Sleep Medicine 16 (2015) 792–795
Contents lists available at ScienceDirect
Sleep Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / s l e e p
Brief Communication
Cytokine gene polymorphisms in obstructive sleep apnoea/hypopnoea syndrome P. Bielicki a, A.K. MacLeod b, N.J. Douglas c, R.L. Riha c,* a b c
Department of Internal Diseases, Pneumology and Allergology, Warsaw Medical University, Warsaw, Poland Medical Genetics Section, Centre for Molecular Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Department of Sleep Medicine, Royal Infirmary Edinburgh, Edinburgh, UK
A R T I C L E
I N F O
Article history: Received 31 March 2014 Received in revised form 21 December 2014 Accepted 13 January 2015 Available online 7 April 2015 Keywords: Sleep apnoea Cytokine gene polymorphisms TNF-alpha haplotypes Interleukin-6 Interleukin-8 Lymphotoxin
A B S T R A C T
Objective: The pro-inflammatory cytokines, TNF-α, IL-6, and IL-8 are elevated in obstructive sleep apnoea/ hypopnoea syndrome (OSAHS). Cytokine gene interactions are complex and haplotype analysis may be more informative. We hypothesized that the effects of TNF-α in OSAHS might be due to linkage disequilibrium of the TNF-α (−308A) single nucleotide polymorphism (SNP) with other polymorphisms within the TNF-α gene, and that predisposition to elevated IL-6 and IL-8 levels in OSAHS might be attributable to pro-inflammatory IL-6 and IL-8 gene promoter polymorphisms. Method: 173 subjects were classified as having definite OSAHS or not on the basis of apnoea– hypopnoea frequency, sex, age, and symptoms. Population controls comprised 192 random UK blood donors. Genotyping was undertaken for the TNF- α promoter polymorphisms (−1031, −863, −857, −238), two lymphotoxin-α polymorphisms (intron 1 and Thr60Asn), the pro-inflammatory IL-6 gene promoter polymorphism (−174), and IL-8 gene promoter polymorphisms (−251; −781). Results: There was no significant difference between groups re: genotype/allelic frequency in the genes investigated. Association between disease status and the TNF-α alleles independently (TNF-103, TNF803, TNF-857, TNF-238) with five haplotypes of TNF-α was not significant (p > 0.05). There was no difference in allelic or genotypic frequencies between obese and non-obese subjects with OSAHS. The TNF- α (−863A) allele alone, was significantly associated with obesity (OR 2.4; CI95% 1.1–5; p = 0.025). Conclusion: Only the TNF- α (308A) SNP appears to be significantly associated with OSAHS. The impact of cytokine gene polymorphisms on phenotypic expression of inflammation in OSAHS is likely to be complex. © 2015 Published by Elsevier B.V.
1. Introduction There is increasing evidence that obstructive sleep apnoea/ hypopnoea syndrome (OSAHS) is associated with hypertension, cardiovascular disease, metabolic derangements, and impaired glucose tolerance [1]. Sleep disruption in OSAHS may contribute to increased susceptibility to cardiovascular diseases. This effect may be exacerbated by an increase in inflammatory activity due to underlying genetic susceptibility. It has been found that tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are elevated in OSAHS independently of obesity and that the circadian rhythm of TNF-α secretion is disrupted [2,3]. Other elevated mediators of inflammation include intercellular adhesion molecule-1 (ICAM-1) and C-reactive protein (C-RP) [4]. TNFα, C-RP, and IL-6 appear to produce harmful effects by inducing
* Corresponding author. Department of Sleep Medicine, Royal Infirmary Edinburgh, 51 Little France Crescent, Little France, Edinburgh EH164SA, UK. Tel.: +44 0131 2423882; fax: +44 (0) 131 2423878. E-mail address: [email protected] (R.L. Riha). http://dx.doi.org/10.1016/j.sleep.2015.01.006 1389-9457/© 2015 Published by Elsevier B.V.
endothelial dysfunction. In particular, TNF-α damages endothelial cells, causes apoptosis, and triggers pro-coagulant activity and fibrin deposition. TNF-α also enhances the production of reactive oxygen species including inducible nitric oxide (NO) [5]. There could be a possible genetic propensity towards increased pro-inflammatory cytokine production in OSAHS. Ryan et al. demonstrated in a cell culture model the selective activation of nuclear factor kappa B (NFκB)-dependent inflammatory pathways through intermittent hypoxia and reoxygenation [6]. In another study, reversing OSAHS using continuous positive airway pressure resulted in a drop in the levels of circulating NFκB-dependent cytokines, specifically TNF-α and IL-8 [7]. However, no consistent associations with OSAHS were found with a number of cytokines, including IL-1, IL-6, IL-8, IL-10, IL-12, and interferon gamma (IFN-γ). These findings are supported by our previously published work [8], which showed an independent association of the pro-inflammatory TNF-α (−308A) allele with the diagnosis of OSAHS. More recently, Bhushan et al. showed significantly higher levels of TNF-α (−308A) and serum TNF-α levels in obese Asian Indians with obstructive sleep apnoea compared to obese controls [9]. Taken together, results from these studies suggest a disease-promoting role for TNF-α in OSAHS.
P. Bielicki et al./Sleep Medicine 16 (2015) 792–795
TNF-α (−308A) is in linkage disequilibrium with human leucocyte antigen (HLA) class I and II alleles; the class III region, which encodes several components of the complement system; and the major histocompatibility (MHC) class IV cluster, which includes lymphotoxin-α (one of five microsatellites within the TNF locus) and lymphotoxin-β [10]. The association of TNF-α (−308 A) with OSAHS may, therefore, be due to the direct influence of the −308 (A-G) single-nucleotide polymorphism (SNP) in question and/or due to linkage disequilibrium with other polymorphisms within the TNF-α gene or other genes within the HLA system. Since the publication of our paper [8], evidence has emerged that SNP cytokine interactions are complex and that haplotype analysis may be more informative. The aims of this study were to investigate the hypothesis that inflammatory consequences associated with TNF-α in OSAHS are due to linkage disequilibrium of the TNF-α (−308A) SNP with the TNF-α promoter polymorphisms (−1031, −863, −857 and −238) and the lymphotoxin-α polymorphisms (intron 1 and Thr60Asn). Based on the studies that have found elevated levels of pro-inflammatory cytokines IL-6 and IL-8 in OSAHS [7,11], we also hypothesized that this might be due to a predisposition attributable to the proinflammatory IL-6 gene promoter polymorphism (−174) and IL-8 gene promoter polymorphisms (−251 and −781). 2. Method Patient identification, recruitment and the study design have been previously reported [8]. A total of 103 Caucasian sibling pairs (index case diagnosed with OSAHS on the basis of symptoms and apnoea– hypopnoea index (AHI) ≥ 15) were recruited between 1997 and 2002 at the Department of Sleep Medicine, Edinburgh. A total of 192 random anonymous UK blood donors were used as population controls. The local research ethics committee approved the study. Anthropometric data of all recruited subjects were measured, and 20 ml of blood samples were collected. All subjects had overnight polysomnography (PSG) using standard techniques (Compumedics™ W-series® and R-series® system; Compumedics, Melbourne, Australia) [12], except for 16 index cases who had limited sleep studies (Edentrace™ EdenTec® Model 3711 Digital Recorder, Nellcor, Eden Prairie, MN, USA). Sleep studies were analysed by one researcher (RR) after blinding of data using standard scoring criteria [13,14]. The OSAHS phenotype was defined using the AHI and sleepiness, as measured by the Epworth Sleepiness Scale (ESS). Each subject’s AHI, based on normative data for Caucasian subjects, was first scored as either definitely abnormal, indeterminate or definitely normal on the basis of sex and age. The ESS (out of a total of 24) was scored as either sleepy (ESS ≥11) or not abnormally sleepy (ESS ≤11), the cut-offs being based on normative data derived for Caucasian subjects. OSAHS was then classified as being definitely present, indeterminate or definitely absent (see Ref. [8] for more detail). 3. Blood donors DNA from UK Caucasian human random control DNA panels (Product No. HRC-1 96 array and No. HRC-2 96 array) produced by ECACC® (European Collection of Cell Cultures; http://www.phe -culturecollections.org.uk/collections/ecacc.aspx) and distributed by Sigma® was used as a control. All donors had given written informed consent for their blood to be used for research purposes. 4. Allelic discrimination analysis using TaqMan® DNA for recruited subjects was extracted using either the Wizard ® R Genomic DNA Purification Kit (Part # TM050; Promega™) or the Nucleon Extraction and Purification Protocol (Product Code:
793
Nucleon BACC3 RPN 8512; © Amersham International plc). The TaqMan system was used to study the SNP. Assay-by-Design® (ABI™) was used to design the probe and primers. Alleles were read and classified on the plots by two readers independently blinded to subject status. 4.1. Statistical analysis Between group, comparison was performed using the chisquared test (Fisher’s exact test where any expected cell value was 80% for all TNF-α polymorphisms, under the dominant model for the minor allele. Significance was taken at p = 0.05. 5. Results Table 1 shows the characteristics of the recruited population in this study (n = 173; subjects with an indeterminate diagnosis of OSAHS were excluded). Table 2 shows no significant difference across the groups in terms of genotype or allelic frequency in any of the genes investigated. Using the FBAT programme, analysis of the association between disease status and the TNF-α alleles independently (TNF-103, TNF-803, TNF-857 and TNF-238) and with five haplotypes of TNF-α showed no significance (p > 0.05 in all cases). Likewise, analysis of allele and genotype frequencies between obese and non-obese OSAHS subjects showed no significant difference overall (p > 0.05; data not shown). However, only 16% of the group had a body mass index (BMI) ≥ 30 kg/m2. Logistic regression analysis of the TNF-α (−836) alleles, controlling for obesity, a diagnosis of OSAHS, age and gender, revealed that (−836A) carriers were significantly more likely to be obese: odds ratio (OR) 2.4; 95% confidence interval (CI) 1.1–5; and p = 0.025. 6. Discussion In this study, no specific genotype of the TNF-α promoter polymorphisms (−1031, −863, −857 and −238) nor of the two
Table 1 Characteristics of the recruited subjects (n = 173). Variable
No OSAHS (n = 64)
Definite OSAHS (n = 109)
p-value
Sex ratio (M:F) Age (years) BMI (kg/m2) Neck circumference (cm) SBP (mmHg) DBP (mmHg) Sleep efficiencya REM time (min) NREM time (min) SaO2 (%) awake Lowest SpO2 (%)
34:30 52 ± 10 27 ± 5 37 ± 4 129 ± 17 82 ± 12 77 ± 12 70 ± 26 289 ± 48 97 ± 2 90 ± 7
86:23 52 ± 9 30 ± 6 41 ± 4 137 ± 19 83 ± 11 73 ± 15 65 ± 30 274 ± 62 96 ± 2 82 ± 10
Contents lists available at ScienceDirect
Sleep Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / s l e e p
Brief Communication
Cytokine gene polymorphisms in obstructive sleep apnoea/hypopnoea syndrome P. Bielicki a, A.K. MacLeod b, N.J. Douglas c, R.L. Riha c,* a b c
Department of Internal Diseases, Pneumology and Allergology, Warsaw Medical University, Warsaw, Poland Medical Genetics Section, Centre for Molecular Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Department of Sleep Medicine, Royal Infirmary Edinburgh, Edinburgh, UK
A R T I C L E
I N F O
Article history: Received 31 March 2014 Received in revised form 21 December 2014 Accepted 13 January 2015 Available online 7 April 2015 Keywords: Sleep apnoea Cytokine gene polymorphisms TNF-alpha haplotypes Interleukin-6 Interleukin-8 Lymphotoxin
A B S T R A C T
Objective: The pro-inflammatory cytokines, TNF-α, IL-6, and IL-8 are elevated in obstructive sleep apnoea/ hypopnoea syndrome (OSAHS). Cytokine gene interactions are complex and haplotype analysis may be more informative. We hypothesized that the effects of TNF-α in OSAHS might be due to linkage disequilibrium of the TNF-α (−308A) single nucleotide polymorphism (SNP) with other polymorphisms within the TNF-α gene, and that predisposition to elevated IL-6 and IL-8 levels in OSAHS might be attributable to pro-inflammatory IL-6 and IL-8 gene promoter polymorphisms. Method: 173 subjects were classified as having definite OSAHS or not on the basis of apnoea– hypopnoea frequency, sex, age, and symptoms. Population controls comprised 192 random UK blood donors. Genotyping was undertaken for the TNF- α promoter polymorphisms (−1031, −863, −857, −238), two lymphotoxin-α polymorphisms (intron 1 and Thr60Asn), the pro-inflammatory IL-6 gene promoter polymorphism (−174), and IL-8 gene promoter polymorphisms (−251; −781). Results: There was no significant difference between groups re: genotype/allelic frequency in the genes investigated. Association between disease status and the TNF-α alleles independently (TNF-103, TNF803, TNF-857, TNF-238) with five haplotypes of TNF-α was not significant (p > 0.05). There was no difference in allelic or genotypic frequencies between obese and non-obese subjects with OSAHS. The TNF- α (−863A) allele alone, was significantly associated with obesity (OR 2.4; CI95% 1.1–5; p = 0.025). Conclusion: Only the TNF- α (308A) SNP appears to be significantly associated with OSAHS. The impact of cytokine gene polymorphisms on phenotypic expression of inflammation in OSAHS is likely to be complex. © 2015 Published by Elsevier B.V.
1. Introduction There is increasing evidence that obstructive sleep apnoea/ hypopnoea syndrome (OSAHS) is associated with hypertension, cardiovascular disease, metabolic derangements, and impaired glucose tolerance [1]. Sleep disruption in OSAHS may contribute to increased susceptibility to cardiovascular diseases. This effect may be exacerbated by an increase in inflammatory activity due to underlying genetic susceptibility. It has been found that tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are elevated in OSAHS independently of obesity and that the circadian rhythm of TNF-α secretion is disrupted [2,3]. Other elevated mediators of inflammation include intercellular adhesion molecule-1 (ICAM-1) and C-reactive protein (C-RP) [4]. TNFα, C-RP, and IL-6 appear to produce harmful effects by inducing
* Corresponding author. Department of Sleep Medicine, Royal Infirmary Edinburgh, 51 Little France Crescent, Little France, Edinburgh EH164SA, UK. Tel.: +44 0131 2423882; fax: +44 (0) 131 2423878. E-mail address: [email protected] (R.L. Riha). http://dx.doi.org/10.1016/j.sleep.2015.01.006 1389-9457/© 2015 Published by Elsevier B.V.
endothelial dysfunction. In particular, TNF-α damages endothelial cells, causes apoptosis, and triggers pro-coagulant activity and fibrin deposition. TNF-α also enhances the production of reactive oxygen species including inducible nitric oxide (NO) [5]. There could be a possible genetic propensity towards increased pro-inflammatory cytokine production in OSAHS. Ryan et al. demonstrated in a cell culture model the selective activation of nuclear factor kappa B (NFκB)-dependent inflammatory pathways through intermittent hypoxia and reoxygenation [6]. In another study, reversing OSAHS using continuous positive airway pressure resulted in a drop in the levels of circulating NFκB-dependent cytokines, specifically TNF-α and IL-8 [7]. However, no consistent associations with OSAHS were found with a number of cytokines, including IL-1, IL-6, IL-8, IL-10, IL-12, and interferon gamma (IFN-γ). These findings are supported by our previously published work [8], which showed an independent association of the pro-inflammatory TNF-α (−308A) allele with the diagnosis of OSAHS. More recently, Bhushan et al. showed significantly higher levels of TNF-α (−308A) and serum TNF-α levels in obese Asian Indians with obstructive sleep apnoea compared to obese controls [9]. Taken together, results from these studies suggest a disease-promoting role for TNF-α in OSAHS.
P. Bielicki et al./Sleep Medicine 16 (2015) 792–795
TNF-α (−308A) is in linkage disequilibrium with human leucocyte antigen (HLA) class I and II alleles; the class III region, which encodes several components of the complement system; and the major histocompatibility (MHC) class IV cluster, which includes lymphotoxin-α (one of five microsatellites within the TNF locus) and lymphotoxin-β [10]. The association of TNF-α (−308 A) with OSAHS may, therefore, be due to the direct influence of the −308 (A-G) single-nucleotide polymorphism (SNP) in question and/or due to linkage disequilibrium with other polymorphisms within the TNF-α gene or other genes within the HLA system. Since the publication of our paper [8], evidence has emerged that SNP cytokine interactions are complex and that haplotype analysis may be more informative. The aims of this study were to investigate the hypothesis that inflammatory consequences associated with TNF-α in OSAHS are due to linkage disequilibrium of the TNF-α (−308A) SNP with the TNF-α promoter polymorphisms (−1031, −863, −857 and −238) and the lymphotoxin-α polymorphisms (intron 1 and Thr60Asn). Based on the studies that have found elevated levels of pro-inflammatory cytokines IL-6 and IL-8 in OSAHS [7,11], we also hypothesized that this might be due to a predisposition attributable to the proinflammatory IL-6 gene promoter polymorphism (−174) and IL-8 gene promoter polymorphisms (−251 and −781). 2. Method Patient identification, recruitment and the study design have been previously reported [8]. A total of 103 Caucasian sibling pairs (index case diagnosed with OSAHS on the basis of symptoms and apnoea– hypopnoea index (AHI) ≥ 15) were recruited between 1997 and 2002 at the Department of Sleep Medicine, Edinburgh. A total of 192 random anonymous UK blood donors were used as population controls. The local research ethics committee approved the study. Anthropometric data of all recruited subjects were measured, and 20 ml of blood samples were collected. All subjects had overnight polysomnography (PSG) using standard techniques (Compumedics™ W-series® and R-series® system; Compumedics, Melbourne, Australia) [12], except for 16 index cases who had limited sleep studies (Edentrace™ EdenTec® Model 3711 Digital Recorder, Nellcor, Eden Prairie, MN, USA). Sleep studies were analysed by one researcher (RR) after blinding of data using standard scoring criteria [13,14]. The OSAHS phenotype was defined using the AHI and sleepiness, as measured by the Epworth Sleepiness Scale (ESS). Each subject’s AHI, based on normative data for Caucasian subjects, was first scored as either definitely abnormal, indeterminate or definitely normal on the basis of sex and age. The ESS (out of a total of 24) was scored as either sleepy (ESS ≥11) or not abnormally sleepy (ESS ≤11), the cut-offs being based on normative data derived for Caucasian subjects. OSAHS was then classified as being definitely present, indeterminate or definitely absent (see Ref. [8] for more detail). 3. Blood donors DNA from UK Caucasian human random control DNA panels (Product No. HRC-1 96 array and No. HRC-2 96 array) produced by ECACC® (European Collection of Cell Cultures; http://www.phe -culturecollections.org.uk/collections/ecacc.aspx) and distributed by Sigma® was used as a control. All donors had given written informed consent for their blood to be used for research purposes. 4. Allelic discrimination analysis using TaqMan® DNA for recruited subjects was extracted using either the Wizard ® R Genomic DNA Purification Kit (Part # TM050; Promega™) or the Nucleon Extraction and Purification Protocol (Product Code:
793
Nucleon BACC3 RPN 8512; © Amersham International plc). The TaqMan system was used to study the SNP. Assay-by-Design® (ABI™) was used to design the probe and primers. Alleles were read and classified on the plots by two readers independently blinded to subject status. 4.1. Statistical analysis Between group, comparison was performed using the chisquared test (Fisher’s exact test where any expected cell value was 80% for all TNF-α polymorphisms, under the dominant model for the minor allele. Significance was taken at p = 0.05. 5. Results Table 1 shows the characteristics of the recruited population in this study (n = 173; subjects with an indeterminate diagnosis of OSAHS were excluded). Table 2 shows no significant difference across the groups in terms of genotype or allelic frequency in any of the genes investigated. Using the FBAT programme, analysis of the association between disease status and the TNF-α alleles independently (TNF-103, TNF-803, TNF-857 and TNF-238) and with five haplotypes of TNF-α showed no significance (p > 0.05 in all cases). Likewise, analysis of allele and genotype frequencies between obese and non-obese OSAHS subjects showed no significant difference overall (p > 0.05; data not shown). However, only 16% of the group had a body mass index (BMI) ≥ 30 kg/m2. Logistic regression analysis of the TNF-α (−836) alleles, controlling for obesity, a diagnosis of OSAHS, age and gender, revealed that (−836A) carriers were significantly more likely to be obese: odds ratio (OR) 2.4; 95% confidence interval (CI) 1.1–5; and p = 0.025. 6. Discussion In this study, no specific genotype of the TNF-α promoter polymorphisms (−1031, −863, −857 and −238) nor of the two
Table 1 Characteristics of the recruited subjects (n = 173). Variable
No OSAHS (n = 64)
Definite OSAHS (n = 109)
p-value
Sex ratio (M:F) Age (years) BMI (kg/m2) Neck circumference (cm) SBP (mmHg) DBP (mmHg) Sleep efficiencya REM time (min) NREM time (min) SaO2 (%) awake Lowest SpO2 (%)
34:30 52 ± 10 27 ± 5 37 ± 4 129 ± 17 82 ± 12 77 ± 12 70 ± 26 289 ± 48 97 ± 2 90 ± 7
86:23 52 ± 9 30 ± 6 41 ± 4 137 ± 19 83 ± 11 73 ± 15 65 ± 30 274 ± 62 96 ± 2 82 ± 10
