1. Introduction
In April 2009, the Mexican Secretary of Health reported the emergence of the influenza A/H1N1 pdm09 virus, which shares molecular characteristics of swine, avian, and human influenza viruses, and rapidly spread throughout the world [
1,
2]. In Mexico, the lethality rate was estimated to be 5% for confirmed cases of influenza A/H1N1, with several comorbidities such as immunosuppression, pre-existing respiratory and cardiovascular diseases, diabetes, tobacco smoking, and obesity, some of the most important predictors of the disease severity [
2,
3,
4]. The most frequent signs and symptoms are fever, dyspnea, cough, myalgias, arthralgias, headache, and dyspnea. The most frequent adverse outcomes are mechanical ventilation, intensive unit care (ICU) admission, and death [
1,
5].
Additionally, previous studies have described several clinical and laboratory predictors of mortality or disease outcome. These parameters include sex (male), delayed medical attention and treatment onset, intensive care unit admission, low lymphocyte and platelet count, high creatine phosphokinase (CPK), serum creatine, blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) [
6,
7,
8].
Previous studies have determined that one of the most critical pathological mechanisms in this viral infection is systemic dysregulation of the inflammatory response correlated with disease severity [
9,
10,
11]. In this sense, genetic variants in proinflammatory genes have been associated with host susceptibility for influenza A/H1N1 infection. For example, single nucleotide polymorphisms (SNPs) in
TNF,
LTA,
IL1B, and
IL6 are associated with a higher risk for influenza A/H1N1 infection in the Mexican population [
11,
12].
However, antigen presentation by influenza-infected cells through Human leukocyte antigens (HLA) class I is necessary to mount an effective antiviral immune response. It has been described that cytotoxic T lymphocytes (CTL) kill influenza-infected cells upon recognition of diverse HLA class I peptide complexes at the cell surface, including short viral peptides of 8–12 aa in length [
13,
14]. The assembly of these peptides occurs in the endoplasmic reticulum (ER) of infected cells and involves the transporter associated with antigen processing (TAP) and Tapasin (encoded by
TAPBP gene), as well as ER chaperones. TAP is a critical factor comprising two subunits, TAP1 and TAP2 (encoded by
TAP1 and
TAP2 genes, respectively), required for translocation of peptides from the cytosol into the ER. On the other hand, Tapasin is another critical cofactor required for the assembly of HLA class I with exogenous peptides, which were obtained by intracellular degradation through proteasome, formed by proteasome subunit beta type 8 (PSMB8, encoded by
PSMB8 gene) and 9 (PSMB9, encoded by
PSMB9 gene) (previously known as large multifunctional peptidase 7 (LMP7) and 2 (LMP2), respectively) subunits [
15,
16,
17,
18].
Various studies have demonstrated that different HLA class I alleles are associated with a higher risk for influenza A/H1N1 infection in several populations [
19,
20]. Moreover, the role of SNPs in processing and antigen presentation-related genes has been studied in hypersensitivity pneumonitis (HP) [
21], ankylosing spondylitis [
22], and human papillomavirus (HPV) infection [
23,
24], but not in influenza A/H1N1 infection.
For all mentioned above, the study aimed to evaluate the association between processing and antigen presentation-related gene (TAP1, TAP2, TAPBP, PSMB8, and PSMB9) SNPs with the genetic susceptibility for influenza A/H1N1 infection in a Mexican mestizo population.
2. Materials and Methods
A retrospective case-control study was performed between May 2009 and March 2012 using nasopharyngeal swab samples from patients hospitalized with clinical symptoms of influenza and confirmed diagnosis by PCR techniques as previously stated [
19].
2.1. Ethics Statements
This study was reviewed and approved by the Institutional Committees for Investigation, Ethics in Research, and Biosecurity of the Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER) (approbation number: B05–10, approved on 2 March 2010). All participants were informed about the protocol’s aim after being given a detailed description of the study and invited to participate as volunteers. All of them signed an informed consent paper and supplied a privacy statement describing the legal protection of personal data; both documents approved by the institutional Research and Ethics in Research Committees.
A trained and responsible physician informed the patient about a possible diagnosis of influenza. All participants were invited to participate voluntarily by a trained researcher. All participants received medical care even if they chose not to participate in the research protocol. A clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. All experiments were performed following the relevant guidelines and regulations. The STREGA (STrengthening the REporting of Genetic Association) guidelines were considered to design this genetic association study [
25].
2.2. Diagnosis
All patients underwent the QuickVue Influenza A + B test (Quidel, San Diego, CA, USA). Additionally, peripheral blood from patients and asymptomatic healthy contacts (HC) was collected; these interventions adhered to the guidelines and recommendations of the Centers for Disease Control and Prevention of the United States (CDC) and the World Health Organization (WHO). Subsequently, the RespiFinder assay was done, and the presence of the influenza A/H1N1 virus was confirmed.
2.3. Population
Our study’s subjects were evaluated according to a clinical questionnaire, which contained questions regarding influenza vaccination. The inclusion period was from May 2009 to March 2012, so the subjects examined during the study belonged to three different influenza waves in Mexico. A total of 261 subjects with clinical symptoms and signs of influenza were enrolled, of which 128 were positive (INF-P) and 133 were negative for influenza A/H1N1 infection. On admission to the hospital, all patients with suspected influenza were treated with oseltamivir. In this study, most patients sought medical attention 3–4 days after the onset of symptoms. All clinical data were collected retrospectively; nonetheless, blood samples and nasopharyngeal swabs were collected at the hospital during the first 24 h after admission. We also included the variable “died” during the patients’ hospitalization to perform a mortality analysis.
In the healthy contacts (HC) group, 111 biologically unrelated, intrahousehold contacts were enrolled who were asymptomatic and in close, continuous contact with influenza A/H1N1 patients before and during hospital admission. None of the HCs developed symptoms or was hospitalized for influenza virus infection. Peripheral blood from these individuals was tested for anti-A/H1N1 antibody titers by hemagglutination inhibition (HI) assay according to the method described by Julkunen I, 1985 [
18] to determine exposure to the virus. All subjects included in this group had HI titers greater than 1:16, indicating direct contact with the virus. According to the samples’ availability in the biobank generated since 2009 in the HLA Laboratory, the participants were selected.
2.4. DNA Extraction
Firstly, we obtained 15 mL of peripheral blood via venipuncture in two EDTA tubes (as anticoagulant) from all 472 subjects. After that, the DNA extraction was performed using the commercial BDtract Genomic DNA isolation kit (Maxim Biotech, San Francisco, CA, USA). The DNA was quantified by UV absorption spectrophotometry at the 260-nm wavelength employing a NanoDrop 2000 device (Thermo Scientific, Wilmington, DE, USA). Contamination with organic compounds and proteins was determined by measuring the ratio absorbance at 260/280. Samples were considered of good quality when the ratio was ~1.8. All samples were adjusted to 15 µg/µL for subsequent genotyping.
2.5. SNP Selection
Four SNPs (rs1057149, rs2127679, rs4148882, and 41561219) in
TAP1; four (rs13501, rs241433, rs241441, and rs2071544) in
TAP2; two (rs2071888 and rs2282851) in
TAPBP; four (rs2071542, rs2071543, rs3763365, and rs9276810) in
PSMB8; and three (rs17587, rs241418, and rs2071534) in
PSMB9 genes were selected based on a bibliographic search in PubMed (NCBI), identifying polymorphisms previously associated with other virus infectious and respiratory diseases. The molecular characteristics of the SNPs evaluated are described in
Supplementary Table S1 (frequencies were obtained from the 1000 genomes project [
26]).
2.6. SNP Genotyping
The allele discrimination was performed using commercial TaqMan probes (Applied Biosystems, San Francisco CA, USA), employing qPCR in a 7300 Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific Inc., Singapore), and the analysis performed by sequence detection software version 1.4 (Applied Biosystems, San Francisco, CA, USA). Four controls without template (contamination controls) were included for each genotyping plate, and 5% of the genotyped in duplicate as controls for allele assignment.
2.7. Bioinformatic Analysis and in Silico Analysis
HaploReg v4.1 [
27] was consulted to determine the associated SNPs’ putative biologic effect. In addition, with the RNAfold web server [
26], we introduce the section’s sequence that includes the SNPs to elucidate for changes in the secondary RNA structures. We also conducted an in silico analysis with the associated SNPs to evaluate the possible function or biological consequences across diverse software, considering if they are coding or noncoding SNPs.
Supplementary Figure S1 shows the algorithm used for the in silico analysis of the associated SNPs.
2.8. Hardy–Weinberg Equilibrium and Haplotypes
The Hardy–Weinberg Equilibrium (HWE) was assessed using SNPStats online software for the 17 SNPs evaluated [
28]. The haplotype analysis was performed using Haploview software v. 4.2 [
29], using the Gabriel et al. [
30] established criteria.
2.9. Statistical Analysis
Clinical quantitative variables were analyzed using SPSS for Mac Os, v20.0 (SPSS software, IBM, Chicago, IL, USA). Kolmogorov–Smirnov normality test was used, and parametric or nonparametric tests were used as appropriate. Genotype analysis and genetic association models were carried out with Pearson’s chi-squared and Fisher’s exact tests using Epi Info v. 7.1 [
31] and Epi Dat v.3.1 software, and 2 × N and 2 × 2 contingency tables were made to estimate the genetic association for Influenza A/H1N1 infection and mortality. A
p-value < 0.05 was considered statistically significant. Bonferroni correction (multiple testing) was applied to adjust the significant values of the genetic association results. Comparisons were made between case and control groups. After that, a logistic regression analysis was performed to adjust for potential confounding variables (sex and body mass index (BMI)) using Plink v. 1.07 [
32].
4. Discussion
Clinical features of influenza A/H1N1 pdm09 infection associated with higher risk (hospitalization, disease severity, and mortality) have been widely investigated. Our study found that patients with influenza A/H1N1 infection were predominantly smoker men with higher BMI. These findings agree with previous reports, where they are added to the mentioned risk factors—pre-existing pulmonary diseases, diabetes, cardiovascular diseases, immunosuppression, and pregnancy are included [
3,
4,
33].
Additionally, previous studies in the Mexican population have reported that intensive care unit admission; low lymphocyte and platelet count; and high CPK, BUN, and LDH are associated with a higher risk for influenza A/H1N1 virus infection mortality [
6,
7,
8], coinciding with our results.
The HWE was assessed for all the SNPs evaluated. Unfortunately, six of the 17 selected SNPs (in
TAP2 and
TAPBP) did not meet it (
p > 0.05). However, we decided to continue with the subsequent analyses due to the SNPs position at the gene structure and their possible biological implications. After allele and genotype frequencies and adjustment results by covariates (sex and BMI), we found that rs2071888/GG genotype in
TAPBP is associated with increased risk for influenza A/H1N1 infection. Conversely, rs241433/AG genotype in
TAP2 is associated with reduced risk for influenza A/H1N1 infection. Nevertheless, both SNPs did not meet the HWE. Although this could seem a limitation, some authors have described that mestizo populations (such as Mexican) have a rich genetic variability, a product of the years of genetic recombination between ancestral populations (Amerindian, Caucasian, African, and Asian descendants), which could result in some SNPs having a different behavior from those described in the scientific literature [
34,
35].
TAP complex (shaped by TAP1 and TAP2 subunits) is a necessary transporter for translocating peptides, which were obtained via proteasome degradation, from the cytosol to ER, where HLA class I assembly takes place. Previous studies have demonstrated that in mice and humans, the TAP complex can play a crucial role in the translocation of viral epitopes to ER [
36,
37], and mutations in
TAP1 or
TAP2 result in structural changes that can alter antigen recognition and presentation. In this sense, SNPs in
TAP2 have been previously associated with a higher risk for ankylosing spondylitis [
22] and cervical carcinoma associated with HPV [
24]. It has been proposed that the genetic variation in this gene may cause functional alterations, possibly by affecting the ATP-binding capacity and transport efficiency of TAP, as well as the expression and structural stability of the mRNA and protein [
38]. The rs241433 has previously only been associated with a higher risk for rheumatoid arthritis [
39]. Our work is the first in which this SNP is associated with reduced risk for influenza A/H1N1 infection, and more functional and genetic studies are required to establish its role in the pathogenesis of this infectious disease. In the bioinformatic analysis, the rs241433/A allele has reduced affinity with the myeloid and B-cell development regulatory protein UP1; and on the other hand, the C allele has a high affinity for this protein. Additionally, no differences in predicted secondary mRNA structures were found with the rs241433. The UP1 protein (also known as heterogeneous nuclear ribonucleoprotein A1 (
HNRNPA1)) plays a crucial role in the regulation of alternative splicing, and multiple alternatively spliced transcript variants have been found [
40]. However, there are no previous studies in literature where the biological and pathological influence has been reported. In silico analysis for rs241433 shows no sites of alternative splicing or miRNA generation. However, functional studies are required to establish the specific biological participation of this SNP.
Tapasin is encoded by the
TAPBP gene. This protein plays an essential function in the peptides assembly previously translocated by TAP complex to HLA class I into the ER of cells [
17]. It has been reported that some viral products compromise the antigen presentation by interacting with the Tapasin protein [
41]. For example, the molluscum contagiosum virus encodes MC80 protein, which generates ER and Tapasin degradation, accompanied by a TAP loss, limiting HLA class I access to cytosolic peptides and a deficient antigen presentation [
42]. Moreover, in a Chinese population, the rs9277972 was associated with an increased risk for hepatitis C virus [
43]. On the other hand, the rs2071888 (SNP-associated in our study) has only been linked with gastrointestinal stromal tumors [
44] and aspirin-exacerbated respiratory disease [
45].
There are no previous studies where rs2071888 has been associated with influenza A/H1N1 or another viral infection. The rs2071888 is a missense variant in the
TAPBP exon 4, which generates a Threonine > Arginine change, which could alter the polarity of the protein structure, thereby modulating interactions with other molecules such as the HLA class I, leading to an altered antigen presentation and consequently, deficient infected cells clearance by CD8+ T cells, higher viral replication, and disease development [
45]. In this sense, we conducted a bioinformatics analysis where we found that the rs2071888/G allele decreases the affinity for a transcriptional factor, affecting the TAPBP expression, probably leading to a deficient antigen presentation. In addition, this SNP modifies the secondary structure of the mRNA, which could alter the protein function. Interestingly, in the in silico analysis, we corroborate that—as we described before—the rs2071888/G allele change from medium size and polar amino acid (threonine) change to large size and basis (arginine), which modifies the polarity of the protein structure [
46,
47].
We did not find significant associations with the three SNPs associated with influenza A/H1N1 infection in the mortality association analysis, possibly due to the reduced sample size. Other studies have evaluated different SNPs with disease severity (where mortality is included as a severe characteristic). García-Ramírez et al. [
11] described the rs361525 in
TNF associated with greater disease severity in the same Mexican population. In addition, Chen and colleagues [
48] found that the IFITM3 rs12252 SNP is associated with an increased risk of severe and mild influenza in Asian and Caucasian populations.
Furthermore, in the haplotype analysis, we found an increased frequency of the GG haplotype in the INF-P group, shaped by the two common alleles of rs3763365 and rs9276810 (both in the
PSMB8 gene), as well as a higher frequency of the AG haplotype, shaped by minor alleles of rs3763365 (
PSMB8) and rs4148882 (
TAP1) in HC group. Previously, it was established that catalytic proteasome subunits—PSMB8 and PSMB9—were closely linked to the
TAP1 and
TAP2 genes, suggesting a critical role in processing and antigen presentation [
41,
49]. To the best of our knowledge, to date, no studies have determined whether the products of different PSMB alleles differ concerning the peptides produced. A previous study demonstrated that an SNP (rs2071543) in
PSMB8 is associated with a higher risk for HP [
21]. Additionally, a study where rs2071543 was evaluated in cervical cancer showed that a common allele is associated with a higher risk for developing the oncological disease [
38], which is in the same sense as our haplotype results, suggesting an essential role of the proteasome in viral clearance and antigen presentation.
Other studies have evaluated single-nucleotide variants in diverse genes. For example, Zúñiga et al. [
50] found four polymorphisms associated with severe pneumonia in Influenza A/H1N1, including SNPs in the Fc fragment of immunoglobulin (Ig) G; low-affinity IIA; receptor (
FCGR2A), RPA interacting protein (
RPAIN); and complement component 1, q subcomponent binding protein (
C1QBP) genes. In this sense, Chatzopoulou and colleagues [
51] also found positive associations with SNPs in complement-associated genes (
FCGR2A,
C1QBP,
CD55).
Our study is not free of limitations. Firstly, we included a relatively small sample size for both cases and control; however, our study was done with samples from our laboratory’s biobank and exclusively with samples from patients with a confirmed influenza A/H1N1 diagnosis. Secondly, we carried out a mortality analysis; however, we have a reduced sample size. Thirdly, we did not measure the expression for any of the genes evaluated. Despite the associated SNPs not meet HWE, we conducted bioinformatics and in silico analysis, where we found data supporting our genetic-association study.