Asthma Phenotypes in Asthma: A Genetic Study
Asthma Phenotypes in Asthma: A Genetic Study
Patients with asthma (n = 131) and healthy controls (n = 75) who presented to the Third Chest Diseases Outpatient Clinic at the Yedikule Chest Diseases and Surgery Training and Research Hospital between January 2010 and December 2011 were included in this study. A power analysis was performed for each gene polymorphism (CD14 -159, TLR4 299, TLR4 399) at the beginning of the study (to determine the necessary sample size). We determined the allele frequencies of all polymorphisms (CD14 -159, TLR4 299, TLR4 399) according to the results of the studies from our region and a meta-analysis (that included European, East Asian and Indian populations). The allele frequency of CD14 C-159 T was approximately 75% in the asthma group, 53% in the control group. The power analysis was performed according to these values. For the C-159 T polymorphism, the required sample size was 74 for each group (Approximate Test). The allele frequencies for TLR4 299 and TLR4 399 were approximately 20% in the asthma group, 5% in the control group, an again the reguired sample size was determined to be 70 for each group (Approximate Test). The number of patients in our study met these criteria.
Asthma was diagnosed according to the Global Initiative for Asthma (GINA) guidelines in patients with recurrent dyspnoea, wheezing attacks and positive airway reversibility test (12% or > 200 mL increase in FEV1 after 400 μg salbutamol inhalation). Patients who had an attack in the previous month and/or were treated with systemic steroids, took specific allergen immunotherapy, had a personal or family history of tuberculosis, or had active pathology or sequelae upon chest X-ray examination were excluded. The control group was composed of healthy individuals with no personal or family history of atopy or tuberculosis. Ninety six patients with allergic symptoms or a history of allergy and who showed a positive reaction to at least one allergen on skin prick testing were classified as having atopic asthma, and 35 patients with no allergic symptoms, no personal history and negative skin test results were classified as having non-atopic asthma. This study was performed in accordance with the principles of the Declaration of Helsinki (2008) and approved by the ethics committee of Yeditepe University. Written and signed informed consent was obtained from all participants.
Spirometric evaluations were performed with a MasterScope-PC (Jaeger, Germany), total IgE levels were measured with a Unicel D × I 800 analyser (Beckman Coulter, (US) and eosinophil counts were determined using an ABX Pentra 120 system (Impedance&Optical, Minnesota, USA). All patients with asthma underwent a skin prick test with 15 standard aero-allergens and five food allergens (ALK-Abellò, Hørsholm, Denmark). Oedema of >3 mm was accepted as positive.
A total of 131 EDTA containing blood samples were collected from patients with asthma and stored at −80°C until nucleic acid purification. Residual EDTA containing blood samples collected for routine biochemistry tests were used as the control group. Nucleic acids were isolated from these samples using a commercial genomic DNA extraction kit (Thermo Scientific, MA, USA).
The Rotor-Gene Q 5plex high resolution melting (HRM) platform and Rotor-Gene 6000 series software v1.7 (Qiagen, Hilden, Germany) were used for real time PCR amplification and HRM analysis. 5x Hot Fire Pol Evagreen HRM mix (Solis BioDyne, Tartu, Estonia) used for PCR amplification and PCR reactions were performed in a 20 μL final volume containing 5 μL of purified genomic DNA. 0.1 mM of primers ( Table 1 ) used for amplification. Amplification was performed 95°C for 10 minutes for initial denaturation followed by 30 cycles at 95°C for 10 seconds, 53°C for 45 seconds and 72°C for 45 seconds. After amplification, denaturation was performed from 72°C to 90°C for HRM analysis. Representative samples of genotypes were confirmed by DNA sequencing for TLR4 polymorphisms and via the restriction fragment length polymorphism (RFLP) method for the CD14 polymorphism. Representative samples were also used controls for further tests ( Table 1 ).
Statistical analyses were performed using the SPSS 16 package software (SPSS Inc., Chicago, IL, USA). Patient demographics and disease characteristics were summarised using descriptive statistics. All numeric data were expressed as the mean ± standard deviation, and non-numeric data as frequencies and percentages. Statistical differences between group means were analysed using Student's t-test. One- way analysis of variance (ANOVA) was used to compare means among more than two groups, and Tukey's HSD test was used in the post-hoc analysis as necessary. A chi-square test was used to determine whether allele and genotype frequencies in the asthma and healthy control groups deviated from the Hardy–Weinberg equilibrium. The chi-square test was used for non-numeric data. A p value of <0.05 was accepted as statistically significant.
Methods
Study Subjects
Patients with asthma (n = 131) and healthy controls (n = 75) who presented to the Third Chest Diseases Outpatient Clinic at the Yedikule Chest Diseases and Surgery Training and Research Hospital between January 2010 and December 2011 were included in this study. A power analysis was performed for each gene polymorphism (CD14 -159, TLR4 299, TLR4 399) at the beginning of the study (to determine the necessary sample size). We determined the allele frequencies of all polymorphisms (CD14 -159, TLR4 299, TLR4 399) according to the results of the studies from our region and a meta-analysis (that included European, East Asian and Indian populations). The allele frequency of CD14 C-159 T was approximately 75% in the asthma group, 53% in the control group. The power analysis was performed according to these values. For the C-159 T polymorphism, the required sample size was 74 for each group (Approximate Test). The allele frequencies for TLR4 299 and TLR4 399 were approximately 20% in the asthma group, 5% in the control group, an again the reguired sample size was determined to be 70 for each group (Approximate Test). The number of patients in our study met these criteria.
Asthma was diagnosed according to the Global Initiative for Asthma (GINA) guidelines in patients with recurrent dyspnoea, wheezing attacks and positive airway reversibility test (12% or > 200 mL increase in FEV1 after 400 μg salbutamol inhalation). Patients who had an attack in the previous month and/or were treated with systemic steroids, took specific allergen immunotherapy, had a personal or family history of tuberculosis, or had active pathology or sequelae upon chest X-ray examination were excluded. The control group was composed of healthy individuals with no personal or family history of atopy or tuberculosis. Ninety six patients with allergic symptoms or a history of allergy and who showed a positive reaction to at least one allergen on skin prick testing were classified as having atopic asthma, and 35 patients with no allergic symptoms, no personal history and negative skin test results were classified as having non-atopic asthma. This study was performed in accordance with the principles of the Declaration of Helsinki (2008) and approved by the ethics committee of Yeditepe University. Written and signed informed consent was obtained from all participants.
Measurements
Spirometric evaluations were performed with a MasterScope-PC (Jaeger, Germany), total IgE levels were measured with a Unicel D × I 800 analyser (Beckman Coulter, (US) and eosinophil counts were determined using an ABX Pentra 120 system (Impedance&Optical, Minnesota, USA). All patients with asthma underwent a skin prick test with 15 standard aero-allergens and five food allergens (ALK-Abellò, Hørsholm, Denmark). Oedema of >3 mm was accepted as positive.
Genotyping
A total of 131 EDTA containing blood samples were collected from patients with asthma and stored at −80°C until nucleic acid purification. Residual EDTA containing blood samples collected for routine biochemistry tests were used as the control group. Nucleic acids were isolated from these samples using a commercial genomic DNA extraction kit (Thermo Scientific, MA, USA).
The Rotor-Gene Q 5plex high resolution melting (HRM) platform and Rotor-Gene 6000 series software v1.7 (Qiagen, Hilden, Germany) were used for real time PCR amplification and HRM analysis. 5x Hot Fire Pol Evagreen HRM mix (Solis BioDyne, Tartu, Estonia) used for PCR amplification and PCR reactions were performed in a 20 μL final volume containing 5 μL of purified genomic DNA. 0.1 mM of primers ( Table 1 ) used for amplification. Amplification was performed 95°C for 10 minutes for initial denaturation followed by 30 cycles at 95°C for 10 seconds, 53°C for 45 seconds and 72°C for 45 seconds. After amplification, denaturation was performed from 72°C to 90°C for HRM analysis. Representative samples of genotypes were confirmed by DNA sequencing for TLR4 polymorphisms and via the restriction fragment length polymorphism (RFLP) method for the CD14 polymorphism. Representative samples were also used controls for further tests ( Table 1 ).
Statistical Analysis
Statistical analyses were performed using the SPSS 16 package software (SPSS Inc., Chicago, IL, USA). Patient demographics and disease characteristics were summarised using descriptive statistics. All numeric data were expressed as the mean ± standard deviation, and non-numeric data as frequencies and percentages. Statistical differences between group means were analysed using Student's t-test. One- way analysis of variance (ANOVA) was used to compare means among more than two groups, and Tukey's HSD test was used in the post-hoc analysis as necessary. A chi-square test was used to determine whether allele and genotype frequencies in the asthma and healthy control groups deviated from the Hardy–Weinberg equilibrium. The chi-square test was used for non-numeric data. A p value of <0.05 was accepted as statistically significant.
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