1pdm09 virus-infected patients. In contrast, a study from Mexican population showed no association between rs1800629 and disease severity (7). Another study from Mexico which compared rs1800629 genotypes between severe A/H1N1pdm09 patients and healthy controls reported that G/A genotype of TNFA rs1800629 was associated with protection against severe A/H1N1pdm09 virus infection (12). The differences between different studies could be due to the population-specific prevalence of risk alleles and gene–environment interactions.

Independent studies have shown the association of TNFA rs1800629 “A” allele with higher TNF-α production compared with “G” allele (9,21,24). Another study has reported elevated blood urea nitrogen levels in influenza A/H1N1pdm09-infected patients with TNFA rs1800629 “G/A” genotype (15). It is possible that TNFA rs1800629 “G/A” genotype would contribute to higher levels of TNF-α leading to inflammation affecting multiple organs and hence enhanced disease severity and fatality in subjects with influenza A/H1N1pdm09 virus infection.

The present study also revealed the association of IL10 rs1800896 “G” allele in a dominant mode with protection to severe disease and IL10 rs1800872 “C/A” genotype with susceptibility to fatality. The protective effect of IL10 rs1800896 “G” allele was further corroborated by the observation of higher frequency of IL10 rs1800896-rs1800872 A-C haplotype and lower frequency of G-C haplotype in the severe and fatal cases. Similarly, a study from Mexico also reported the association between rs1800896 “G” allele and protection to severe disease in A/H1N1pdm09 patients. The same study also reported an association between higher IL10 production and IL10 rs1800896 “G” allele (12). Higher IL10 production might contribute to reduction in inflammation and hence less severe disease.

The present study also reported an association between the IL10 rs1800896-rs1800872 rare haplotype “G-A” and severe disease. IL10 production is influenced by three SNPs (IL10 rs1800896-rs1800871-rs1800872) in the promoter region of IL10 gene, and different haplotypes involving all the three SNPs might have variable effect on IL10 production. Hence, it is possible that the rare haplotype “G-A” might be associated with altered IL10 production and hence susceptibility to severe disease. However, due to the lower frequency of this haplotype, the confidence intervals for the association reported were large and needs confirmation in larger studies. Lower levels of IL10 might lead to severe inflammation, whereas higher levels of IL10 might reduce the inflammation but may also affect antiviral activity.

A delicate balance between the proinflammatory and anti-inflammatory cytokines determines the optimal immune response and greater control of the disease. This delicate balance might be further affected by age, gender, coinfections, and other environmental factors, which will have an overall impact on the disease outcome. The present study also revealed no association between SNPs in the IFNG and CCL2 genes and disease severity. Similar results have been reported earlier (12). The sample size of the present study may be limited to delineate the minor effects conferred by these SNPs.

In the context of lower minor allele frequency of TNFA rs1800629, the sample size of the present study may not be sufficient, particularly, with regard to the number of fatal cases. The level of cytokines were not investigated and hence cannot be correlated with patient genotypes. Moreover, apart from age and gender, the information on other factors that might influence disease severity in influenza A/H1N1pdm09 virus-infected subjects such as the presence of type 2 diabetes, chronic obstructive pulmonary disease, and other chronic diseases were not available.

Despite the limitations, this study reports an association between SNPs in TNFA and IL10 genes and disease severity for the first time in an Indian population. These SNPs can serve as markers to predict disease severity in influenza A/H1N1pdm09 virus-infected subjects. Further studies in different populations with larger sample size investigating the association of SNPs in the cytokine genes and cytokine levels with influenza A/H1N1pdm09 virus-infected subjects may help to understand the role played by these SNPs in determining disease severity and might help in understanding the interindividual variability in disease outcomes. The study might also provide clues to design appropriate therapeutic strategies to treat influenza A/H1N1pdm09 virus-infected subjects.

Footnotes

Acknowledgments

The authors would like to thank Vasant Walkoli for help in blood collection. The authors acknowledge Dr. Jayati Mullick and Dr. B.V. Tandale for their critical review and suggestions. The study was funded by a grant from the Department of Biotechnology, Government of India (BT/PR8957/MED/12/630/2013 dated February 8, 2017).

Author Disclosure Statement

No competing financial interests exist.

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