Abstract
Modifier genes, as the TNF-α gene, can modulate the cystic fibrosis (CF) severity. Thus, -238G>A and -308G>A polymorphisms of TNF-α gene were analyzed as modifiers of CF. In this context, the present study enrolled 49 CF patients (diagnosis performed by sweat test and complete CFTR screening). The -238G>A polymorphism analysis was performed by ARMS-PCR, and -308G>A, by PCR-RFLP. In our data, the -238G>A polymorphism was not associated with clinical variability. The AA genotype for -308G>A polymorphism was a risk factor for early gastrointestinal symptoms (OR=5.98, 95%CI=1.06-49.68) and protection for the first Pseudomonas aeruginosa (OR=0.05, 95%CI=0.0003-0.007). For the first P. aeruginosa, GA genotype was a risk factor (OR=10.2, 95%CI=1.86-84.09); for the same genotype, the diagnosis was made in minor time than the AA genotype (p=0.031). Considering the -308G>A polymorphism alleles, the G allele was a risk factor for early pulmonary symptoms (OR=3.81, 95%CI=1.13-12.97) and P. aeruginosa (OR=66.77, 95%CI=15.18-482.7); however, the same allele showed better transcutaneous oxygen saturation (OR=9.24, 95%CI=1.53-206.1). The A allele was a protective factor for early pulmonary symptoms (OR=12.26, 95%CI=0.08-0.89) and P. aeruginosa (OR=12.15, 95%CI=0002-0007), however, the same allele was a risk factor for worst transcutaneous oxygen saturation (OR=7.01, 95%CI=1.14-157.4). As conclusion, the -308G>A polymorphism of the TNF-α gene was associated with the CF severity.
Keywords: Cystic fibrosis, genotype, phenotype, polymorphism, TNF-α, TNF-alpha, CFTR, lung disease, inflammation, modifier gene
Introduction
Cystic fibrosis (CF) has high clinical variability conditioned by environmental factors, CFTR and modifier genes [1-3]. Our group has studied the association of modifier genes with clinical markers of CF, among the studied genes, we can highlight: MBL-2, TGF-β1, CD14 [4], GSTM1, GSTT1 [5,6], ACE [7], ADRB2 [8], TCF7L2 [9], COX-2 [10], ADRA2A [11], IFRD1 [12], GSTP1 and GCLC [6].
Among the genes with possible modifier effect of the CF severity, the TNF-α (region 6p21.3) has highlight. TNF-α gene is associated with the expression of tumor necrosis factor alpha by polymorphism in the promoter region, acting on transcriptional and post-transcriptional levels [13,14]. Some polymorphisms were associated with the severity of lung disease in CF [15] and infection by Pseudomonas aeruginosa [16]. The tumor necrosis factor has been found with high concentrations in the lungs of CF patients, suggesting action in the inflammatory lung disease [15,17].
The most studied polymorphisms are characterized by the exchange of a guanine by an adenine at positions -238 (rs361525) and -308 (rs1800629). The -238A>G polymorphism shows conflicting results acting in TNF expression [18,19], including in the CF [20]. While the -308A>G polymorphism was associated with the defense response produced by TNF-α [21]. The G allele (-308A>G polymorphism) is the rare allele and was associated with increased gene transcription and inflammatory process [21,22], being in CF, the allele responsible for the worst clinical presentation [15], lowest pulmonary function and lower body mass index (BMI) [23,24].
TNF-α protein is a multifunctional pro-inflammatory cytokine produced primarily by macrophages, being the main mediator of the acute inflammatory response to microorganisms, as well, responsible for systemic complications [25]. The main function of TNF-α is stimulate the recruitment of neutrophils and monocytes to sites of infection and activate these cells to eradicate microorganisms. TNF-α induces the vascular endothelial cells to express adhesion molecules that become the endothelial surface adhesive for leukocytes in the site of infection [25,26]. The TNF-α protein has function is the regulation of biological processes, such as: (i) cellular proliferation and differentiation, (ii) apoptosis, (iii) metabolism of lipids, and (iv) blood coagulation. Considering the importance of TNF protein and modulation of its expression by polymorphisms in TNF-α gene, the -238G>A and -308G>A polymorphisms in the regulatory region were analyzed and compared with clinical variables of CF patients with CF.
Material and methods
Casuistic
49 CF patients diagnosed by the sweat test were enrolled (sodium and chloride with values above 60 mEq/L). All patients were homozygous or compound heterozygous for mutations Class I or II in the CFTR gene. No patient had diagnosis performed by neonatal screening test. The study was approved by the Ethics Committee from our university (#570/2004).
DNA extraction
Patients’ DNA was obtained by phenol-chloroform extraction. The DNA concentration used for analysis was 50 ng/mL, evaluated using GE NanoVueTM Spectrophotometer (GE Healthcare Biosciences, Pittsburgh, USA).
TNF-α polymorphisms screening
The polymerase chain reaction (PCR) reaction for amplification of the TNF-α polymorphisms was performed with bidistilled water, 10x Taq buffer with (NH4)2SO4, MgCl2 (25 mM), dNTP (25 mM each nitrogenous base), primers (0.2 pmol), Taq polymerase (5U) and genomic DNA (50 ng/mL). For the -238A>G polymorphism was realized an amplification-refractory mutation system (ARMS)-PCR, being respectively for the internal control [5’-GCCCCTCCAGTTCTAGTTCTATC-3’ and 5’-CCGGATCATGCTTTCAGTGC-3’], allele A [5’-GCCCCTCCCAGTTCTAGTTCTATC-3’ and 5’-CACACTCCCATCCTCCCTGGTCT-3’] and allele G [5’-AGACCCCCCTCGGAATCG-3’ and 5’-CCGGATCATGCTTTCAGTGC-3’], observed a fragment of 608, 209 and 447 basis pair. For the -308A>G [primers sense 5’-AGGCAATAGGTTTTGAGGGCCAT-3’ and antisense 5’-GAGCGTCTGCTGGCTGGGTG-3’] polymorphism a fragment of 345 basis pair was amplified, follow by restriction fragment length polymorphism (RFLP) technique.
The PCR digestion was performed with the enzyme NcoI (New England BioLabs, Ipswich, MA). Before the digestion, the purification was performed with 10 μL of the PCR and 80 μL of isopropyl alcohol (75%), follow by 10 min on 25°C, and after centrifuged at 13,000 rpm for 30 min. The tube was inverted and was added 150 μL of ethyl alcohol (70%) at room temperature and again centrifuged at the same speed for 10 min. After centrifugation, the tube was inverted and allowed to dry at room temperature, and 10 μL of water was added. From this volume, was added 2.0 uL of enzyme specific buffer (10 mM Tris-HCl pH 7.5; 50 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.2 mg/mL BSA and 50% glycerol), 7.5 mL of water and 0.5 mL of enzyme (NcoI) (5U). Digestion was carried out at 37°C. The G allele is not digested. The A allele represents the restriction site (GG - 345 basis pair, GA - 345, 320 and 25 basis pair, AA - 320 and 25 basis pair).
Clinical variables
The clinical data included in the study were: CFTR mutations, age at diagnosis, age at onset of pulmonary and digestive symptoms, first isolation by P. aeruginosa, spirometry, Shwachman-Kulczycki and Kanga scores, and transcutaneous oxygen saturation of hemoglobin.
For age at diagnosis, age at onset of pulmonary and digestive symptoms, and time for the first isolation of P. aeruginosa, the following groups were included: under 12 months, from 13 to 36 months, above 36 months. In the case of digestive symptoms was considered meconium ileus as an additional risk factor. For P. aeruginosa, some patients were decolonized until the beginning of the study period, being not included in the statistical analysis.
In the spirometry test was considered severe obstruction, values for the forced expiratory volume in one second (FEV1%) minor than 40% of the predicted, moderate obstruction, values between 40 to 60%, mild obstruction, values between 60 to 80%, and normal lung function, values greater than 80%. In data analysis, the FEV1% mean was used for the classification of severity, being compared minor and greater values than the median.
The Spirometry proof was performed using a speedometer model CPFS/D (Med Graphics, Saint Paul, Minnesota, USA). Data were recorded by the BREEZE PF Version 3.8 B software for Windows 95/98/NT.
The Shwachman-Kulczycki score is graded as excellent (86-100), good (71-85), mild (56-70), moderate (41-55) and severe (40 or less), taking into account the number of points. In the study, the categories were compared between the polymorphisms analyzed directly, considering the possible genotypes and alleles, and reassembled, as follow: excellent and good scores became mild, the mild score became moderate, and moderate and severe scores were defined as severe. The distribution of scores in three categories was performed to allow statistical analysis considering the number of patients included in the study. The score was evaluated by two professionals of the Pediatric Clinic of the Clinical Hospital of the Faculty of Medical Sciences, to better evaluation, being a subjective analysis. In case of disparity between evaluators, third evaluation was performed by a qualified professional [27].
The score Kanga was analyzed considering the presence or absence of exacerbation through the points obtained in the analysis [27].
Transcutaneous oxygen saturation of hemoglobin was used in the classification: saturation greater than 95% - normal value, between 91 to 95 - mild hypoxemia, between 85 to 90 - moderate hypoxemia and minor than 8% - severe hypoxemia.
Statistical analysis
The statistical analysis was performed by Statistical Package for Social Sciences version 21.0 (SPSSv.21). The Kruskal-Wallis test and T-student test were conducted to assess the interaction between the phenotypic criteria with numerical distribution, and the χ2 test or Fisher’s exact test for categorical variables, with polymorphisms of the TNF-α gene, taking into account the genotypes and alleles. In the data analysis was considered α=0.05.
Results
Of the 49 subjects enrolled, the age ranged from one to 26 years, with a mean of 9.71 years (± 6.06 years). Distribution by age group showed 34 patients between zero and 10 years, 12 between 11 and 21, and three above 21 years. 25 women (51.02%) was included. The mean age of diagnosis was 29.70 months (± 2.47 months). For the first isolation of P. aeruginosa, the mean age was 38.11 months (± 3.17 months).
Considering CFTR genotype, F508del homozygous predominated (63.26%). The F508del allele had the highest prevalence (79.59%), followed by G542X (9.18%), R1162X (6.12%), N1303K (3.06%) and R553X (2.04%).
The results for TNF-α and CFTR genotype, Shwachman-Kulczycki and Kanga scores, transcutaneous oxygen saturation of hemoglobin and FEV1% are shown in Table 1. The population description for the clinical variables with categorical distribution is shown in Table 2. In Table 3, the frequency of genotypes for the polymorphisms of TNF-α gene is described, including the allele frequency and Hardy-Weinberg equilibrium. In the population studied, both polymorphisms are in balance.
Table 1.
Population characterization for the TNF-α and CFTR genotypes, Shwachman-Kulczycki and Kanga scores, transcutaneous oxygen saturation of hemoglobin and forced expiratory volume in the first second (%) (FEV1) of forced vital capacity
| Patient | TNF-α genotype | CFTR genotype | Shwachman-Kulczycki | Kanga | Oxygen saturation | FEV1% | |
|---|---|---|---|---|---|---|---|
|
| |||||||
| -238G>A | -308G>A | ||||||
| MVG | G/G | G/A | F508del/F508del | Moderate | Normal | Normal | Normal |
| JPD | G/G | G/A | F508del/F508del | **** | Normal | Normal | Mild |
| RSR | G/A | A/A | F508del/F508del | Excellent | Normal | Mild | Normal |
| EVM | G/G | A/A | F508del/R1162X | Excellent | Normal | Mild | Severe |
| IFM | G/G | A/A | F508del/F508del | **** | **** | **** | **** |
| MLA | G/G | A/A | F508del/F508del | **** | Exacerbate | Normal | Moderate |
| VLPC | G/G | A/A | F508del/F508del | Moderate | Normal | Normal | Normal |
| MAB | G/G | A/A | F508del/F508del | Severe | Exacerbate | Mild | Severe |
| EVSMS | G/G | A/A | F508del/F508del | **** | Normal | Normal | Mild |
| YBK | G/G | A/A | F508del/F508del | Moderate | Normal | Normal | Normal |
| HB | G/A | A/A | F508del/G542X | **** | Normal | Severe | Severe |
| WSP | G/G | A/A | F508del/G542X | Moderate | Normal | Mild | Severe |
| DRG | G/G | A/A | F508del/F508del | **** | **** | **** | **** |
| BBK | G/G | G/A | F508del/N1303K | **** | **** | **** | **** |
| AO | G/G | A/A | F508del/F508del | **** | Exacerbate | Mild | Mild |
| LSM | G/A | A/A | F508del/F508del | Excellent | Normal | Normal | Moderate |
| VAL | G/G | A/A | F508del/F508del | Good | Normal | Mild | Mild |
| LFSA | G/G | A/A | F508del/F508del | **** | Normal | Mild | Mild |
| IBN | G/G | A/A | F508del/R553X | Excellent | Normal | Normal | Normal |
| CAQ | G/G | G/G | F508del/G542X | **** | Normal | Normal | Mild |
| MEMZ | G/A | A/A | F508del/F508del | **** | Normal | Normal | Normal |
| TMG | G/G | G/G | F508del/F508del | Moderate | Normal | Normal | Normal |
| JMGR | G/G | G/A | F508del/N1303K | **** | Normal | Normal | **** |
| MAP | G/A | G/G | F508del/F508del | **** | **** | **** | **** |
| AX | G/G | A/A | F508del/F508del | Good | Normal | Normal | Normal |
| LPOL | G/G | G/A | F508del/N1303K | Good | Normal | Normal | **** |
| EG | G/G | A/A | F508del/R1162X | Good | Normal | Normal | Normal |
| NCB | G/G | G/A | F508del/R553X | **** | Normal | Mild | Severe |
| FVV | G/G | A/A | F508del/G542X | Excellent | Normal | Normal | Normal |
| LSS | G/G | A/A | F508del/F508del | **** | Exacerbate | Moderate | **** |
| RNC | G/G | A/A | F508del/F508del | **** | Normal | Normal | **** |
| FEL | G/G | A/A | F508del/G542X | Moderate | Normal | Normal | Moderate |
| GOV | G/G | G/A | F508del/F508del | **** | Normal | Normal | **** |
| CVAR | G/G | A/A | F508del/G542X | **** | Normal | Normal | **** |
| EAG | G/G | A/A | F508del/F508del | **** | Normal | Mild | **** |
| AVM | G/G | G/G | DF508/G542X | Excellent | Normal | Normal | Normal |
| BSG | G/G | A/A | G542X/R1162X | Good | Exacerbate | Mild | Mild |
| GPNS | G/A | A/A | F508del/F508del | **** | Normal | Normal | **** |
| CAL | G/G | A/A | DF508/G542X | Good | Normal | Normal | Normal |
| MOS | G/G | G/G | F508del/F508del | Moderate | Normal | Normal | **** |
| FSC | G/G | A/A | F508del/F508del | Good | Normal | Normal | Normal |
| ITVS | G/G | G/A | F508del/F508del | **** | Normal | Normal | Normal |
| JRL | G/G | G/G | F508del/F508del | Mild | Normal | Normal | **** |
| GPT | G/A | A/A | F508del/F508del | Excellent | Normal | Normal | Normal |
| ASR | G/A | G/A | F508del/R1162X | **** | **** | **** | **** |
| VAF | G/G | A/A | F508del/F508del | **** | Normal | Normal | **** |
| IRO | G/A | G/G | F508del/F508del | **** | Normal | Normal | **** |
| BMSL | G/G | A/A | R1162X/R1162X | **** | **** | **** | **** |
| BSC | G/A | A/A | F508del/F508del | Moderate | Normal | Normal | Mild |
****: no data; CFTR = Cystic Fibrosis Transmembrane Regulator; TNF-α = tumor necrosis factor.
Table 2.
Patients distribution according with the clinical characteristics taking into account categorical data
| Clinical variable | Category | Number of patients (%) |
|---|---|---|
| Age at diagnosis | <12 months | 27 (56.25) |
| 13 to 36 months | 8 (16.66) | |
| >36 months | 13 (27.08) | |
| Onset of pulmonary symptoms | <12 months | 33 (70.21) |
| 13 to 36 months | 7 (14.89) | |
| >36 months | 5 (10.64) | |
| No clinical symptom | 2 (4.25) | |
| Onset of digestive symptoms | <12 months | 32 (68.08) |
| 13 to 36 months | 4 (8.51) | |
| >36 months | 4 (8.51) | |
| Meconium ileus | 7 (14.89) | |
| No clinical symptoms | 1 (2.12) | |
| Age for the first infection by Pseudomonas aeruginosa | <12 months | 13 (27.66) |
| 13 to 36 months | 14 (29.79) | |
| >36 months | 15 (31.91) | |
| Without bacteria | 5 (10.64) | |
| Kanga score | Non exacerbation | 38 (88.37) |
| Exacerbation | 5 (11.36) | |
| Shwachman-Kulczycki score | Excellent | 7 (29.16) |
| Good | 7 (29.16) | |
| Moderate | 8 (33.33) | |
| Mild | 1 (4.16) | |
| Severe | 1 (4.16) | |
| Transcutaneous oxygen saturation of hemoglobin | Normal | 32 (72.72) |
| Mild | 10 (22.72) | |
| Moderate | 1 (2.27) | |
| Severe | 1 (2.27) | |
| Forced expiratory volume in first second of the FVC | Normal | 15 (51.72) |
| Mild | 8 (27.59) | |
| Moderate | 3 (10.34) | |
| Severe | 3 (10.34) |
FVC = forced vital capacity.
Table 3.
Genotypes for the -238G>A and -308G>A polymorphisms of the TNF-α gene in cystic fibrosis patients
| Polymorphism | Genotype | Number of patients (%) |
|---|---|---|
| -238G>A* | G/G | 39 (79.59) |
| A/A | 0 | |
| G/A | 10 (20.41) | |
| Total | 49 | |
| -308G>A# | GG | 7 (14.28) |
| GA | 9 (18.36) | |
| AA | 33 (67.35) | |
| Total | 49 |
TNF-α = tumor necrosis factor.
Hardy-Weinberg equilibrium - p>0.05; χ2=2.0. Allelic frequency: G allele = 0.90, A allele = 0.10.
Hardy-Weinberg equilibrium - p>0.05; χ2=2.57. Allelic frequency: G allele = 0.23, A allele = 0.77.
In Tables 4,5 and 6 are described the associations of the -238G>A polymorphisms of the TNF-α gene, considering, respectively, clinical categorical variables and genotypes for the polymorphism, clinical categorical variables and allele frequency for the polymorphism, and numeric clinical variables and genotypes/allele for the polymorphism. For the polymorphism -238G>A, no association with clinical variables was observed.
Table 4.
Association of -238G>A polymorphism (genotypes) of the TNF-α gene with clinical variables of cystic fibrosis patients
| Clinical variables | Groups | Genotypic distribution | |||
|---|---|---|---|---|---|
|
| |||||
| G/G | OR (95%CI) | G/A | OR (95%CI) | ||
| Age at diagnosis | <12 months | 4 | 0.44 (0.09-1.89) | 23 | 2.26 (0.53-10.49) |
| 13 to 36 months | 4 | 5.39 (0.98-30.7) | 4 | 0.19 (0.03-1.02) | |
| >36 months | 2 | 0.62 (0.08-3.22) | 11 | 1.61 (0.31-12.67) | |
| Onset of pulmonary symptoms | <12 months | 6 | 0.53 (0.12-2.56) | 26 | 1.90 (0.39-8.64) |
| 13 to 36 months | 2 | 1.49 (0.17-9.18) | 5 | 0.67 (0.11-5.81) | |
| >36 months | 2 | 1.91 (0.21-12.84) | 4 | 0.52 (0.08-4.72) | |
| No clinical symptom | 0 | - | 2 | - | |
| Onset of digestive symptoms | <12 months | 7 | 0.88 (0.15-7.54) | 24 | 1.14 (0.13-6.89) |
| 13 to 36 months | 2 | 3.82 (0.35-42.28) | 2 | 0.26 (0.02-2.88) | |
| >36 months | 0 | - | 4 | - | |
| No clinical symptom | 0 | - | 1 | - | |
| Age for the first infection by Pseudomonas aeruginosa | <12 months | 4 | 1.93 (0.39-9.10) | 11 | 0.52 (0.11-2.54) |
| 13 to 36 months | 3 | 1.22 (0.22-5.87) | 11 | 0.82 (0.17-4.63) | |
| >36 months | 2 | 0.71 (0.09-3.79) | 11 | 1.42 (0.26-11.3) | |
| No clinical symptom | 0 | - | 5 | - | |
| Forced expiratory volume in first second of the FVC | Normal (>80%) | 3 | 1 (0.14-6.90) | 12 | 1 (0.14-6.90) |
| Mild (60-80%) | 1 | 0.61 (0.02-5.66) | 6 | 1.64 (0.18-45.91) | |
| Moderate (40-60%) | 1 | 2.13 (0.06-33.11) | 2 | 0.47 (0.03-15.98) | |
| Severe (<40%) | 1 | 1 (0.03-10.34) | 4 | 1 (0.10-29.45) | |
| Shwachman-Kulczycki score | Excellent (86-100) | 4 | 2.35 (0.37-16.63) | 3 | 1.36 (0.19-8.90) |
| Good (71-85) | 1 | 0.16 (0.006-1.39) | 6 | 6.26 (0.72-172.2) | |
| Mild (56-70) | 0 | - | 1 | - | |
| Moderate (41-55) | 4 | 1.63 (0.27-10.02) | 4 | 0.61 (0.10-3.69) | |
| Severe (≤40) | 1 | - | 0 | - | |
| Kanga score | Exacerbated | 2 | 2.86 (0.29-22.87) | 3 | 0.35 (0.04-3.41) |
| Non exacerbated | 7 | 0.35 (0.04-3.41) | 31 | 2.86 | |
| Transcutaneous oxygen saturation of hemoglobin | Normal (>95%) | 9 | 4.38 (0.60-107.9) | 22 | 0.23 (0.009-1.66) |
| Mild (91-95%) | 1 | 0.30 (0.01-2.26) | 9 | 3.30 (0.44-82.39) | |
| Moderate (85-90%) | 0 | - | 1 | - | |
| Severe (<85%) | 0 | - | 1 | - | |
TNF-α = tumor necrosis factor; FVC = forced ventilator capacity. Statistical analysis performed by χ2 test and Fisher’s exact test.
Table 5.
Association of -238G>A polymorphism (alleles) of the TNF-α gene with clinical variables of cystic fibrosis patients
| Clinical variables | Groups | Allelic distribution | |||
|---|---|---|---|---|---|
|
| |||||
| G allele | OR (95%CI) | A allele | OR (95%CI) | ||
| Age at diagnosis | <12 months | 50 | 2.07 (0.53-8.86) | 4 | 0.48 (0.11-1.89) |
| 13 to 36 months | 12 | 0.25 (0.06-1.13) | 4 | 4.03 (0.89-17.01) | |
| >36 months | 24 | 1.54 (0.33-11.29) | 2 | 0.65 (0.09-3.05) | |
| Onset of pulmonary symptoms | <12 months | 58 | 17.46 (0.40-6.99) | 6 | 0.57 (0.14-2.50) |
| 13 to 36 months | 12 | 0.71 (0.14-5.40) | 2 | 1.41 (0.19-7.05) | |
| >36 months | 10 | 0.58 (0.11-4.46) | 2 | 1.74 (0.22-8.91) | |
| No clinical symptom | 4 | - | 0 | - | |
| Onset of digestive symptoms | <12 months | 55 | 1.12 (0.15-5.71) | 7 | 0.89 (0.18-6.86) |
| 13 to 36 months | 6 | 0.34 (0.06-2.85) | 2 | 2.94 (0.35-17.31) | |
| >36 months | 8 | - | 0 | - | |
| No clinical symptom | 2 | - | 0 | - | |
| Age for the first infection by Pseudomonas aeruginosa | <12 months | 26 | 0.67 (0.16-3.01) | 4 | 1.5 (0.33-6.42) |
| 13 to 36 months | 25 | 1 (0.23-5.25) | 3 | 1 (0.19-4.37) | |
| >36 months | 24 | 1.64 (0.34-12.25) | 2 | 0.61(0.08-2.98) | |
| No clinical symptom | 10 | - | 0 | - | |
| Forced expiratory volume in first second of the FVC | Normal (>80%) | 27 | 1 (0.16-6.29) | 3 | 1 (0.16-6.29) |
| Mild (60-80%) | 13 | 1.57 (0.19-40.47) | 1 | 0.64 (0.02-5.13) | |
| Moderate (40-60%) | 5 | 0.52 (0.06-14.46) | 1 | 1.93 (0.07-18.17) | |
| Severe (<40%) | 9 | 1 (0.12-26.31) | 1 | 1 (0.04-8.40) | |
| Shwachman-Kulczycki score | Excellent (86-100) | 10 | 0.54 (0.12-2.59) | 4 | 1.84 (0.39-8.22) |
| Good (71-85) | 13 | 4.56 (0.64-110.9) | 1 | 0.22 (0.009-1.56) | |
| Mild (56-70) | 2 | - | 0 | - | |
| Moderate (41-55) | 12 | 0.70 (0.16-3.28) | 4 | 1.43 (0.31-6.26) | |
| Severe (≤40) | 1 | 0.25 (0.006-10.51) | 1 | 3.95 (0.10-164.2) | |
| Kanga score | Exacerbated | 8 | 0.41 (0.07-3.32) | 2 | 2.43 (0.30-13.36) |
| Non exacerbated | 69 | 2.43 (0.30-13.36) | 7 | 0.41 (0.07-3.32) | |
| Transcutaneous oxygen saturation of hemoglobin | Normal (>95%) | 53 | 0.26 (0.01-1.71) | 9 | 3.86 (0.58-89.85) |
| Mild (91-95%) | 19 | 2.97 (0.44-69.6) | 1 | 0.34 (0.01-2.25) | |
| Moderate (85-90%) | 2 | - | 0 | - | |
| Severe (<85%) | 2 | - | 0 | - | |
TNF-α = tumor necrosis factor; FVC = forced ventilator capacity. Statistical analysis performed by χ2 test and Fisher’s exact test.
Table 6.
Association of the -238G>A e -308G>A polymorphisms of the TNF-α gene with clinical variables with numerical distribution
| Clinical variable | -238G>A genotype | N (%) | Mean | Standard deviation | p-value |
| Age at diagnosis (months) | GG | 34 (77.27) | 30.23 | 40.11 | 1.0 |
| GA | 10 (22.72) | 27.09 | 39.39 | ||
| Onset of pulmonary symptoms (months) | GG | 32 (78.04) | 22.22 | 44.31 | 1.0 |
| GA | 9 (21.95) | 13 | 18.43 | ||
| Onset of digestive symptoms (months) | GG | 20 (74.04) | 22.45 | 29.39 | 0.126 |
| GA | 17 (62.96) | 5 | 3.31 | ||
| Forced expiratory volume in first second of the FVC | AG | 6 (19.35) | 75 | 33.97 | 0.4973 |
| GG | 25 (80.64) | 73 | 28.39 | ||
| Shwachman-Kulczycki score | AG | 4 | 85 | 12.90 | 1.0 |
| GG | 20 | 74 | 13.38 | ||
| Clinical variable | -308G>A genotype | N (%) | Mean | Standard deviation | p-value |
| Age at diagnosis (months) | GG | 7 (15.90) | 12.71 | 13.04 | GG vs GA = 1.0 |
| GA | 8 (18.18) | 39.75 | 66.42 | GG vs AA = 1.0 | |
| AA | 29 (65.90) | 31.03 | 33.97 | GA vs AA = 0.031 | |
| Onset of pulmonary symptoms (months) | GG | 7 (17.07) | 9.28 | 15.14 | GG vs GA = 1.0 |
| GA | 8 (19.51) | 31.85 | 62.68 | GG vs AA = 1.0 | |
| AA | 26 (63.41) | 18.30 | 36.96 | GA vs AA = 0.226 | |
| Onset of digestive symptoms (months) | GG | 3 (11.11) | 4.33 | 1.52 | GG vs GA = 1.0 |
| GA | 4 (14.81) | 23.25 | 25.07 | GG vs AA = 1.0 | |
| AA | 20 (74.07) | 18.9 | 28.54 | GA vs AA = 1.0 | |
| Forced expiratory volume in first second of the FVC | GG | 3 (9.68) | 85 | 13.45 | GG vs GA = 1.0 |
| GA | 4 (12.90) | 82 | 48.39 | GG vs AA = 1.0 | |
| AA | 24 (77.42) | 71 | 27.10 | GG vs GA = 0.253 | |
| Shwachman-Kulczycki score | GG | 4 (16.16) | 74 | 11.81 | GG vs GA = 1.0 |
| GA | 4 (16.16) | 62 | 14.14 | GG vs AA = 1.0 | |
| AA | 16 (66.66) | 81 | 15.62 | GA vs GG = 1.0 |
TNF-α = tumor necrosis factor; FVC = forced ventilator capacity; N = number of patients. Positive p-value is in bold. Statistical analysis performed by Kruskal-Wallis test and T-student test.
In Tables 6, 7 and 8 are described associations of the -308G>A polymorphisms of the TNF-α gene, considering, respectively, numeric clinical variables and genotypes/allele for the polymorphism, clinical categorical variables and genotypes for the polymorphism, and clinical categorical variables and allele frequency for the polymorphism.
Table 7.
Association of -308G>A polymorphism (genotypes) of the TNF-α gene with clinical variables of cystic fibrosis patients
| Clinical variable | Groups | Genotype distribution | |||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| G/G | OR (95%CI) | G/A | OR (95%CI) | A/A | OR (95%CI) | ||
| Age at diagnosis | <12 months | 5 | 2.13 (0.37-17.39) | 3 | 0.32 (0.06-1.49) | 19 | 1.45 (0.42-5.03) |
| 13 to 36 months | 1 | 0.81 (0.03-6.80) | 3 | 3.29 (0.53-18.61) | 4 | 0.44 (0.08-2.23) | |
| >36 months | 1 | 0.41 (0.02-3.18) | 3 | 1.44 (0.25-6.98) | 9 | 1.17 (0.30-5.18) | |
| Onset of pulmonary symptoms | <12 months | 4 | 0.41 (0.07-2.55) | 8 | 3.31 (0.45-82.17) | 22 | 2.92 (0.53-17.87) |
| 13 to 36 months | 3 | 6.00 (0.85-41.44) | 1 | 0.63 (0.024-5.17) | 3 | 0.36 (0.06-1.97) | |
| >36 months | 0 | - | 0 | - | 5 | - | |
| No clinical symptom | 0 | - | 0 | - | 1 | - | |
| Onset of digestive symptoms | <12 months | 3 | 0.33 (0.04-3.32) | 7 | - | 21 | 5.98 (1.06-49.68) |
| 13 to 36 months | 1 | 2.50 (0.08-30.47) | 0 | - | 3 | 1.36 (0.13-39.32) | |
| >36 months | 1 | 2.50 (0.08-30.47) | 0 | - | 3 | 1.36 (0.13-39.32) | |
| Age for the first infection by Pseudomonas aeruginosa | <12 months | 7 | - | 7 | 10.2 (1.86-84.09) | 1 | 0.08 (0.0003-0.07) |
| 13 to 36 months | 0 | - | 2 | 1 | 11 | 1 | |
| >36 months | 0 | - | 0 | - | 14 | - | |
| No clinical symptom | 0 | - | 0 | - | 5 | - | |
| Forced expiratory volume in first second of the FVC | Normal (>80%) | 2 | 2.25 (0.16-72.3) | 2 | 1.07 (0.10-11.6) | 11 | 0.54 (0.10-3.78) |
| Mild (60-80%) | 1 | 1.48 (0.04-22.1) | 1 | 0.95 (0.32-10.58) | 6 | 0.84 (0.13-7.65) | |
| Moderate (40-60%) | 0 | - | 0 | - | 3 | - | |
| Severe (<40%) | 0 | - | 1 | 1.87 (0.06-22.97) | 4 | 1.19 (0.12-34.39) | |
| Shwachman-Kulczycki score | Excellent (86-100) | 2 | 1.82 (0.17-15.96) | 2 | 0.74 (0.08-5.15) | 3 | 0.68 (0.10-4.29) |
| Good (71-85) | 2 | 1.82 (0.17-15.96) | 1 | 0.32 (0.01-2.91) | 4 | 1.48 (0.23-10.16) | |
| Mild (56-70) | 1 | - | 0 | - | 0 | - | |
| Moderate (41-55) | 0 | - | 4 | 4.04 (0.60-31.01) | 4 | 1 (0.17-5.97) | |
| Severe (≤40) | 0 | - | 0 | - | 1 | - | |
| Kanga score | Exacerbated | 0 | - | 2 | 4.19 (0.41-35.52) | 3 | 0.62 (0.08-5.81) |
| Non exacerbated | 6 | - | 5 | 0.24 (0.028-2.42) | 27 | 1.62 (0.17-12.31) | |
| Transcutaneous oxygen saturation of hemoglobin | Normal (>95%) | 6 | - | 6 | 2.59 (0.33-66.37) | 19 | 0.15 (0.0006-1.05) |
| Mild (91-95%) | 0 | - | 1 | 0.51 (0.02-4.10) | 9 | 4.99 (0.69-122.5) | |
| Moderate (85-90%) | 0 | - | 0 | - | 1 | - | |
| Severe (<85%) | 0 | - | 0 | - | 1 | - | |
TNF-α = tumor necrosis factor; FVC = forced ventilator capacity. Positive p-value is in bold. Statistical analysis performed by χ2 test and Fisher’s exact test.
Table 8.
Association of -308G>A polymorphism (alleles) of the TNF-α gene with clinical variables of cystic fibrosis patients
| Clinical variables | Groups | Allelic distribution | |||
|---|---|---|---|---|---|
|
| |||||
| G allele | OR (95%CI) | A Allele | OR (95%CI) | ||
| Age at diagnosis | <12 months | 13 | 1.01 (0.39-2.69) | 41 | 0.99 (0.37-2.56) |
| 13 to 36 months | 5 | 1.56 (0.44-5.06) | 11 | 0.64 (0.20-2.29) | |
| >36 months | 5 | 0.69 (0.21-2.05) | 21 | 1.45 (0.48-4.87) | |
| Onset of pulmonary symptoms | <12 months | 16 | 0.75 (0.26-2.25) | 52 | 1.33 (0.45-3.79) |
| 13 to 36 months | 7 | 3.81 (1.13-12.97) | 7 | 0.26 (0.08-0.89) | |
| >36 months | 0 | - | 10 | - | |
| No clinical symptom | 0 | - | 2 | - | |
| Onset of digestive symptoms | <12 months | 13 | 0.80 (0.22-3.29) | 49 | 1.25 (0.30-4.48) |
| 13 to 36 months | 2 | 1.22 (0.16-6.44) | 6 | 0.82 (0.16-6.43) | |
| >36 months | 2 | 1.22 (0.16-6.44) | 6 | 0.82 (0.16-6.43) | |
| Age for the first infection by Pseudomonas aeruginosa | <12 months | 21 | 66.77 (15.18-482.7) | 9 | 0.15 (0.002-0.07) |
| 13 to 36 months | 2 | 0.15 (0.02-0.62) | 24 | 6.69 (1.62-45.66) | |
| >36 months | 0 | - | 28 | - | |
| No clinical symptom | 0 | - | 10 | - | |
| Forced expiratory volume in first second of the FVC | Normal (>80%) | 6 | 2.30 (0.52-12.34) | 24 | 0.44 (0.08-1.94) |
| Mild (60-80%) | 3 | 1.49 (0.27-6.91) | 13 | 0.67 (0.14-3.70) | |
| Moderate (40-60%) | 0 | - | 6 | - | |
| Severe (<40%) | 0 | - | 9 | - | |
| Shwachman-Kulczycki score | Excellent (86-100) | 6 | 2.41 (0.60-9.84) | 8 | 0.41 (0.10-1.68) |
| Good (71-85) | 5 | 1.51 (0.36-6.08) | 9 | 0.66 (0.16-2.77) | |
| Mild (56-70) | 2 | - | 0 | - | |
| Moderate (41-55) | 0 | - | 12 | - | |
| Severe (≤40) | 0 | - | 2 | - | |
| Kanga score | Exacerbated | 2 | 0.87 (0.12-4.20) | 8 | 1.15 (0.24-8.58) |
| Non exacerbated | 17 | 1 | 59 | 1 | |
| Transcutaneous oxygen saturation of hemoglobin | Normal (>95%) | 18 | 9.24 (1.53-206.1) | 44 | 0.11 (0.005-0.66) |
| Mild (91-95%) | 1 | 0.14 (0.006-0.87) | 19 | 7.01 (1.14-157.4) | |
| Moderate (85-90%) | 0 | - | 2 | - | |
| Severe (<85%) | 0 | - | 2 | - | |
TNF-α = tumor necrosis factor; FVC = forced ventilator capacity. Positive p-value is in bold. Statistical analysis performed by χ2 test and Fisher’s exact test.
The AA genotype for -308A>G polymorphism was a risk factor for early onset of digestive symptoms (under 12 months) (OR=5.98, 95%CI=1.06-49.68) and protection for the first isolation of P. aeruginosa (under 12 months) (OR=0.05, 95%CI=0.0003-0.007). For the first P. aeruginosa, the heterozygous genotype was a risk factor for early isolation (under 12 months) (OR=10.2, 95%CI=1.86-84.09); and for the same genotype, the diagnosis was made in minor time than the AA genotype (p=0.031).
Considering the alleles for the -308G>A polymorphism, the G allele was a risk factor for early onset of pulmonary symptoms (OR=3.81, 95%Cl=1.13-12.97), and for the first P. aeruginosa (under 12 months) (OR=66.77, 95%CI=15.18-482.7), however, the same allele was a protective factor for better values of transcutaneous oxygen saturation (>95%) (OR=9.24, 95%CI=1.53-206.1). The A allele was a protective factor for early onset of pulmonary symptoms (OR=12.26, 95%CI=0.08-0.89), and for the first P. aeruginosa (under 12 months) (OR=12.15, 95%CI=0002-0007), however, the same allele was a risk factor for worst values of transcutaneous oxygen saturation (values between 91 to 95%) (OR=7.01, 95%CI=1.14-157.4).
Discussion
The difficulty in establishing association between CFTR genotype and CF phenotype is well known in the scientific literature. Currently, the search for modifier genes of the phenotype has gained prominence, and allowed a better understanding of the clinical phenotype of the CF patients.
Cystic fibrosis population
There was a predominated adolescent population, with a mean age of 9.71 (± 6.6) years. In age group distribution, the most patients were in the group between zero and ten years (69.38%). By the inclusion of only patients with severe mutations, the observed data may be associated with severe prognosis, with low life expectancy associated with severe CFTR mutations.
However, life expectancy, quality of life and prognosis in CF have improved. One of the factors that have been blamed for this fact is the systematic care of patients in specialized centers [28]. In the 30s, when the CF was described, 80% of children died in the first years of life. In 1980, the survival rate increased to 20 years, reaching 28 in the 90s and in 2001 reached 32 years old [29].
Regarding sex, a uniform distribution was observed, with 51.02% of women, a fact associated with autosomal recessive inheritance. However, the literature reports a slight predominance of males compared to females, with the increasing of the patients age, and in Brazil, the same was observed with males with values around 60% [30,31]. Results with different data of our study. However, in our study, only patients with two mutations of Class I and II were included. The lowest prevalence in females may occur by female vulnerability to certain clinical characteristics, such as, the occurrence of diabetes mellitus.
The mean of age at diagnosis (2.47 years) was higher than that found by Dorfman et al. [32] (0.36 years) in a group of 611 patients F508del homozygous. The mean of age for the first infection by the P. aeruginosa (3.17 years) was minor than that described by Dorfman et al. [32] (7.5 years). The difference can be explained by the age of diagnosis of the Dorfman et al. [32] to be minor than the present study, suggesting that treatment of those patients started before our sample, what may have delayed the colonization by P. aeruginosa.
Analysis of polymorphisms in TNF-α gene
Analysis of polymorphism -238G>A in TNF-α gene
Of the 49 CF patients, 79.59% were homozygous for the G allele, and 20.41% heterozygous. The allele frequency found was 0.90 and 0.10 for the G and A allele, respectively. Buranawuti et al. [20] analyzed 101 children and 115 adults with CF, and found the GG genotype in most individuals analyzed (91.1%), and, as in our study, no patients with the AA genotype was found. Yarden et al. [16] studied 180 CF patients with F508del/F508del genotype and the GG genotype was found with higher frequency (88.2%), being the AA genotype detected in 1.2% of the patients.
Age at diagnosis, onset of pulmonary and digestive symptoms were not associated with the polymorphism. It was expected that the age at diagnosis and onset of symptoms occurred in most patients within the first year of life, which denotes a worse prognosis and more severe presentation of the disease, and that these, were associated with more severe allele, however, there was no association.
For the first isolation of P. aeruginosa was not found association with the -238G>A genotype of TNF-α gene. This data is in accordance with Yarden et al. [16] study (p=0.64). As the most patients had early diagnosis, it was expected that the age of first P. aeruginosa infection was delayed, being that the early treatment could delay the infection. But, this was not observed in our study.
In the classification of severity based on FEV1%, 70.96% of patients were classified as having normal and mild phenotype, and the classification was not associated with the polymorphisms examined, as well the mean values of FEV1%. The results are in agreement to that reported by Yarden et al. [16], who found no relationship between FEV1% with the genotype for the polymorphism analyzed (p=0.8).
The distribution of genotypes for the -238G>A polymorphism of TNF-α gene in the sample compared to the classification obtained by the Shwachman-Kulczycki score and the mean values, showed no significant association.
The score Kanga showed that at the time of the examination the most part of the patients (88.37%) were not in exacerbation, and that 81.57% of these patients were homozygous for the G allele, however, the association was not positive. The severity classification by transcutaneous oxygen by hemoglobin showed that 72.09% of patients were normal, and that the majority of these (70.96%) were homozygous for the G allele, however, the association was not positive.
Based on these results, in the evaluated sample, there was no association between the polymorphic variant (-238G>A) and CF severity, as proposed by Yarden et al. [16].
Analysis of polymorphism -308G>A in TNF-α gene
Of the 49 patients studied, 14.28% were GG homozygotes, 67.35% AA homozygotes and 18.36% GA heterozygotes. The allele frequencies were 0.77 and 0.23 for allele A and G, respectively, data contrary to reported in the literature. The G allele in other studies showed higher frequency than the A allele, being the frequency of G allele of 0.7, 0.75 and 0.81 (only patient AA homozygote), respectively, for the study of 53, 261 and 53 CF patients [15,33,34]. The variation can be explained by admixture population analyzed and the kind of colonization occurred in the our country.
Age at diagnosis (except the minor time for the diagnostic for G/A genotype vs AA genotype) and onset of pulmonary symptoms (for genotypic analysis) showed no association. However, the A allele, previously described as risk factor for the severe pulmonary disease [15], was associated with increased risk of early digestive disease (under 12 months) and had lower odds ratios for late-onset pulmonary disease, confirming the previously reported findings.
The colonization by P. aeruginosa was associated with the -308G>A genotype of TNF-α gene, being the AA genotype a protective factor for early colonization and GA a risk. The finding is not in agreement with that described by Arkwright et al. [34] and Yarden et al. [16] studies in homozygotes patients (F508del) from Belgium and Czech Republic. The G allele was a risk factor for early colonization and the A allele a protector factor. This data were also not previously reported in the scientific literature referenced.
In the classification of severity based on FEV1%, 74.19% of patients were classified as normal and mild pulmonary phenotype. It was expected that the A allele of the -308G>A polymorphism of TNF-α gene was associated with greater clinical severity. The majority (73.91%) of patients with greater than of 80% of predicted FEV1 had the AA genotype. The A allele showed a higher odds ratio for worse saturation, and the G allele, higher odds ratio for better saturation. Thus, the data confirmed the greater severity of the lung disease associated with the A allele. By the comparison between the mean of FEV1% was held, for the -308G>A genotype of TNF-α gene, there was no association. Hull and Thompson [15] demonstrated that the mean of FEV1% in patients with GA genotype was lower than in GG group, which reinforces the hypothesis of these authors that the A allele, is associated with severe lung disease. Schmitt-Grohe et al. [33] did not observe the same differences compared to FEV1%, considering -308G>A genotype in the TNF-α gene, the same occurred in the study of Yarden et al. [16].
For the Shwachman-Kulczycki score no association was described, the same was observed in other studies [15,33]. For the Kanga score, 88.37% of the patients without exacerbation, 71.05% were homozygotes for the A allele. However, no significant association was observed.
For transcutaneous hemoglobin oxygen saturation, of 72.09% of the patients considered normal, 63.33% were homozygotes for A allele. This results are contrary to the Hull and Thompson [15] in which the A allele was associated with severe disease. However, it is consistent with other studies [16,33].
Conclusion
The study of polymorphisms in CF modifier genes present importance to promote greater understanding of the factors that influence the clinical variability of the disease. In the present study, for the two polymorphisms of the TNF-α gene, only the -308A>G polymorphism was associated with clinical severity. Thus, it emphasizes the need for more studies for polymorphisms and other possible modifiers genes in CF, considering the large number of studies with conflicting results, and models of sampling.
Acknowledgements
We thank Taís Daiene Russo Hortencio, Kátia Cristina Alberto Aguiar, Aline Gonçalves, Luciana Montes Rezende and Simoni Avansini for assistance in data collection and organization of ideas, Maria Angela Ribeiro for spirometry analysis and Fapesp for the financial support.
Disclosure of conflict of interest
None.
Abbreviations
- ACE
angiotensin I converting enzyme
- ADRA2A
adrenoceptor alpha 2A
- ADRB2R
Beta-2 adrenergic receptor
- ARMS-PCR
amplification refractory mutation system
- BMI
Body mass index
- CF
Cystic Fibrosis
- CFTR
Cystic Fibrosis Transmembrane Regulator
- COX-2
cytochrome c oxidase
- FEV1
Forced Expiratory Volume in the first second
- FVC
forced vital capacity
- GCLC
Glutamate-cysteine ligase
- GSTP1
Glutathione S-transferase pi 1
- GSTM1
Glutathione S-transferase mu 1
- IFRD1
Interferon-related developmental regulator 1
- MBL-2
Mannose-Binding Lectin (Protein C) 2
- PCR
Polymerase chain reaction
- RFLP
restriction fragment length polymorphism
- TCF7L2
transcription factor 7-like 2 (T-cell specific, HMG-box)
- TGF-β1
Transforming Growth Fctor, Beta 1
- TNF-α
tumor necrosis factor
- USA
United States of America
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