IntroductIon
Obstructive sleep apnoea (OSA) is the most frequent clin-ical form of breathing disorders during sleep. The preva-lence of OSA in Poland is estimated to reach 8.7% in men and 2.5% in women aged 40–72 years [1]. There is strong evidence that OSA is an independent risk factor for the development of arterial hypertension (AH) [2, 3]. AH may affect up to 40% of patients with breathing disorders dur-ing sleep [4]. In the recent years, much attention has been focused on the genetic background of AH in patients with OSA.
Among others, 3 aspects had been taken into considera-tion in the search for genes that could influence the
de-velopment of arterial hypertension in patients with OSA: the contribution of inflammatory mediators, oxidative stress and endothelial dysfunction. Research on the role of tumor necrosis factor a (TNF-α) gene polymorphism showed its association with breathing disorders during sleep [5, 6]. The impact of polymorphism of the gene for endothelial synthesis of nitrogen oxide (eNOS) on the increased risk of arterial hypertension was also investi-gated, but the results were equivocal [7]. Research on the oxidative stress factors did not confirm the relationship between the polymorphism of the gene for haptoglobins and hypertension in young patients with OSA [8] or an influence of the gene polymorphism for NADPH oxi-dase and the value of systolic blood pressure in patients
gene polymorphisms related
to an increased incidence of arterial
hypertension in a cohort of Polish patients
with obstructive sleep apnea syndrome
(oSaS)
Związek polimorfizmu wybranych genów ze zwiększoną częstością nadciśnienia tętniczego
– badanie w populacji polskich pacjentów z obturacyjnym bezdechem podczas snu (OSAS)
Piotr Bielicki, MD, PhD
1, Prof. Robert Pływaczewski, MD, PhD
2, Kamil Brzóska, PhD
3,
Piotr Sobieraj, MD
4, Małgorzata Barnaś, MD, PhD
1, Marta Kumor, MD, PhD
1,
Luiza Jonczak, MD, PhD
2, Tomasz M. Stępkowski, PhD
3, Aleksandra Piechuta, MD
1,
Prof. Ryszarda Chazan, MD, PhD
1, Prof. Paweł Śliwiński, MD, PhD
2,
Prof. Marcin Kruszewski
3, 5 1 Department of Internal Medicine, Pulmonary Diseases and Allergy, Medical University of Warsaw, Poland 2 II Department of Respiratory Medicine, Institute for Tuberculosis and Lung Diseases Warsaw, Poland 3 Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warsaw, Poland4 Department of Internal Medicine, Hypertension and Vascular Diseases, Medical University of Warsaw, Poland 5 Department of Molecular Biology and Translational Research, Institute of Rural Health, Lublin, Poland
with OSA [9]. Most of the genes mentioned above are di-rectly related to arterial hypertension. Data on the poly- morphisms of genes which may indirectly impact the risk of arterial hypertension in OSA are scarce. These include, among others, genes which are involved in adipose tissue metabolism, lipid disorders and genes which increase sus-ceptibility to stroke, diabetes mellitus, obesity and meta-bolic syndrome which are frequent comorbidities in pa-tients with AH and in papa-tients with OSA at the same time. We therefore undertook a study aimed at the evaluation of the influence of the polymorphism of selected genes known to be related with cardiovascular diseases, obesity and metabolic syndrome on the diagnosis of arterial hy-pertension in a cohort of Polish patients with OSA.
MaterIal and Methods
study design
This is a cross sectional study which included patients with OSA in all stages of severity. Polymorphisms of genes encoding the following proteins were evaluated:
• arachidonate 5-lipoxygenase-activating protein (ALOX5AP), the variants of which have been shown to be related with the susceptibility to stroke [10] • sterol regulatory element binding transcription factor
2 (SREBF2), a regulator cholesterol metabolism. A re-lationship between this gene polymorphism and myo-cardial infarction has been documented [11]
• adrenergic alpha-2A-receptor (ADRA2A), involved in the regulation of neurotransmission in the central nervous system and the reduction of lipolysis in adi-pose tissue. Genetic variants of the gene encoding this protein may promote the development of obesity [12] • apolipoprotein A-V (APOA5), a regulator of
triglycer-ides level, the polymorphism of which may promote atherosclerosis [13]
• transcription factor 7-like 2 (TCF7L2), related to an increased susceptibility to diabetes type 2 [14].
The study protocol was approved by the Bioethical Com-mittee of the Medical University of Warsaw (KB/2/2010) and received financial support from the Polish National Science Centre (710/N-COST/2010/0).
study participants
Six hundred consecutive adult patients recruited from 2 tertiary care outpatient clinics in Warsaw, Poland,
were enrolled to the study. The inclusion criteria were as follows: 1) age > 18 years, 2) the diagnosis of OSA confirmed in overnight polysomnography, 3) informed consent to participate in the study (to genetic testing in particular). Exclusion criteria were as follows: 1) lack of signed informed consent for genetic testing, 2) secondary arterial hypertension due an underlying condition other than OSA.
Polysomnography
In all patients the diagnosis of OSA was confirmed in
at-tended overnight polysomnography (Embla®S4000 and
Alice 5 Polysomnography) with electroencephalogram, electrooculogram, electromyogram, electrocardiogram, airflow (measured by nasal cannula or a thermistor) and chest and abdominal motion (two piezoelectric belts) re-cording. Blood oxygen saturation during sleep was mon-itored with pulse oximetry. The diagnosis of OSA and its severity were established in accordance with the Ameri-can Academy of Sleep Medicine guidelines [15].
diagnosis of arterial hypertension
The diagnosis of arterial hypertension was established in accordance with the guidelines [16]. In all patients with AH, the diagnosis of arterial hypertension had been es-tablished prior to the study onset and the data on AH were taken from the patients’ medical history.
Genetic testing
Gene polymorphisms for genes encoding ALOX5AP, SREBF2, ADRA2A, APOA5 and TCF7L2 were analyzed in whole blood samples. Genomic DNA was isolated us-ing QIAamp DNA Blood Mini Kit (Qiagen) accordus-ing to the manufacturer’s protocol. Gene polymorphisms were assessed using TaqMan® SNP Genotyping Assays (Life Technologies) and TaqMan® Universal Master Mix II, no UNG (Life Technologies). Thermal cycling was per-formed on the 7500 Real-Time PCR System (Life Tech-nologies) using 100 ng DNA as a template. Assay IDs are given in table 1. Initial denaturation cycle was at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The data were collected and ana-lyzed using SDS 2.4 software (Life Technologies).
statistical analysis
Statistical analysis was performed with Statistica for Windows (Statsoft Inc. STATISTICA version 9, www. statsoft.com). Normality of the continuous variables was
verified with Shapiro-Wilk test. The comparison of con-tinuous variables between 2 groups were performed us-ing Mann-Whitney test. Chi-square tests were employed for comparison of discrete data. The differences between analysed groups were considered as significant when p-value was < 0.05.
results
Patient characteristics
Arterial hypertension was diagnosed in 427 of the 600 study participants (71.2%). Although patients with AH had a higher value of oxygen desaturation index (ODI) and lower values of minimal and mean oxygen satura-tion during sleep, there was no difference in the degree of daily sleepiness and OSA severity when compared to normotensive OSA patients. The characteristics of the patients with and without arterial hypertension are pre-sented in table 2.
relationship between the polymorphisms of the selected genes and the incidence of arterial hypertension
Analysis of the relationship between polymorphism of selected genes and diagnosis of arterial hypertension in the group as a whole showed such a relationship only for TCF7L2, finding the lowest incidence of arterial hy-pertension in TT homozygotes, particularly low in the group of patients with AHI > 30 (74% CC vs. 48% TT, p < 0.05).
In men, there was a higher risk of hypertension in AA ho-mozygotes of the gene for ALOX5AP (84% AA vs. 66%
table 1. TaqMan® SNP Genotyping Assays used in the study.
Gene Polymorphism taqMan® snP Genotyping assay Id
ALOX5AP [A/G] rs9579646 C___29375_20
SREBF2 [C/G] rs2228314 C___2189943_10
ADRA2A [C/G] rs1800544 C___7611979_10
APOA5 [C/G] rs3135506 C___25638153_10
TCF7L2 [C/T] rs7903146 C___29347861_10
ALOX5 AP – arachidonate 5-lipoxygenase-activating protein, SREBF2 – sterol regulatory element binding transcription factor 2, ADRA2A – adrenergic α-2A-receptor, APOA5 – apolipoprotein A-V, TCF7L2 – transcription factor 7-like 2, ID – identification.
table 2. Characteristics and comparison of the investigated
hypertensive and normotensive patients with obstructive sleep apnea syndrome (OSA).
Parameter osa ah(-)(n = 173) osa ah(+)(n = 427) p
Age (years) 57.0 (47–63) 59.0 (53–64) p < 0.01 BMI (kg/m2) 29.7 (26.4–33.3) 32.8 (28.9–37.3) p < 0.0001 Men (n, %) 129 (74.6) 320 (74.9) ns Hip circumfer-ence (cm) 106 (100–114) 111 (104–120) p < 0.0001 Waist circumfer-ence (cm) 102 (95–110) 110 (100–123) p < 0.0001 Neck circumfer-ence (cm) 42 (39–44) 43 (40–45) p = 0.0001 ESS (points) 10 (8–15) 11 (7–15) ns Cholesterol (mg/dl) 197 (181–223) 181 (155–209) p < 0.0001 Triglycerides (mg/dl) 122 (79–173) 142.5 (99–198) p < 0.01 LDL (mg/dl) 120 (103–137) 104.5 (80–133) p < 0.01 HDL (mg/dl) 48 (40–56) 44 (37–54) ns HbA1c (%) 5.9 (5.5–6.3) 6.1 (5.7–6.6) p < 0.001 AHI (n/hour) 36.9 (23–57) 39.0 (24–60) ns ODI (n/hour) 25.6 (14–44) 32.7 (17–59) p < 0.01 Mean SpO2 during sleep (%) 93 (91–94) 92 (89–93) p < 0.01 Minimum SpO2 during sleep (%) 79 (73–84) 76 (67–82) p < 0.01
Data presented as median and interquartile range. OSA AH(-) – patients with obstructive sleep apnea without co-existing hypertension, OSA AH(+) – patients with obstructive sleep apnea with co-existing hyper-tension, BMI – body mass index, ESS – Epworth Sleepiness Scale, LDL – low density lipoprotein cholesterol, HDL – high density lipoprotein
cholesterol, HbA1c – glycated hemoglobin (hemoglobin A1C), AHI –
ap-nea/hypopnea index, ODI – oxygen desaturation index, SpO2 – blood
oxygen saturation measured by pulse oximetry, ns – not significant.
GG, p < 0.05). A relationship between ALOX5AP gene polymorphism was also found in a group of patients with AHI > 15 and < 30.
Analysis of the polymorphisms of the other genes showed a higher incidence of arterial hypertension in: GG homozygotes of SREBF gene in a group of persons with BMI > 30 and < 35 (74% GG vs. 29% CC, p < 0.05), CG heterozygotes for ADRA2A gene in a group of
pa-table 3. Frequency of arterial hypertension in groups with polymorphisms of the selected genes.
a
ll gr
oup
n (%) Men n (%) Women n (%)
BMI < 25 n (%) BMI 25–30 n (%) BMI 30–35 n (%) BMI > 35 n (%) ahI < 15 n (%) ahI 15–30 n (%) ahI > 30 n (%)
a ge < 50 y ears n (%) a ge 50–65 y ears n (%) a ge > 65 y ears n (%) al o X5 a P a/a n = 43 33(76%) 27(84%) 6(55%) 2(50%) 9(69%) 8(73%) 14(93%) 6(75%) 5(56%) 22(85%) 8(80%) 21(72%) 4(100%) a/G n = 226 170(75%) 130(76%) 40(74%) 13(59%) 47(69%) 48(73%) 62(89%) 20(69%) 37(80%) 113(75%) 28(67%) 107(78%) 35(75%) G/G n = 331 224(68%) 163(67%) 61(71%) 10(48%) 57(55%) 77(70%) 80(83%) 22(69%) 48(57%) 154(72%) 42(51%) 135(72%) 47(78%) p ns p < 0.05 ns ns ns ns ns ns p < 0.05 ns ns ns ns sre BF2 c/c n = 26 14(54%) 12 (55%) 2 (50%) 1 (100%) 4 (50%) 2 (29%) 7 (70%) 2 (50%) 3 (50%) 9 (56%) 3 (50%) 8 (53%) 3 (60%) c/G n = 218 151(69%) 106 (69%) 45 (68%) 8 (47%) 40 (59%) 51 (71%) 52 (85%) 21 (81%) 25 (52%) 105 (73%) 22 (50%) 96 (75%) 33 (72%) G/G n = 356 262(74%) 202(74%) 60(74%) 16(55%) 69(64%) 80(74%) 97(87%) 25(64%) 62(72%) 175(76%) 56(62%) 159(75%) 50(83%) p ns ns ns ns ns p < 0.05 ns ns ns ns ns ns ns adr a 2a c/c n = 362 251(69%) 186 (68%) 65 (72%) 17 (57%) 61 (57%) 78 (68%) 95 (86%) 31 (76%) 51 (58%) 169 (732%) 41 (50%) 156 (74%) 54 (77%) c/G n = 204 148(73%) 112 (74%) 36 (68%) 6 (43%) 42 (67%) 49 (75%) 51 (82%) 13 (54%) 32 (80%) 103 (74%) 32 (67%) 87 (74%) 29 (76%) G/G n = 34 28(82%) 12 (84%) 6 (75%) 2 (67%) 10 (77%) 6 (75%) 10 (100%) 4 (100%) 7 (68%) 17 (94%) 5 (100%) 20 (77%) 3 (100%) p ns ns ns ns ns ns ns ns p < 0.05 ns p < 0.05 ns ns a Po a 5 c/c n = 3 2 (67%) 2(67%) - - 1(50%) - (100%)1 1(50%) - 1(100%) 1(100%) 1(50%) -c/G n = 60 47(78%) 35(83%) 12(67%) 4(100%) 11(64%) 17(77%) 15(88%) 7(100%) 12(86%) 28(72%) 9(69%) 27(82%) 11(79%) G/G n = 537 378(71%) 283 (70%) 95 (71%) 21 (49%) 101 (61%) 116 (70%) 140 (85%) 40 (67%) 78 (62%) 260 (74%) 68 (56%) 235 (74%) 75 (77%) p ns ns ns p < 0.05 ns ns ns ns ns ns ns ns ns tc F7 l2 c/c n = 354 252(71%) 192(71%) 60(71%) 12(57%) 67(63%) 77(69%) 96(83%) 27(69%) 58(66%) 167(74%) 48(59%) 151(73%) 53(80%) c/t n = 217 160(74%) 118 (74%) 42 (72%) 11 (52%) 43 (62%) 50 (77%) 56 (90%) 18 (72) 30 (61%) 112 (78%) 27 (61%) 102 (78%) 31 (74%) t/t n = 29 15(51%) 10(50%) 5(56%) 2(40%) 3(38%) 6(55%) 4(80%) 3(60%) 2(67%) 10(48%) 3(30%) 10(69%) 2(67%) p p < 0.05 ns ns ns ns ns ns ns ns p < 0.05 ns ns ns
BMI – body mass index, ESS – Epworth Sleepiness Scale, AHI – apnea/hypopnea index, ALOX5 AP – arachidonate 5-lipoxygenase-activating protein, SREBF2 – sterol regulatory element binding transcription factor 2, ADRA2A – adrenergic α-2A-receptor, APOA5 – apolipoprotein A-V, TCF7L2 – transcription factor 7-like 2, ns – not significant, p = 0.05.
tients with AHI > 15 and < 30 (80% CG vs. 58% CC and GG, p < 0.05) and in CG heterozygotes of APOA5 gene in normal weight patients with OSA. The incidence of sepa-rate gene polymorphisms in subjects with arterial hyper-tension in the tested groups is presented in table 3.
dIscussIon
Our results indicate essential link of TCF7L2 gene poly-morphism and the incidence of arterial hypertension in the Polish population with OSAS. We also found a lower AH incidence in GG homozygotes of the gene for SRBEF
among OSA patients with a value of BMI between 30 and 35 kg/m2. Our results suggest significant contribution of
the polymorphism of the genes related to cardiovascular diseases and lipid disorders in the pathogenesis of AH in OSA patients. Our study presents the analysis of gene polymorphism in the Polish population which is con-sidered to be relatively genetically uniform; 94.8% of the population is Polish [Central Statistical Office of Poland.
www.stat.gov.pl, accessed May 8th 2016] and may
there-fore be an interesting contribution in the research on the role genetic factors in OSA. The genes investigated in the
present study have not been widely studied in the context of OSA and arterial hypertension.
There are numerous studies confirming that OSA is an independent risk factor for the development of arterial hypertension [2, 3]. High daytime blood pressure, both systolic and diastolic, and a higher variability of arterial blood pressure during sleep are found in the majority of patients with OSAS [17, 18].
There is evidence that the relationship between arterial hypertension and OSA is independent of obesity [13]. In our study, hypertensive subjects had a higher BMI and a larger neck, hip and waist circumference. They also had higher values of triglycerides. Surprisingly, other
li-pid parameters and glycated hemoglobin (HbA1c) were
higher in normotensive patients. This may probably be explained by the fact that hypertensive OSA patients had already been receiving treatment for hyperlipidemia and diabetes, while patients without arterial hypertension were not being treated.
In the development of arterial hypertension in patients with OSA the following mechanisms play a role: chronic intermittent hypoxia during sleep, stimulation of sympa-thetic nervous system, a higher expression of adhesion molecules and proinflammatory cytokines activation. In the investigated group, persons with arterial hyper-tension had a much higher value of desaturation index (ODI) during sleep and a higher serum CRP than normo-tensive patients. However, no influence of OSA severity (expressed in AHI value) on the incidence of arterial hy-pertension was found, what concurs with the observa-tions of other authors [2]. This may confirm that ODI is superior to AHI as an index of potential cardiovascular complications in OSA.
Studies regarding genetic aspects in patients with OSA are still scarce. The majority of studies published up to date concerned the impact of TNF-α gene polymorphism on the diagnosis of OSA. Although, some studies con-firmed this relationship, the influence of this polymor-phism on the occurrence of cardiovascular complications in these patients was not shown [5]. However, evaluation of genetic variability for oxidative stress factors showed a correlation of haptoglobin and oxidase NADPH gene polymorphism with arterial hypertension [8, 9].
As the aim of our study was the search for genes with al-lelic variability that may influence the diagnosis of arteri-al hypertension in patients with OSA, we selected genes with a confirmed relationship between with their poly-morphism and the diagnosis of cardiovascular or meta-bolic diseases. 5-lipoxygenase gene polymorphism is re-lated with the occurrence of stroke [10]. Apolipoprotein A-V (APOA5) and SREBF2 gene polymorphism is related with an increased risk of myocardial infarction and ather-osclerosis [11, 13]. There is a link between ADRA2A and leptin receptor gene polymorphism and obesity [12]. To our knowledge, there are no available data on the poly-morphisms of these genes in patients with OSAS.
The analysis of the selected genes in a large group of 600 OSA patients from Poland showed a connection between the diagnosis of arterial hypertension and the genetic var-iability only for the TCF7L2 gene known to be associated with diabetes mellitus type 2 [19]. This observation was particularly pronounced in subjects with the highest de-gree of OSA severity. The large number of the investigat-ed subjects is, in our opinion, a big advantage of our study and increases the credibility our findings. However, the group of TT homozygotes in whom the incidence of ar-terial hypertension was low comprised only of 29 patients, thus the results for this subgroup must be interpreted with caution.
The second important finding is the lower incidence of arterial hypertension in the group of male apolipoprotein A-V GG homozygotes. As far as other genes are con-cerned, we found an impact of their polymorphism on the diagnosis of arterial hypertension in the subgroups in which AHI, BMI and age were the discriminating crite-ria, however, the small number of patients in these groups also requires caution while interpreting the results. Our results show the need for further investigations in this field to unequivocally determine the role of gene poly-morphism on the occurrence of arterial hypertension in OSA patients. The analyses regarding impact of genes polymorphism in relationship with traditional AH risk factors should be performed. These studies should also be extended to a group of persons with genotypes known to be rare in the population.
To conclude, among the investigated gene polymor-phisms, only TCF7L2 polymorphism was related to an increased incidence of AH in a cohort of Polish patients
aBstract
Introduction: Obstructive sleep apnoea (OSA) in an independent risk factor for arterial hypertension development.
Both OSA and arterial hypertension frequently coexist, thus common genetic background can be suspected.
Material and methods: We enrolled 600 participants with diagnosis of OSA in all stages of severity from 2 tertiary
care outpatient clinics in Warsaw, Poland.
All patients undergone polysomnography and were evaluated for the diagnosis of hypertension. Gene polymor-phisms for genes encoding ALOX5AP, SREBF2, ADRA2A, APOA5 and TCF7L2 were analyzed in blood samples. Chi-square and Mann-Whitney tests were used for comparisons.
results: Data of 449 (74.8%) men and 151 (25.2%) women were analyzed. Arterial hypertension was diagnosed in
427 (71.2%) participants. In the hypertensive patients with OSA median age was 59 (53–64) years, BMI 32.8 (28.9– 37.3) kg/m2, AHI 39 (24–60)/hour. Among tested genes only TCF7L2 polymorphism ([C/T] rs7903146) was linked to
increased incidence of arterial hypertension.
conclusions: Results of our analysis suggest potential relationship of TCF7L2 polymorphism ([C/T] rs7903146) and
development of hypertension in patients with OSA, although further studies regarding impact of genetic in compari-son with traditional risk factor for hypertension are needed.
Key words: obstructive sleep apnoea, arterial hypertension, genetic factors
streszczenIe
Wprowadzenie: Obturacyjny bezdech podczas snu (OSA) jest niezależnym czynnikiem ryzyka nadciśnienia
tętnicze-go (NT). Ze względu na to, że OSA i NT często ze sobą współwystępują, można podejrzewać, że mają one wspólne podłoże genetyczne.
Materiał i metody: W badaniu wzięło udział 600 uczestników z rozpoznanym OSA, we wszystkich stopniach
nasile-nia choroby. Badanie przeprowadzono w 2 ośrodkach III stopnasile-nia referencyjności w Warszawie.
U wszystkich pacjentów przeprowadzono badanie polisomnograficzne oraz diagnostykę w kierunku NT. Analizowa-no polimorfizm genów kodujących ALOX5AP, SREBF2, ADRA2A, APOA5 i TCF7L2 w próbkach krwi. W analizie wyko-rzystano test Chi2 oraz Manna-Whitneya.
Wyniki: Dane 449 (74,8%) mężczyzn oraz 151 (25,2%) kobiet zostały poddane analizie. NT rozpoznano u 427 (71,2%)
uczestników. W grupie chorych z NT i OSA mediana wieku wynosiła 59 (53–64) lat, BMI 32,8 (28,9–37,3) kg/m2, AHI 39
(24–60)/h. Spośród analizowanych polimorfizmów tylko TCF7L2 ([C/T] rs7903146) okazał się związany ze zwiększo-nym ryzykiem NT.
Wnioski: Wyniki naszej analizy wskazują na potencjalny związek pomiędzy polimorfizmem TCF7L2 ([C/T] rs7903146)
i NT, konieczne są jednak dalsze badania uwzględniające wpływ genetycznych oraz tradycyjnych czynników ryzyka.
słowa kluczowe: obturacyjny bezdech podczas snu, nadciśnienie tętnicze, czynniki genetyczne
corresPondence
Piotr sobieraj, Md
Department of Internal Medicine, Hypertension and Vascular Diseases, Medical University of Warsaw
02-097 Warsaw, Banacha 1a phone: (22) 599-28-28 fax: (22) 599-28-18 e-mail: piotrsob@vp.pl Authors’ contributions:
Bielicki P.: 20%; Pływaczewski R.: 10%; Brzóska K.: 15%; Sobieraj P.: 15%; Barnaś M.: 5%; Kumor M.: 5%; Jonczak L.: 5%; Stępkowski T.M.: 5%; Piechuta A.: 5%; Chazan R.: 5%; Śliwiński P.: 5%; Kruszewski M.: 5%.
Conflict of interests: None. Financial support:
with OSA. This relationship was particularly expressed in patients with severe OSA.
The study received financial support from the Polish Na-tional Science Centre (710/N-COST/2010/0).
None. Ethics: The contents presented in this paper are compatible with the rules the
Declaration of Helsinki, EU directives and standardized requirements for medical journals.
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