Association of the −14C/G MET and the −765G/C COX-2 Gene Polymorphisms with the Risk of Chronic Rhinosinusitis with Nasal Polyps in a Polish Population

Abstract

Chronic rhinosinusitis with nasal polyps is strongly associated with other diseases, including asthma and allergy. The following study tested the association of the −765G/C polymorphism of cyclooxygenase-2 (COX-2) encoding gene and the −14C/G polymorphism of protooncogen MET (MET) encoding gene with a risk of chronic rhinosinusitis with nasal polyps in a Polish population. One hundred ninety-five patients of chronic rhinosinusitis with nasal polyps as well as 200 sex-, age-, and ethnicity-matched control subjects without chronic sinusitis and nasal polyps were enrolled in this study. Among the group of patients, 63 subjects were diagnosed with allergy and 65 subjects with asthma, respectively. DNA was isolated from peripheral blood lymphocytes of patients as well as controls, and gene polymorphisms were analyzed by restriction fragment length polymorphism–polymerase chain reaction (RFLP-PCR). Ten percent of the samples have been confirmed by a second method single-strand conformation polymorphism (SSCP)-PCR. We reported that the −765G/C COX-2 (odds ratio [OR] 7.79; 95% confidence interval [CI] 4.88–12.4, p<0.001) and the −14C/G MET (OR 2.83; 95% CI 1.74–4.61, p<0.001) were associated with an increased risk of chronic rhinosinusitis with nasal polyps among analyzed group of patients. Moreover, the group of patients without allergy or asthma indicated the association of the −765C/G (OR 7.25; 95% CI 4.38–12.1, p<0.001 and OR 7.61; 95% CI 4.47–12.6, p<0.001) genotype of the COX-2 as wells as the −14C/G (OR 2.47; 95% CI 1.46–4.17, p<0.001 and OR 2.59; 95% CI 1.54–4.37, p<0.001) genotype of MET with an increased risk of chronic rhinosinusitis with nasal polyps. Finally, it was also found that the selected group of patients with allergy or asthma indicated a very strong association of the −765G/C (OR 5.64; 95% CI 2.91–10.9 and OR 4.74; 95% CI 2.49–9.03, p<0.001, respectively) genotype of the COX-2 with an increased risk of chronic rhinosinusitis with nasal polyps. Thus, our results suggest that COX-2 and MET gene polymorphisms may have deep impact on the risk of rhinosinusitis nasal polyp formation, which may also depend on asthma or allergy. Our results showed that the −765G/C polymorphism of COX-2 gene and the −14C/G polymorphism of the MET gene may be associated with the risk of chronic rhinosinusitis with nasal polyps in a Polish population.

Introduction

T he Pathophysiology of the chronic sinusitis (nasal polyps) is an inflammatory process involving the nasal mucosa and paranasal sinuses, a complex and multifactorial etiology. This is a group of conditions where the precise incidence in the population is difficult because of the variability of these disorders. Occurrence of nasal polyps in the United States is estimated at about 15.5% of the population, which places them in second place among the chronic diseases (31 million patients per year) (Collins et al., 1997). The inflammatory process persists in the nasal polyps by more than 12 weeks. About 20% of patients with this disease have nasal polyps (Settipane et al., 1997). Chronic sinusitis with nasal polyps (65%–90% of cases) is often associated with the presence of eosinophilic infiltrates in the mucosa of the nose and paranasal sinuses. Mucosal eosinophilic inflammation is the ground on which easily arise, in particular, nasal polyps. Much less chronic rhinosinusitis with nasal polyps proceeds from the domination of the neutrophil infiltration in inflammatory mucosa of the nose and paranasal sinuses (Bachert et al., 2003). Although the role of allergy in chronic rhinosinusitis remains controversial, it has been postulated that the swelling of the nasal mucosa in allergic rhinitis may restrict ventilation and obstruct the sinus ostia, leading to mucus retention and an infection (Fokkens et al., 2005). Inflammation associated with allergic rhinitis and rhinosinusitis can reduce the physical size of the nasal passages by inducing vasodilatation, increasing blood flow and vascular permeability. Nasal polyps are found in about 36%–72% of patients with intolerance to nonsteroidal antiinflammatory drugs (aspirin triad: asthma, intolerance to nonsterodial antiinflammatory drugs, and nasal polyps), 13% of patients with nonallergic asthma, 5% of people with allergic asthma, 20%–50% of patients with cystic fibrosis, and 85% of people with allergic fungal sinusitis. Nasal polyps also occur in patients with primary ciliary dyskinesia syndrome (Kartagenera and Young syndrome) as well as in one-third of patients with nasal polyps, in which also occurs asthma or allergy, where asthma is associated with hypersensitivity to aspirin (Hamad et al., 2004). Previous studies show that the chronic sinusitis, asthma, allergy, and nasal polyp formation may be associated with a specific expression of such factors as COX-2 and MET. COX-2 is induced during inflammation, and takes an active part in the synthesis of pro-inflammatory prostanoids. In patients with intolerance to aspirin, there are large amounts of eosinophils from both peripheral blood and lavage fluid from the nose, sinuses, and bronchial tree. However, the number of lymphocytes in these samples did not deviate from the norm. Additionally, recent studies indicate the ability of the MET receptor to induct the expression of the prostaglandin synthesis of COX-2 (Lee et al., 2004). Searches for genetic determinants of chronic sinusitis may contribute to the efficient selection of high-risk patients who are likely to develop nasal polyps. Finally, these studies are able to explore the still unexplained descent. Therefore, this study aimed to investigate an association of the −765G/C polymorphism of COX-2 gene (rs20417) and the −14C/G polymorphism of MET gene (rs78116323) with a risk of chronic rhinosinusitis with nasal polyps in Polish population.

Materials and Methods

Patients

Peripheral blood samples were obtained from 195 patients with chronic rhinosinusitis and nasal polyps (109 men and 86 women, median age 57, quartiles: 32 and 74 years) treated during the study period (2009–2010) at the Department of Otolaryngology and Oncology of Medical University of Lodz in Poland. Among the group of 65 patients with chronic allergic rhinitis (sensitive to mites/Der. p., Der. f.), 63 subjects were diagnosed with mild controlled asthma. Symptoms of allergy in these patients have been present for 4–10 years. The patients were subjected to pharmacological treatment (inhaled steroids, antileukotrienes, and antihistaminic drugs). Before blood sample collection, each of the patients was clinically characterized according to physical examination, ear, nose, throat (ENT) examination, allergy skin test, and point prick test.

Two hundreds of sex-, age-, and ethnicity-matched control subjects without allergy, nasal polyps, chronic sinusitis, and any other chronic inflammations within ENT diagnosed due to hearing impairment in the Department of Otolaryngology and Laryngological Oncology were enrolled in this study. All patients and controls were Caucasian. The protocol of the study was reviewed and approved by the Ethic Committee of the Medical University of Lodz in Poland.

Genotype determination

Genomic DNA was isolated from blood samples of patients and controls using the QIAamp DNA Blood Mini. Gene was analyzed by restriction fragment length polymorphism–polymerase chain reaction (RFLP-PCR) was employed to analyze polymorphisms of COX-2 and MET genes. Each 20 μL of the PCR reaction contained 10 ng genomic DNA, 1.25 U Taq polymerase (Qiagen, Chatsworth, CA) in 1×PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 11 mM MgCl₂, and 0.1% gelatin), 1.5 mM MgCl₂, 50 mM dNTPs, and 250 nM each primer. Thermal cycling conditions for the −765G/C polymorphism of COX-2 gene were as follows: initial denaturation step at 94°C for 35 cycles, at 94°C for 30 s, at 56°C annealing temperature for 30 s, and at 72°C for 30 s. The final extension was performed at 72°C for 5 min. Thermal cycling conditions for the −14C/G polymorphism of MET gene were as follows: initial denaturation step at 95°C for 6 min, 35 cycles at 95°C for 30 s, at 57°C annealing temperature for 30 s, and at 72°C for 1 min. The final extension was performed at 72°C for 8 min. The PCR was carried out in an MJ Research, Inc., thermal cycler, model PTC-100 (Waltham, MA). The −765G/C polymorphism of COX-2 was determined using the following primers (Sigma–Aldrich, St. Louis, MO): sense, 5′-CATTAACTATT TACAGGGTAACTGCTT-3′; antisense, 5′-TGCAGCACA TACATACATAGCTTTT-3′. Primers were designed in the Primer3plus program (www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). The 238-bp PCR product was digested overnight with 5 U of the restriction enzyme SsiI. The GG allele was digested into 168- and 70-bp fragments whereas the CC variant remained intact (Fig. 1). The −14C/G polymorphism was determined using the following primers: sense, 5′-GAGCGCCTCAGTCTGGTC-3; antisense, 5′-CGCCTCCTCTCAGCAAGTC-3′. The 290-bp product was digested overnight with 5 U of the restriction enzyme BsaJI. The GG allele was digested into 220- and 67-bp fragments and the CC also remained intact (Fig. 2). Restriction fragments were separated on an 8% polyacrylamide gel and visualized by ethidium bromide staining. Blinded replicate samples were used for quality control of PCR-RFLP method. More than 10% of the samples were repeated by a second method single-strand conformation polymorphism (SSCP)-PCR, and the results were 100% concordant.

FIG. 1.

Representative restriction fragment length polymorphism–polymerase chain reaction (RFLP-PCR) analysis of the −765G/C polymorphism of the COX-2 gene. Lane M: DNA marker 50 bp; lanes 1, 3, and 9: the G/G homozygote is not cleaved by SsiI and remains a single 238-bp band; lanes 2, 5, and 6: the C/C homozygote is cleaved by SsiI and yields a 168-bp band; lanes 4, 7, and 8: the G/C heterozygote contains both bands (168 and 70 bp) following restriction digestion. Digested PCR product was electrophoresed on 8% polyacrylamide gel containing ethidium bromide. COX-2 RFLP-PCR band sizes are indicated on the right side of the panel.

FIG. 2.

Representative RFLP-PCR analysis of the −14C/G polymorphism of the MET gene. Lane M: DNA marker 100 bp; lanes 1, 4, and 9: the C/C homozygote is not cleaved by BsaJI and remains a single 290-bp band; lanes 3, 5, and 8: the G/G homozygote is cleaved by BsaJI and yields a 220-bp band; lanes 2, 6, and 7: the C/G heterozygote contains both bands (220 and 67 bp) following restriction digestion. Digested PCR product was electrophoresed on 8% polyacrylamide gel containing ethidium bromide. MET RFLP-PCR band sizes are indicated on the right side of the panel.

SSCP-PCR method

More than 10% of the samples were repeated by a second method SSCP-PCR, and the results were 100% concordant. The electrophoretic analysis was carried out on PhastSystem using PhastGel^® Homogeneous 12.5 polyacrylamide gels and PhastGel Native Buffer Strips. Samples were applied using PhastGel Sample Applicator 8/1 μL. The resulting PCR products were diluted in Elga water at 1/4 and mixed with an equal volume (2 μL) of denaturing solution (99% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol). The DNA samples were denatured at 95°C for 5 min followed by rapid cooling on ice. Running conditions were performed as follows: Prerun 100 V, 7 mA, 1.0 W, 18°C, and 10 Vh. Separation was performed under the following conditions: 200 V, 10 mA, 2.5 W, 18°C, and 75 Vh. The gels were stained using PhastGel DNA Silver Staining Kit according to the instructions (Figs. 3 and 4).

FIG. 3.

PCR-SSCP analysis of the COX-2 gene. Mutated factors: 1, 2, 4, 6, and 7 (homozygous); normal factors: 3 and 5 (heterozygous).

FIG. 4.

PCR-SSCP analysis of the MET gene. Mutated factors: 1, 2, 4, 5, and 7 (homozygous); normal factors: 3 and 6 (heterozygous).

Data analysis

The allele frequencies were estimated by gene counting and genotypes were scored. The χ² test was used to compare the observed numbers of genotypes with those expected for a population in the Hardy–Weinberg equilibrium and to test the significance of the differences of observed alleles and genotypes between groups. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. To calculate the probability, Pearson correction was used, and if the expected cell values were less than 5, then Fisher's exact test was used. A p-value of <0.05 was taken as statistically significant. An analysis of variance test was used to identify parameters that would make significant differences between more than two groups; Scheffe's test was then used to assess the significance of difference in each identified parameter between any two groups. STATISTICA 6.0 software (Statsoft, Tulsa, OK) was used to perform analyses.

Results

The genotypes of patients and controls were scored according to −765G/C polymorphism of the COX-2 gene and −14C/G polymorphism of the MET gene. All distributions in patients and control group did not differ significantly from those predicted by the Hardy–Weinberg equilibrium. The genotype distributions and allele frequencies of analyzed polymorphisms for COX-2 gene and MET gene in patients and controls are displayed in Table 1. It was shown that there is an increased risk of chronic rhinosinusitis with nasal polyp occurrence in association with the G/C genotype of the COX-2 gene (OR 7.79; 95% CI 4.88–12.4, p<0.001) and the C/G genotype (OR 2.83; 95% CI 1.74–4.61, p<0.001, respectively) of the MET gene. It was also observed that there are differences in the frequency of the C allele between the group of patients and controls for the −765G/C COX-2 gene polymorphism (OR 6.05; 95% CI 4.15–8.83, p<0.001) and the G allele between the group of patients and controls for the −14C/G MET gene polymorphism (OR 5.52; 95% CI 3.68–8.27, p<0.001).

Table 1.

Distribution of Genotypes and Frequency of Alleles of the −765G/C of the COX-2 Gene, and the −14C/G Polymorphism of the MET Gene and Odds Ratio with 95% Confidence Interval in Patients with Chronic Rhinosinusitis and Nose Polyps and Controls

	Patients (n=195)		Controls (n=200)
Genotype or allele	Number	Frequency	Number	Frequency	OR (95% CI)	p-Value
−765G/C
G/G	56	0.28	158	0.79	1.00 Ref.
G/C	116	0.59	42	0.21	7.79 (4.88–12.4)	p<0.001
C/C	23	0.11	0	0	–
G	228	0.58	358	0.88	1.00 Ref.
C	162	0.41	42	0.10	6.05 (4.15–8.83)	p<0.001
−14C/G
C/C	98	0.50	165	0.82	1.00 Ref.
C/G	59	0.30	35	0.17	2.83 (1.74–4.61)	p<0.001
G/G	38	0.19	0	0	–
C	255	0.65	365	0.91	1.00 Ref.
G	135	0.34	35	0.08	5.52 (3.68–8.27)	p<0.001

CI, confidence interval; OR, odds ratio.

Bold values indicate an increased risk of chronic rhinosinusitis with nasal polyps.

The genotype distributions and allele frequencies of analyzed polymorphisms for COX-2 gene and MET gene in patients without allergy or asthma and group of healthy controls are displayed in Tables 2 and 3. The selected group of patients without allergy or asthma indicated the association of the −765C/G (OR 7.25; 95% CI 4.38–12.1, p<0.001 and OR 7.61; 4.57–12.6, p<0.001, respectively) genotypes of the COX-2 as well as −14C/G (OR 2.47; 95% CI 1.46–4.17, p<0.001 and OR 2.59; 95% CI 1.54–4.37, p<0.001) genotypes with an increased risk of chronic rhinosinusitis with nasal polyps. It was also observed that there are differences in the frequency of the C allele between the group of patients without allergy or asthma and group of healthy controls for the −765G/C COX-2 gene polymorphism (OR 5.11; 95% CI 3.41–7.67, p<0.001 and OR 5.24; 95% 3.49–7.86, p<0.001, respectively). It was also observed that there are differences in the frequency of the G allele between the group of patients without allergy or asthma and group of healthy controls for the allele of the −14C/G MET (OR 3.09; 95% CI 1.97–4.96, p<0.001 and OR 3.19; 95% CI 2.03–5.01, p<0.001, respectively).

Table 2.

Distribution of Genotypes and Frequency of Alleles of the −765G/C of the COX-2 Gene, and the −14C/G Polymorphism of the MET Gene and Odds Ratio with 95% Confidence Interval in Rhinosinusitis Patients with Nose Polyps Without Allergy and Controls

	Patients (n=132)		Controls (n=200)
Genotype or allele	Number	Frequency	Number	Frequency	OR (95% CI)	p-Value
−765G/C
GG	42	0.31	158	0.79	1.00 Ref.
C/G	81	0.61	42	0.21	7.25 (4.38–12.1)	p<0.001
CC	9	0.06	0	0	–
G	165	0.62	358	0.88	1.00 Ref.
C	99	0.37	42	0.10	5.11 (3.41–7.67)	p<0.001
−14C/G
C/C	80	0.61	165	0.82	1.00 Ref.
C/G	42	0.32	35	0.17	2.47 (1.46–4.17)	p<0.001
G/G	9	0.06	0	0	–
C	202	0.77	365	0.91	1.00 Ref.
G	60	0.22	35	0.08	3.09 (1.97–4.96)	p<0.001

Bold values indicate an increased risk of chronic rhinosinusitis with nasal polyps.

Table 3.

Distribution of Genotypes and Frequency of Alleles of the −765G/C of the COX-2 Gene, and the −14C/G Polymorphism of the MET Gene and Odds Ratio with 95% Confidence Interval in Rhinosinusitis Patients with Nose Polyps Without Asthma and Controls

	Patients (n=130)		Controls (n=200)
Genotype or allele	Number	Frequency	Number	Frequency	OR (95% CI)	p-Value
−765G/C
GG	40	0.30	158	0.79	1.00 Ref.
C/G	81	0.62	42	0.21	7.61 (4.57–12.6)	p<0.001
CC	9	0.06	0	0	–
G	161	0.61	358	0.88	1.00 Ref.
C	99	0.38	42	0.10	5.24 (3.49–7.86)	p<0.001
−14C/G
C/C	78	0.60	165	0.82	1.00 Ref.
C/G	43	0.33	35	0.17	2.59 (1.54–4.37)	p<0.001
G/G	9	0.06	0	0	–
C	199	0.76	365	0.91	1.00 Ref.
G	61	0.23	35	0.08	3.19 (2.03–5.01)	p<0.001

Bold values indicate an increased risk of chronic rhinosinusitis with nasal polyps.

The genotype distributions and allele frequencies of analyzed polymorphisms for COX-2 gene and MET gene in patients with allergy or asthma and group of healthy controls are displayed in Tables 4 and 5. The selected group of patients with allergy or asthma indicated the association of the −765C/G (OR 5.64; 95% CI 2.91–10.9, p<0.001 and OR 4.74; 95% CI 2.49–9.03, p<0.001, respectively) genotypes of the COX-2 with an increased risk of chronic rhinosinusitis with nasal polyps. It was also observed that there are differences in the frequency of the C allele between the group of patients with allergy or asthma and group of healthy controls for the −765G/C COX-2 gene polymorphism (OR 6.81; 95% CI 4.20–10.9, p<0.001 and OR 6.25; 95% 3.89–10.2, p<0.001, respectively). It was also observed that there are differences in the frequency of the G allele between the group of patients with allergy or asthma and group of healthy controls for the −14C/G MET polymorphism (OR 9.48; 95% CI 5.79–15.5, p<0.001 and OR 8.93; 95% CI 5.48–14.5, p<0.001, respectively). Gene–gene combined interaction analysis was displayed in Table 6. A significant association was observed between the combined genotypes G/C-C/G (OR 2.53; 95% CI 1.20–5.33, p=0.021) and G/C-G/G (OR 4.07; 95% CI 1.97–8.40, p<0.001) of the COX-2 gene and MET gene polymorphisms in chronic rhinosinusitis with nasal polyps. These genotype combinations may increase the risk of chronic rhinosinusitis with nasal polyps in Polish population.

Table 4.

	Patients (n=63)		Controls (n=200)
Genotype or allele	Number	Frequency	Number	Frequency	OR (95% CI)	p-Value
−765G/C
GG	20	0.31	158	0.79	1.00 Ref.
C/G	30	0.47	42	0.21	5.64 (2.91–10.9)	p<0.001
CC	13	0.20	0	0	–
G	70	0.55	358	0.88	1.00 Ref.
C	56	0.44	42	0.10	6.81 (4.20–10.9)	p<0.001
−14C/G
C/C	27	0.42	165	0.82	1.00 Ref.
C/G	12	0.19	35	0.17	2.09 (0.96–4.53)	p<0.001
G/G	24	0.38	0	0	–
C	66	0.52	365	0.91	1.00 Ref.
G	60	0.47	35	0.08	9.48 (5.79–15.5)	p<0.001

Bold values indicate an increased risk of chronic rhinosinusitis with nasal polyps.

Table 5.

Distribution of Genotypes and Frequency of Alleles of the −765G/C of the COX-2 Gene, and the −14C/G Polymorphism of the MET Gene and Odds Ratio with 95% Confidence Interval in Rhinosinusitis Patients with Nose Polyps and Asthma and Controls

	Patients (n=65)		Controls (n=200)
Genotype or allele	Number	Frequency	Number	Frequency	OR (95% CI)	p-Value
−765G/C
GG	23	0.35	158	0.79	1.00 Ref.
C/G	29	0.44	42	0.21	4.74 (2.49–9.03)	p<0.001
CC	13	0.20	0	0	–
G	75	0.57	358	0.88	1.00 Ref.
C	55	0.42	42	0.10	6.25 (3.89–10.2)	p<0.001
−14C/G
C/C	29	0.44	165	0.82	1.00 Ref.
C/G	12	0.18	35	0.17	1.95 (0.90–4.19)	p<0.001
G/G	24	0.36	0	0	–
C	70	0.53	365	0.91	1.00 Ref.
G	60	0.46	35	0.08	8.93 (5.48–14.5)	p<0.001

Bold values indicate an increased risk of chronic rhinosinusitis with nasal polyps.

Table 6.

The Combined Genotype and Allele Distribution and Odds Ratios of the −765G/C Polymorphism of the COX-2 Gene and the −14C/G Polymorphism of the MET Gene in Patients with Chronic Rhinosinusitis and Nose Polyps and Controls

Polymorphism	Patients (n=195)	Controls (n=200)
Genotype combination	Number	Number	OR (95% CI)	p-Value
−765G/C−14C/G
G/G-G/G	31	65	1.00 Ref.
G/G-C/G	23	19	2.53 (1.20–5.33)	p=0.021
G/G-C/C	11	0
C/C-C/C	6	0	–
C/C-C/G	5	0	–
C/C-G/G	7	0	–
G/C-C/C	13	0	–
G/C-C/G	33	17	4.07 (1.97–8.40)	p<0.001
G/C-G/G	66	99	1.39 (0.82–2.37)	p=0.267

Bold values indicate an increased risk of chronic rhinosinusitis with nasal polyps.

Discussion

Nasal polyps are strongly associated with a risk of chronic rhinosinusitis development as well as other obstruction, including asthma and allergy. It is suggested that COX-2 and MET might contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps; however, their role is not fully elucidated. The functional variant polymorphism of −765G/C in the COX-2 gene has been described (Vane et al., 1998; Fritsche et al., 2001; Huuskonen et al., 2008; Hoff et al., 2009) and related with several pathologies, such as colorectal cancer risk (Brown et al., 2005), breast cancer (Langsenlehner et al., 2006; Shen et al., 2006), coronary artery disease (Linton et al., 2004), head and neck cancer (Peters et al., 2009), prostate cancer (Shahedi et al., 2006), and lung cancer (Yuan et al., 2005; Grimminger et al., 2009). It was demonstrated by Papafili et al. that −765C allele had lower COX-2 promoter activity than the −765G allele (Ishibashi et al., 1998). Kristinsson et al. reported an association of the presence of the COX-2-765G/C genotype and a higher risk of esophageal adenocarcinoma and squamous cell carcinoma (Rho et al., 2006). Sitarz et al. (2008) showed that the COX-2-765G allele promoter polymorphism is significantly associated with gastric cancer (Horton et al., 1999). Other reports have suggested a significant genetic association between variants in COX-2 and prostate cancer risk in South African men (Fernandez-Morata et al., 2000). Increased genetic risk between COX-2 variants and prostate cancer has been shown in African American (Mullol et al., 2002; Roca-Ferrer et al., 2006) and Nigerian men (Mullol et al., 2002). The other study that was found by Xing et al. (2008) showed that the COX-2-765G/C polymorphism is not associated with an increased risk of colorectal cancer (CRC), whereas −765GG genotype appears to be related to an increased risk in the presence of smoking and higher body mass index. In contrast, Tan et al. obtained contradictory positive results that the increased risk for CRC was associated with the COX-2-765G/C genotype in Chinese population (Coste et al., 1996). Moreover, Zhang et al. (2005) reported that the −765GC genotype was associated with an increased risk of esophageal squamous cell carcinoma and CRC in Chinese population (Weidner et al., 1993). Xing et al. (2008) showed that the COX-2-765G/C allele promoter polymorphism is significantly associated with colorectal cancer risk (Bachert et al., 2003). In addition, the −765GC and 765CC genotypes correlate with decreased risk of myocardial infarct and stroke and decreased COX-2 expression in atherosclerotic plaques compared with −765GG (Coste et al., 2000). In a Dutch study, Brosens et al. (2005) reported that the polymorphism −765G/C of COX-2 gene was significantly higher in the normal-appearing mucosa of patients with familial adenomatous polyposis (Petruson et al., 1988). However, Huuskonen et al. (2008) demonstrated that the functional C variant of the −765G/C polymorphism in the human COX-2 gene associates with the severity of coronary atherosclerosis measured at the coronary artery level in Finnish population (Bachert et al., 2003). Little data evidenced an association between nose polyps and this polymorphism. Our study has demonstrated that G/C variant of the COX-2-765G/C polymorphism may increase the risk of nose polyps.

Recently, an increase in COX-2 expression was demonstrated in chronic rhinosinusitis with polyp formation (Mullol et al., 2002; Rho et al., 2006; Roca-Ferrer et al., 2006). According to literature data, a key phenomenon of this specific type of chronic inflammatory disease in nasal respiratory mucosa is remarkable edema. Vascular permeability/vascular endothelial growth factor (VPF/VEGF) plays an important role in inducing angiogenesis and modulating capillary permeability. The markedly increased expression in nasal polyps, as opposed to healthy nasal mucosa, suggests that VPF/VEGF may play a significant role in both the formation of nasal polyps and in the induction of heavy tissue edema. This finding is discussed with respect to the differential expression of cyclooxygenase isoenzyme-2 (COX-2) in nasal polyps; where in the group of patients the expression of COX-2 in nasal mucosa was significantly stronger than in specimens of healthy nasal mucosa of controls (Gosepath et al., 2005). COX-2 is expressed in nose polyps, and as prostanoids are known to control chemotaxis of inflammatory cells, vascular tone, vascular permeability, and mucous secretion (all hallmarks of nose polyps) (Horton et al., 1999; Fernandez-Morata et al., 2000), their possible involvement in the pathogenesis of nose polyps has long been studied. As COX-2 is presumed to be induced under pro-inflammatory conditions, most studies focused on the role of this enzyme in the pathogenesis of nasal polyposis. This suggests that the deregulation of COX-2 expression may play a role in the risk of chronic rhinosinusitis with polyp formation. Our study is the first one that reports the analysis of an association between the −765G/C polymorphism of COX-2 gene with the nasal polyp development in patients with chronic rhinosinusitis. The −765G/C polymorphism is located in the promoter region of COX-2 gene, which may result in its expression level variation. We have shown that the −765G/C (OR 7.79) genotype of the COX-2 gene is strongly associated with an increased risk of the chronic rhinosinusitis with nasal polyps in the Polish patients. We did not find an association between the −765G/C polymorphism of the COX-2 gene and a prevalence of chronic rhinosinusitis with nasal polyps in the group of patients with allergy or asthma; however, the selected group of patients without allergy or asthma still indicated association of the −765G/C genotypes of the COX-2 gene with an increased risk of the chronic rhinosinusitis with nasal polyps. This indicates that COX-2 may have an influence on rhinosinusitis nasal polyps' formation that is not depending on asthma or allergy. The expression of various growth factors in nasal polyps has been examined, and several of these have been implicated in the pathogenesis, formation, and growth of polyps (Coste et al., 1996; Ishibashi et al., 1998; Petruson et al., 1988; Coste et al., 2000). Hepatocyte growth factor (HGF), also known as scatter factor, is a pleiotropic polypeptide growth factor with several biological activities, including epithelial cell proliferation, stimulation of cell motility, mitogenesis, morphogenesis, angiogenesis, and cellular invasiveness (Weidner et al., 1993; Balkovetz et al., 1999). In addition, HGF plays a key role in directing the integrated phases of wound repair, which consists of inflammation, epithelialization, formation of granulation tissue, and tissue remodeling (Wilson et al., 1999; Cowin et al., 2001; Toyoda et al., 2001). These diverse biological effects of HGF are mediated via interaction with the transmembrane tyrosine kinase receptor MET. HGF and its receptor MET have been identified in various neoplastic tissue types (Weidner et al., 1993; Balkovetz et al., 1999). In this respect, HGF and c-MET may play a role in the development of nasal polyps. Further, it has been hypothesized that HGF might play an important role in regulating the regeneration of the nasal epithelia (Puchelle et al., 2000). Therefore, it is assumed that HGF and MET levels in normal nasal mucosa can be modulated by injury and by factors associated with wound healing. However, there are no literature data concerning an association between the −14C/G polymorphism of the MET gene and nasal polyp formation. The biological effect of this polymorphism is yet to be elucidated and will be important to investigate. The −14C/G polymorphism is located in the promoter region of the MET gene and could affect transcription efficiency, leading to altered product levels, which could influence the biological function of HGF dependent on interaction with the receptor MET. In our study we observed a statistically significant difference between patients of rhinosinusitis with nasal polyps and healthy controls in Polish population. Our study has demonstrated that the −14C/G genotype of MET may increase the risk of chronic rhinosinusitis with nasal polyps (OR 2.83). The high frequency of the G allele in the patient group suggests that it may be involved in the increase of their susceptibility to chronic rhinosinusitis with nasal polyps, which was verified in this study (OR 5.52).

In our study we analyzed the correlation of the −765G/C polymorphism of COX-2 gene as well as −14C/G polymorphism of MET gene with a prevalence of allergies and asthma in patients with chronic rhinosinusitis and the risk of nasal polyp development. We evaluated that the group of patients without allergy or asthma still indicated the association of the −765G/C (OR 7.6, p<0.001 and OR 7.25, p<0.001) genotype of the COX-2 as well as −14C/G (OR 2.47, p<0.001 and OR 2.59, p<0.001) genotype of MET with an increased risk of chronic rhinosinusitis with nasal polyps. Most interestingly, it was found that the selected group of patients with allergy or asthma indicated stronger association of the −765G/C heterozygote (OR 5.64, p<0.001 and OR 4.74, p<0.001) with an increased risk of chronic rhinosinusitis with nasal polyps than patients without allergy or asthma. Thus, our results suggest that COX-2 and MET gene polymorphisms may have deep impact on the risk of rhinosinusitis nasal polyps' formation, which may also depend on asthma or allergy.

Finally, we indicated that the combined genotypes of COX-2 gene and MET may influence the risk of chronic rhinosinusitis with nasal polyps. We found that the genotype interactions of the G/C-C/G (OR 2.53, p=0.021), and the G/C-G/G (OR 4.07, p<0.001) may increase the risk of chronic rhinosinusitis with nasal polyps. The results obtained in our work indicate that the −765G/C polymorphism of the COX-2 gene and the −14C/G polymorphism of the MET gene may be associated with the risk of chronic rhinosinusitis with nasal polyps in Polish population. Further in vitro studies are required to better understand the mechanism of nasal polyp formation in patients with chronic rhinosinusitis and the role of COX-2 and MET gene polymorphisms in their pathogenesis.

Conclusions

The present study showed that the −765G/C polymorphism of COX-2 gene and the −14C/G polymorphism of MET gene may result in an increased risk of developing nose polyps in chronic rhinosinusitis in the Polish population. We have shown that the combined genotypes of the COX-2 and MET genes can modulate the risk of nose polyps in chronic rhinosinusitis in the Polish population. In accordance with our knowledge, this is the first report showing an association between COX-2 and MET gene polymorphisms with susceptibility to nose polyps in chronic rhinosinusitis among the Polish population. To clarify the pathogenesis of nose polyps in chronic rhinosinusitis, further studies are required.

Footnotes

Acknowledgments

This work was supported by the grant N N402 422138 from Polish Ministry of Science and Higher Education as well as Higher Education and Young Scientists grants No. 502-03/7-124-04/502-54-020 and No. 502/03/5-120-02/502-54-012 from Medical University of Lodz.

Disclosure Statement

The authors have declared that no conflict of interest exists.

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