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Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a rather frequent disorder affecting 2%–4% of the general population.1 There is increasing evidence that genetic determinants are important in the pathogenesis of OSAHS;2 however, the exact identity of susceptibility genes in OSAHS remains elusive.
Oxidative stress has emerged as an important pathogenic factor in OSAHS,3 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has been identified as a major contributor to the production of reactive oxygen species (ROS),4 which is composed of a number of subunits and appears to be widely distributed in neutrophils, fibroblasts, vascular smooth muscle cells, endothelial and mesangial cells.5,6 The gene coding is polymorphic for the p22phox subunit of NADPH oxidase, and includes a C242T transition that results in the replacement of histidine by tyrosine at amino acid 72 of the putative heme binding site. These genetic variants have been found to be associated with the activity of NADPH oxidase closely.7 It is possible that functional polymorphisms in the NADPH oxidase p22phox subunit may contribute to the imbalance of ROS in OSAHS. It was thus hypothesized that OSAHS might be associated with the p22phox gene polymorphism.
In this study, we investigated the frequency of the p22phox C242T variants and quantified NADPH oxidase subunit p22phox mRNA expression, along with markers of oxidative stress such as superoxide dismutase (SOD) in OSAHS patients and non-OSAHS controls.
METHODS
Subjects
A total of 107 unrelated male subjects of the Han population in southern region of China with habitual snoring and daytime sleepiness, consecutively referred to Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from July 2007 to April 2008 were enrolled in the study. The subjects were internalized according to the recommended diagnostic criteria of the American Association of Sleep Medicine (AASM) in 1999.8 With the inquiry of case history and related physical examination, all of the subjects had excluded chronic or acute liver, kidney and nerve-muscle disorders, acute and chronic infections, autoimmune diseases, cancer, diabetes, etc. Sixty-nine unrelated subjects of the south region Han population of China were recruited as non-OSAHS control subjects. The control subjects were chosen on the basis of the following criteria to ensure that they were free from sleep-related breathing disorders: absence of sleep disturbance, no symptoms related to disordered breathing during sleep, apnea-hypopnea index (AHI) <5 events/hour, and oxygen saturation by pulse oximetry (SpO2) nadir >90% in an overnight polysomnography. There were no significant differences in the age between the OSAHS (age from 22 to 73 years) and control groups (age from 18 to 65 years). This study was approved by the research ethics committee of Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, and informed consent was obtained from all patients and controls.
Data collection
All subjects were subjected to the measurement of their height, weight, neck circumference (NC), waist circumference, hip circumference and blood pressure. High blood pressure was defined as systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg according to the WHO/ISH standards in 1999.
Polysomnography
All subjects with OSAHS and control subjects underwent overnight polysomnography (Polysmith2004; Neurotronic, America). Polysomnography consisted of a continuous polygraphic recording from surface leads for an EEG, bilateral electrooculography, chin and lower leg electromyography, ECG, thermistors for nasal and oral airflow, thoracic and abdominal impedance belts for respiratory efforts, SpO2, tracheal microphone for snoring, and sensors to detect the position during sleep. Polysomnography records were staged manually according to the standard criteria. Apnea episodes were defined as complete cessation of airflow lasting at least 10 seconds. Hypopnea was defined as at least a 50% reduction in airflow for at least 10 seconds accompanied by a reduction in SpO2 of at least 4%. AHI was defined as the number of events of apnea or hypopnea per hour during sleep time, based on the results of the overnight polysomnography.
Determination of superoxide dismutase activity
Venous blood samples (5 ml) were obtained from all subjects, and heparin used as anticoagulant. Collected blood was centrifuged at 2000 ×g for 15 minutes and plasma was separated. Then, the plasma was deproteinized with 25% trichloroacetic acid by continuous mixing for 5 minutes and centrifuged at 2000 ×g for 15 minutes. The superoxide dismutase activity was assayed using SOD kits according to the manufacturer′s instructions. SOD kits were purchased from Nanjing JianCheng Institute of Biological Engineering, China.
RNA extraction and RT-PCR for p22phox mRNA expression
Peripheral blood mononuclear cell (PBMC) was segregated from blood by isolation of lymphocytes (DATA SHEET) according to the instructions. Total RNA from PBMC was extracted using Trizol reagent (Sigma, USA) according to the manufacturer′s instructions. To check the integrity of the total RNA, 1 μg was fractionated on a 2% agarose gel. RNA concentration was quantified spectrophotometrically and had a 280/260 optical density ratio between 1.8 and 2.0. After extraction, 2 μg of total RNA was reverse-transcribed to cDNA using reverse transcription reagents (TOYOBO, Japan). The PCR primers were designed by Shanghai Sangon Biological Engineering Technology Corporation, China. The primers used for p22phox gene expression were 5′-TTG TGG CGG GCG TGT TTG 3′and 5′-TCC TCG CTG GGC TTC TTG C 3′. The PCR mixture contained 100 ng genomic DNA template, 0.2 μmol/L of each primer, 0.8 mmol/L of each deoxynucleoside triphosphate, 1.5 U Taq polymerase (Fermentas Corporation), and reaction buffer in a total volume of 25 μl. PCR was performed for 35 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 63°C, and extension for 1 minute at 72°C, with initial denaturation at 95°C for 10 minutes, and a final extension at 72°C for 10 minutes. Amplification resulted in a 382-bp fragment. The housekeeping GAPDH PCR products were used as an internal control, their sequences were 5′-TCG ACA ACG GCT CCG GCA T 3′ and 5′-AAG GTG TGG TGC CAG ATT TTC 3′. The PCR and conditions were the same as mentioned above except for annealing at 50°C. Amplification resulted in a 241-bp fragment. PCR products were run in 2% agarose gel (Invitrogen) along with 100 bp ladder markers. Amplified products were visualized by staining with ethidium bromide and were analyzed using gel scanner.
Preparation of DNA and identification of the p22phox C242T polymorphism
High-molecular weight DNA was prepared from 3 ml peripheral blood using Nucleon extraction kits (Tigen Corporation; Beijing, China). The p22phox primer sequences were designed as reported previously.9 The amplification reaction for p22phox C242T was performed in 30 μl volumes containing the amplimers 5-TGC TTG TGG GTA AAC CAA GGC CGG TG-3 and 5- AAC ACT GAG GTA AGT GGG GGT GGC TCC TGT-3, 10 mmol/L dNTPs , 10 µl buffer solution, 10 mmol/l MgCl2, and 1 unit Taq polymerase (Fermentas). The samples were subjected to an initial denaturation at 96°C for 2 minutes, followed by 30 cycles of amplification in a three-step reaction consisting of denaturation for 30 seconds at 94°C, annealing for 1 minute at 56°C, and extension for 1 minute at 72°C in the Cycler Thermal Cycler. Amplification resulted in a 348-bp fragment that included the RsaI polymorphic site. The C242T polymorphism results in the creation of the RsaI recognition site through a cysteine-to-thymine transition. Ten units of RsaI (Fermentas) were used to digest the PCR products for 12 hours at 37°C. The digested fragments were separated by gel electrophoresis on a 1.5% agarose gel (Invitrogen). The bands were visualized under ultraviolet light and scored. The presence of the RsaI site resulted in fragments of 160 and 188-bp in size; if the RsaI site was not present, a fragment of 348-bp was visualized. The selection of restriction enzyme and assessment of genotype were determined as reported previously.9
Statistical analysis
Statistical analysis was made using SPSS version 13.0. Descriptive characteristics of group variables are expressed as mean ± standard deviation (SD). The significance of variables between the two groups was tested by unpaired Student′s t test. The chi-square test was used to test the deviation of genotype distribution from the Hardy-Weinberg equilibrium and to determine whether there were any significant differences in allele or genotype frequencies between the OSAHS and non-OSAHS control groups. P values <0.05 were considered statistically significant.
RESULTS
The clinical characteristics of the 107 patients with OSAHS and 69 control subjects are presented in Table 1. The two groups were matched for age. Body mass index (BMI), NC and waist-to-hip ratio (WHR) in control subjects were significantly different from the corresponding parameters in patients with OSAHS.
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Table 1. Characteristics of the recruited subjects |
Plasma SOD activity in the patients with OSAHS ((59.65±11.61) U/ml) was significantly lower than in the control subjects ((85.31±9.23) U/ml) (t=10.18, P <0.01) (Figure 1).
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Figure 1. Superoxide dismutase activity (SOD) in the study group: the result shows that SOD activity in plasma of patients with OSAHS (59.65±11.61) U/ml) was significantly lower (t=10.18, P <0.01) than that of the control subjects ((85.31±9.23) U/ml). The result is reported as means±SD, P <0.05 was considered statistically significant. |
The mRNA expression patterns of p22phox and GAPDH were illustrated representatively in the study groups (Figure 2). In Figure 2, p22phox mRNA expression was significantly higher in the OSAHS subjects (3.06±0.64) than in the control subjects (2.49±0.54) (t=4.61, P <0.01).
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Figuer 2. mRNA expression in the study groups: representative pattern of gene expression of p22phox and GAPDH in control subjects (lane 2) and patients with OSAHS (lane 1). Lane M shows the DNA ladder (100–600 bp). The result shows that p22phox in peripheral blood mononuclear cell of patients with OSAHS (3.06±0.64) was significantly higher (t=4.61, P <0.01) than that of the control subjects (2.49±0.54). The result is reported as means±SD, and P <0.05 was considered statistically significant. |
The frequency of the C242T genotypes and alleles of the NADPH p22phox gene in the study subjects are shown in Table 2. For the C242T locus, two genotypes were identified and designated homozygous C/C242, and heterozygous C/T242 (The homozygous T/T242 was not found in this study, and this result may be limited to our hands on a sample of content and population). The distribution of allelic frequencies and genotypes was significantly different between subjects with a definite diagnosis of OSAHS subjects and controls, and there was a marked increase in the frequency of T allele in those patients with OSAHS compared with the control subjects (Table 2). Allele distribution in the control group was in Hardy-Weinberg equilibrium (χ2=0.10, P=0.75); similarly, allele distribution in the OSAHS group was also in Hardy-Weinberg equilibrium (χ2=1.55, P=0.21).
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Table 2. Allelic and genotype frequencies for p22phox C242T in recruited subjects |
There were independent effects of p22phox polymorphism on BMI, NC, WHR in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, SBP, DBP and AHI (t=2.09, 2.33, 3.31, 2.76, 2.30 respectively, P <0.05). But the carriers of the T allele had lower SOD (t=2.85, P <0.01) and LSaO2 (t=2.10, P=0.04). There was no significant difference in p22phox mRNA expression between the OSAHS groups with or without T allele (t=0.78, P=0.45) (Table 3).
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Table 3. Clinical characteristic effects of OSAHS patients with or without T allele |
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Figure 3. Representative agarose gel showing the polymerase chain reaction (PCR) amplified fragments of deoxyribonucleic acid (DNA) corresponding to the different p22phox genotypes (C/C: 348 bp, C/T: 166/180/348 bp). Lanes 2, 4, 5, 6: homozygous C/C242; lanes 1, 3: heterozygous C/T242; lane M: the DNA ladder (100–600 bp). |
DISCUSSION
OSAHS is thought to be a complex disorder that involves multiples genes, environmental influences, and developmental factors.10 Traits such as facial and head form, ventilatory chemosensitivity, load compensation, sympathetic nervous system activity, connective tissue laxity, and obesity are candidate factors in the development of OSAHS.11 There is growing evidence linking the pathogenesis of OSAHS with oxidative stress3,12 and NADPH oxidase has been identified as a major contributor to the production of ROS.4 This stimulated us to look at these parameters of OSAHS subjects in the south region Han population of China. Our findings showed that compared with control subjects, the gene expression of NADPH subunit p22phox was increased in leucocytes of the patients with OSAHS. Consistent with the mRNA data, the SOD activity was decreased in blood plasma from the patients with OSAHS. Thus, the increased production of mRNA for p22phox may play a role in the pathogenesis of OSAHS.
In addition, epidemiological investigations showed that the patient with OSAHS had a significant family aggregation and genetics, and even the symptoms of OSAHS area and sleep AHI are certain hereditary.13 Previous studies showed that certain genetic polymorphism may be associated with the pathogenesis of OSAHS.14,15 Our early studies16,17 also indicated that the pathological mechanisms of OSAHS were associated with the genetic polymorphism in Chinese subjects. We investigated the p22phox C242T polymorphism of the NADPH oxidase complex in patients with OSAHS. Present studies18-21 showed that there were racial differences in the p22phox C242T polymorphisms: the Caucasus and the Indians have similar gene frequency, but the Japanese are significantly different. The C242T was chosen because it results in a switch in amino acid (His/Tyr) at residue 72, which is located in the putative heme-binding site and has been closely associated with the activity of NADPH oxidase.7 The carriers of the T allele of p22phox polymorphism have been associated with lower basal as well as stimulated NADPH superoxide production in human blood vessels.22 Neutrophil oxidative bursts in healthy human volunteers increase incrementally from the TT to CC genotype.23 Thus, it is conceivable that the C allele, by virtue of its propensity for greater ROS production, would confer a higher risk for OSAHS. However, our study suggests that the T allele of p22phox contributes to the susceptibility to OSAHS, while the C allele may have a protective role.
On the other hand, obesity is one of the most commonly identified OSAHS risk factors,24 which show multiple pathophysiologic mechanisms such as insulin resistance, hyperleptinemia, and hypoventilation.25 Family studies suggest that human obesity is highly heritable, but the genetic determinants are still largely unidentified. Recent studies have shown that the level of oxidative stress of obese patients increased, while the activity of the body′s antioxidants such as Cu-Zn superoxide dismutase decreased significantly.26-28 The famous Framingham Heart Study Center′s subgroup also found that in obese patients there was a significantly positive correlation of body mass index with creatinine for correcting the urine 8 -iso-prostaglandin F2 α levels.29 Moreover, the present study revealed that the excessive activation of NADPH oxidase may play a major role in obese patients with elevated levels of oxidative stress.25,30 Similarly, Houstis et al31 have reported that ROS is the common pathway with a variety of factors leading to insulin resistance, and that the insulin resistance of 3 T3L1 fat cells was induced by dexamethasone and tumor necrosis factor-α (TNF-α), both of which lead to increased ROS level of cells. Furukawa et al27 used apocynin (the NADPH oxidase inhibitors) in KKay mice of obesity for six weeks, and found that the fatty tissue of lipid peroxidation and H2O2 showed a significant drop. Compared with the control group, there was no significant difference in the weight of mice in the obese group, but the levels of adiponectin in fat tissue increased significantly, and the levels of plasma insulin, glucose and triglyceride were significantly decreased. These results revealed that ROS may be involved in the formation of obesity. In this study, there were independent effects of p22phox polymorphism on BMI, NC, WHR in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC and WHR, but lower SOD and LSaO2. There was no significant difference in p22phox mRNA expression between the OSAHS groups with or without T allele. We speculate that C → T gene mutation in the fourth exon region of the p22phox gene would not change the expression of the gene in transcription level, but merely improve the efficiency of the translation. These changes lead to the increase of ROS which participates in the formation of central obesity, leading to the occurrence of OSAHS.
Our study has some limitations. First, although we have chosen important genetic polymorphism based on understanding of its biology, we have not investigated all the genetic variants of p22phox. Moreover, we also need to study the genetic polymorphism of the other NADPH oxidase subunits. Our future plans aim to study the other genetic variants of p22phox in OSAHS. Second, we may not have thoroughly addressed the issue of confounding by population admixture in genetic association.32 However, confounding by population admixture is more likely to result in a false positive association. Furthermore, we only studied the Chinese subjects with the Han ethnic background from southern region of China, thereby minimizing the population stratification.
In summary, this is the known study to examine the NADPH oxidase subunit p22phox C242T in Chinese with Han ethnic background suffering from obstructive sleep apnea-hypopnea syndrome. We found a significant association of the p22phox C242T with OSAHS. Because all the subjects were assessed by overnight polysomnography, an established diagnostic method for OSAHS, we assume that these findings are valid. However, OSAHS is a disease related to multiple genes, the polymorphism of one particular gene is not valid to explain the mechanism of this complicated disease. Therefore, genotype analysis of more markers for this disorder will be required in the study of OSAHS.
Acknowledgments: We are grateful to ZHANG Zhen-xiang for clinical coordination. This work was supported by Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.
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