上海交通大学学报（医学版）

上海交通大学学报(医学版), 2024, 44(12): 1490-1503 doi: 10.3969/j.issn.1674-8115.2024.12.002

论著 · 基础研究

NOS1基因变异与失眠、睡眠时长以及阻塞性睡眠呼吸暂停临床数量性状的相关性

袁灏琳,, 李念念, 胡珺晖, 沈锦虹, 高振飞, 关建, 刘峰, 殷善开,

上海交通大学医学院附属第六人民医院耳鼻咽喉头颈外科，上海市睡眠呼吸障碍疾病重点实验室，上海 200233

Association of genetic variations in the NOS1 gene with insomnia, sleep duration and obstructive sleep apnea-related clinical quantitative traits

YUAN Haolin,, LI Niannian, HU Junhui, SHEN Jinhong, GAO Zhenfei, GUAN Jian, LIU Feng, YIN Shankai,

Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Sleep Disordered Breathing, Shanghai 200233, China

通讯作者: 殷善开，电子信箱：skyin@sjtu.edu.cn。

编委: 包玲

收稿日期: 2024-04-12   接受日期: 2024-05-21   网络出版日期: 2024-12-25

基金资助: 科技创新2030—重大项目.  2021ZD0201900
国家自然科学基金.  82100105
上海市科学技术委员会项目.  18DZ2260200

Corresponding authors: YIN Shankai, E-mail:skyin@sjtu.edu.cn.

Received: 2024-04-12   Accepted: 2024-05-21   Online: 2024-12-25

作者简介 About authors

袁灏琳（1995—），女，硕士生；电子信箱：hlyuan78@126.com。 E-mail：hlyuan78@126.com。

摘要

目的·探索神经型一氧化氮合酶（nitric oxide synthase 1，NOS1）遗传变异rs7305526和rs11615756与失眠、睡眠时长和阻塞性睡眠呼吸暂停（obstructive sleep apnea，OSA）临床数量性状的相关性。方法·利用Allen Human Brain Atlas数据集分析NOS1基因在人类全脑水平的表达模式，通过表达数量性状位点分析rs7305526和rs11615756对NOS1基因表达的影响。利用英国生物银行（United Kingdom Biobank，UKB）的全基因组关联研究（Genome Wide Association Study，GWAS）数据集，采用回归分析探讨rs7305526和rs11615756与失眠及睡眠时长2种睡眠性状的相关性。利用上海睡眠健康队列研究（Shanghai Sleep Health Study Cohort，SSHS）基于标准多导睡眠监测（polysomnography，PSG）的临床监测数据，分析rs7305526和rs11615756与OSA临床数量性状（包括呼吸、血氧和睡眠结构性状）的相关性。结果·NOS1基因在睡眠调节脑区（杏仁核、基底前脑、纹状体和丘脑）和呼吸中枢（间脑和脑桥被盖部）的部分核团呈较高的表达水平，而在大脑和小脑皮层、脑桥等区域呈低水平的表达或不表达。rs7305526和rs11615756与NOS1在大脑皮层的表达水平均呈显著负相关，此外rs11615756同样与杏仁核中NOS1的表达水平呈显著负相关。UKB GWAS数据显示rs7305526与失眠、睡眠时长无显著相关，而rs11615756仅与睡眠时长呈显著负相关。SSHS临床监测数据显示，rs7305526与最低血氧饱和度（lowest pulse blood oxygen saturation，LSpO2）、呼吸暂停低通气指数和非快速眼动睡眠（non-rapid eye movement，NREM）2期时间占比等OSA临床数量性状的变化显著相关。虽然rs11615756仅与NREM 2和3期次数呈显著负相关，但在根据OSA严重程度分层后，rs11615756和rs7305526与部分呼吸血氧性状存在显著相关性。结论·NOS1基因变异与人类睡眠时长性状和OSA呼吸、血氧、睡眠结构性状存在显著关联，rs7305526（C>A）对睡眠性状的调节独立于对呼吸、血氧的调节。

关键词： 阻塞性睡眠呼吸暂停 ; NOS1基因 ; 人类睡眠 ; 临床数量性状 ; 遗传变异

Abstract

Objective ·To explore the correlation between the genetic variations rs7305526 and rs11615756 of nitric oxide synthase 1 (NOS1) and the human sleep traits, including insomnia, sleep duration, and clinical quantitative traits related to obstructive sleep apnea (OSA). Methods ·The NOS1 gene expression pattern at the whole-brain level using the Allen Human Brain Atlas dataset was analyzed. Subsequently, we performed expression quantitative trait locus (eQTL) analysis to investigate the impact of rs7305526 and rs11615756 on NOS1 gene expression. Regression analysis was conducted to assess the associations between rs7305526 and rs11615756 with insomnia and sleep duration based on the United Kingdom Biobank (UKB) Genome-Wide Association Study (GWAS) dataset. Furthermore, we explored the relationships between rs7305526 and rs11615756 with clinical quantitative traits of OSA, such as respiratory events, oxygen levels, and sleep traits, using clinical monitoring data from the Shanghai Sleep Health Study Cohort (SSHS) based on standard polysomnography (PSG). Results ·The NOS1 genedemonstrated elevated levels of expression in various brain regions crucial for regulating sleep, namely the amygdala, basal forebrain, striatum, and thalamus, as well as in the respiratory center, including the mesencephalon and pontine tegmentum. In contrast, the expression level of NOS1 gene was significantly reduced or absent in areas such as the cerebral cortex and cerebellum. Variants rs7305526 and rs11615756 were significantly negatively correlated with the expression levels of NOS1 in the cerebral cortex. Additionally, rs11615756 was also significantly negatively correlated with the expression level of NOS1 in the amygdala. Analysis of the UKB GWAS data revealed that the variant rs7305526 was not significantly associated with either insomnia or sleep duration, while rs11615756 demonstrated a noteworthy negative correlation specifically with sleep duration. Data obtained from the SSHS indicated a significant association between rs7305526 and alterations in clinical quantitative traits of OSA, including lowest pulse blood oxygen saturation (LSpO2), apnea-hypopnea index (AHI), and the ratio of non-rapid eye movement (NREM) stage 2 sleep duration. Although rs11615756 showed a notable negative correlation solely with the quantity of NREM stages 2 and 3, both rs11615756 and rs7305526 showed significant correlations with some respiratory events and oxygen traits after stratification according to the severity of OSA. Conclusion ·Genetic variants of NOS1 gene are respectively associated with human sleep duration traits and OSA-related variables, suggesting that NOS1 gene plays a crucial regulatory role in human sleep and clinical quantitative traits of OSA. The regulation of sleep traits by rs7305526 (C>A) is independent of its regulation of respiratory events and oxygen traits.

Keywords： obstructive sleep apnea (OSA) ; nitric oxide synthase 1 (NOS1)gene ; human sleep ; clinical quantitative trait ; genetic variation

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袁灏琳, 李念念, 胡珺晖, 沈锦虹, 高振飞, 关建, 刘峰, 殷善开. NOS1基因变异与失眠、睡眠时长以及阻塞性睡眠呼吸暂停临床数量性状的相关性. 上海交通大学学报(医学版)[J], 2024, 44(12): 1490-1503 doi:10.3969/j.issn.1674-8115.2024.12.002

YUAN Haolin, LI Niannian, HU Junhui, SHEN Jinhong, GAO Zhenfei, GUAN Jian, LIU Feng, YIN Shankai. Association of genetic variations in the NOS1 gene with insomnia, sleep duration and obstructive sleep apnea-related clinical quantitative traits. Journal of Shanghai Jiao Tong University (Medical Science)[J], 2024, 44(12): 1490-1503 doi:10.3969/j.issn.1674-8115.2024.12.002

阻塞性睡眠呼吸暂停（obstructive sleep apnea，OSA）是最常见的睡眠障碍之一，其特点是睡眠中上气道发生反复塌陷阻塞，从而导致呼吸暂停、通气不足、血氧下降及睡眠结构紊乱。OSA同时影响睡眠和呼吸两大生理功能，常常继发循环、中枢神经、代谢等多系统损害，增加全因死亡率，是全球范围急需解决的公共卫生问题［1-2］。

一氧化氮（nitric oxide，NO）是一种气体信号分子，在生物体中扮演多种重要的生理和病理角色。由于其独特的化学特性和广泛的生物学功能，NO在心血管、神经、免疫等多个系统中都起到重要的调节作用［3-4］。在神经系统中，NO可作为神经递质或调节剂，参与神经信号的传递和神经系统的各种功能，包括记忆形成和疼痛感知；此外，NO可作为一种促眠递质参与睡眠周期调节。然而其在睡眠中的广泛作用及在OSA发生发展中的作用仍然未知［5-6］。

NO是由不同的一氧化氮合酶（nitric oxide synthase，NOS）亚型产生的，这些亚型包括神经型NOS（NOS1）、诱导型NOS（NOS2）和内皮型NOS（NOS3）。每种亚型的表达具有独特的组织特异性，从而在不同的生理和病理条件下介导特定的NO的生物学功能。其中NOS1是神经系统中主要的NOS亚型，介导NO在神经系统中的生理效应［7-8］。既往研究［9-10］表明，NOS1的单核苷酸多态性（single nucleotide polymorphism，SNP）rs11615756增加人类日间小睡行为的发生概率（OR=1.018，95%CI 1.016~1.021，P=1.40×10-49），而其另一SNP rs7305526则与OSA患者最低血氧饱和度（lowest pulse blood oxygen saturation，LSpO2）呈显著正相关（OR=1.110，95%CI 1.071~1.149，P=1.41×10-8），提示NOS1基因可能在人类睡眠以及OSA临床数量性状调节中发挥关键作用。本研究拟利用英国生物银行（United Kingdom Biobank，UKB；https：//www.ukbiobank.ac.uk/）全基因组关联研究（Genome Wide Association Study，GWAS）的数据集和上海睡眠健康队列研究（Shanghai Sleep Health Study Cohort，SSHS）的临床监测数据，进一步探索rs7305526和rs11615756遗传变异与人类失眠、睡眠时长性状和OSA临床数量性状的相关性，旨在揭示NOS1及NO信号对人类睡眠和OSA临床数量性状的潜在调节作用。

1 材料与方法

1.1 纳入数据

1.1.1 UKB GWAS数据集

本研究基于UKB不同时期GWAS数据集。通过检索欧洲生物信息学研究所（European Bioinformatics Institute，EMBL-EBI）GWAS数据库（https：//www.ebi.ac.uk/gwas/）和荷兰神经基因组学与认知研究中心（Center for Neurogenomics and Cognitive Research，CNCR；https：//cncr.nl/），获取概括统计（Summary_statistics）数据文件，其中失眠相关数据集包括HAMMERSCHLAG等［11］2017年发表的数据集GCST004695和SCHOELER等［12］2023年发表的数据集GCST90267286；睡眠时长数据集包括JONES等［13］2016年发表的数据集GCST003839、DOHERTY等［14］2018年发表的数据集GCST006914和DASHTI等［15］2019年发表的数据集GCST007561。

1.1.2 SSHS队列临床监测数据

SSHS队列的参与者来自2007年起就诊于上海交通大学医学院附属第六人民医院耳鼻咽喉头颈外科的OSA疑似患者。所有参与者都填写了全面的问卷调查，涉及主观睡眠质量、人口学信息、健康状况、家族病史、医疗史和生活方式因素（吸烟和饮酒）。主观睡眠质量由Epworth嗜睡量表（Epworth Sleepiness Scale，ESS）［16］和鼾声评分（Snoring Scale Score，SSS）［17］测评得出。睡眠数据采集自标准的多导睡眠监测（polysomnography，PSG）。PSG采集数据包括呼吸暂停低通气指数（apnea hypopnea index，AHI）、LSpO2、氧减指数（oxygen desaturation index，ODI）、血氧饱和度低于90%/80%/70%总时长（length of time with SpO2<90%/80%/70%，LT90/80/70）、总在床时间（time in bed，TIB）、睡眠周期时间（sleep period time，SPT）、总睡眠时间（total sleep time，TST）、非快速眼动期（non-rapid eye movement，NREM）、NREM 1/2/3期（N1/2/3）、快速眼动期（rapid eye movement，REM）、觉醒期（wakefulness，WK）。后续进一步计算TIB、SPT或TST期间REM、WK，以及N1、N2和N2期持续时间占比。所有参与者全基因组水平的SNP基因分型通过Affymetrix Genome-Wide Human SNP Array 6.0和Affymetrix AxiomTM Genome-Wide CHB Array芯片确定。

1.2 数据分析方法

1.2.1 UKB GWAS数据集分析

将获取的数据集标准化和统一化后进行后续分析。利用R软件（4.0.5版）及forestplot包根据计算出的OR、95%CI和P值绘制森林图，比较特定位点与性状的相关性。

1.2.2 SSHS临床监测数据分析

本研究共纳入SSHS中5 255例病例和764例对照的标准PSG数据。利用PLINK（1.90版）软件对基因型数据质量控制后，进行基因型与各OSA临床数量性状之间的线性回归分析，计算OR及其95%CI和P值。分析的OSA临床数量性状包括呼吸、血氧以及睡眠结构性状。利用LocusZoom（https：//locuszoom.org）绘制区域图，分析指定区域的SNP标记位点与性状的关联强度，左侧纵轴为-log10（P value），右侧纵轴为重组率（recombination rate，RR），横轴表示染色体位置。

1.3 人脑基因表达和表达数量性状位点分析

利用Allen Human Brain Atlas（AHBA）人脑基因表达数据库（https：//portal.brain-map.org/），获取基于NOS1的2个探针在6位捐赠者169个脑区核团的表达数据阵列，利用Graphpad Prism 10.1.2软件绘制NOS1基因表达热图。通过Ensembl数据库（https：//asia.ensembl.org/index.html）获取rs7305526和rs11615756的表达数量性状位点（expression quantitative trait locus，eQTL）数据，分析不同组织中2个位点与NOS1表达情况的相关性。P<0.05表示显著相关。

2 结果

2.1 NOS1 在人类全脑169个解剖结构的表达模式

为明确NOS1在人类全脑中的表达模式，利用AHBA数据库分析6位捐赠者169个脑区及核团的基因表达情况，发现NOS1在睡眠调节脑区（杏仁核、基底前脑、纹状体和丘脑）和呼吸中枢（间脑和脑桥被盖部）的部分核团均呈较高表达水平（图1A）；在大脑和小脑皮层、背侧丘脑、脑桥、胼胝体及扣带回中，NOS1呈低水平表达或不表达（图1B）。

图1

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图1   AHBA数据库6位捐赠者169个核团中 NOS1 基因表达情况热图

Note： A. Brain regions with relatively high levels of NOS1 gene expression. B. Brain regions with relatively low or no NOS1 expression.

Fig 1   Heatmap of the expression levels of the NOS1 gene in 169 nuclei from 6 donors in the AHBA database


2.2 基因功能注释

日间小睡行为显著相关位点rs11615756位于NOS1基因上游142 kb处，而LSpO2显著相关位点rs7305526位于NOS1基因第13个内含子中。为深入分析位于非编码区SNP在不同组织中对邻近基因表达的影响，eQTL分析发现，rs7305526在大脑皮层和肺组织中与NOS1基因的表达呈显著负相关，而与胫神经中NOS1的表达呈显著正相关。rs11615756同样与大脑皮层中NOS1的表达显著负相关，此外该SNP还与启动REM睡眠的杏仁核［18］中NOS1的表达呈显著负相关（表1）。

表1   rs7305526和rs11615756的eQTL分析

Tab 1  eQTL analysis of rs7305526 and rs11615756

SNPTissueEffect sizeP value
rs7305526Brain cortex-0.1270.044
Lung-0.136<0.001
Tibial nerve0.178<0.001
Colon sigmoid-0.0610.039
Heart atrial appendage-0.1550.014
Minor salivary gland0.1410.033
Thyroid0.0990.049
rs11615756Brain amygdala-0.2240.008
Brain cortex-0.1510.028
Esophagus mucosa-0.0840.031
Thyroid0.1040.049
Skeletal muscle0.0610.017
Pancreas0.1140.020

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2.3 rs7305526和rs11615756与人类睡眠性状的关联

为了解NOS1遗传变异与人类睡眠性状的相关性，本研究获取了不同时间段的基于UKB队列的失眠和睡眠时长的GWAS数据集，分析rs7305526和rs11615756分别与2个睡眠性状的相关性。结果表明，rs7305526（C>A）与失眠和睡眠时长无显著相关；rs11615756（T>C）与失眠无显著相关，但与睡眠时长呈显著负相关，并且该相关性随着样本量的增加更加明显及稳定（表2、图2）。

表2   本研究使用的UKB睡眠性状相关GWAS数据集

Tab 2  GWAS datasets of sleep traits from UKB used in data mining

Sleep traitGWAS catalog accessionPMIDAuthor and yearPopulationNumber
InsomniaGCST00469528604731HAMMERSCHLAG 2017European (UK)113 006
GCST9026728637106081SCHOELER 2023European (UK)283 595
Sleep durationGCST00383927494321JONES 2016European (UK)127 573
GCST00691430531941DOHERTY 2018European (UK)91 105
GCST00756130846698DASHTI 2019European (UK)446 118

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图2

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图2   rs7305526和rs11615756与失眠和睡眠时长的相关性

Fig 2   Associations of genetic variants rs7305526 and rs11615756 with insomnia and sleep duration


2.4 rs7305526和rs11615756与OSA临床数量性状的关联

为深入探究NOS1与OSA发生发展和临床数量性状的关联，利用SSHS临床监测数据进行相关性研究。

2.4.1 OSA发病风险关联分析

首先，NOS1基因区域内SNP与OSA发病风险的相关性均未达到全基因组显著性临界值（P<5×10-8）和建议显著性临界值（P<5×10-5）（图3）。尽管rs7305526和rs11615756的各个基因型在总体人群中的分布有差异，但非OSA人群和OSA人群中的各个基因型比率无明显差异（表3~5）。因此NOS1的SNP与OSA发病风险无显著相关性。

图3

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图3   LocusZoom分析 NOS1 SNP与OSA发病风险的关联

Note： TESC—tescalcin; FBXO21—F-box protein 21; KSR2—kinase suppressor of RAS 2; Mb—megabase; cM—centimorgan.

Fig 3   LocusZoom analysis of the associations between NOS1 SNPs with OSA occurrence


表3   rs7305526各基因型特征

Tab 3  Characteristics of each genotype of rs7305526

VariableOverall (n=6 026)AA (n=1 941)CA (n=2 867)CC (n=1 077)P value
Genotype/n(%)<0.001
AA1 941 (32.2)1 941 (100.0)0 (0)0 (0)
CA2 867 (47.6)0 (0)2 867 (100.0)0 (0)
CC1 077 (17.9)0 (0)0 (0)1 077 (100.0)
NA141 (2.3)0 (0)0 (0)0 (0)
Gender/n(%)0.175
Male5 325 (88.4)1 699 (87.5)2 538 (88.5)967 (89.8)
Female701 (11.6)242 (12.5)329 (11.5)110 (10.2)
Smoking status/n(%)0.924
Never3 926 (65.2)1 262 (65.0)1 872 (65.3)708 (65.7)
Ever2 100 (34.8)679 (35.0)995 (34.7)369 (34.3)
Drinking status/n(%)0.862
Never3 637 (60.4)1 181 (60.8)1 722 (60.1)650 (60.4)
Ever2 389 (39.6)760 (39.2)1 145 (39.9)427 (39.6)
AHI (events/h)①/n(%)<0.001
AHI< 5 (Non-OSA)764 (12.7)212 (10.9)360 (12.6)173 (16.1)
5 ≤AHI < 15 (Mild-OSA)318 (5.3)90 (4.6)161 (5.6)63 (5.9)
15 ≤AHI < 30 (Moderate-OSA)1 245 (20.7)407 (21.0)577 (20.1)235 (21.9)
≥30 (Severe-OSA)3 692 (61.3)1 231 (63.4)1 766 (61.6)603 (56.1)
T2DM status/n(%)0.787
No5 580 (92.6)1 790 (92.2)2 658 (92.7)999 (92.8)
Yes446 (7.4)151 (7.8)209 (7.3)78 (7.2)
Hypertension status/n(%)0.761
No5 141 (85.3)1 649 (85.0)2 452 (85.5)925 (85.9)
Yes885 (14.7)292 (15.0)415 (14.5)152 (14.1)

Note: T2DM—diabetes mellitus type 2; ①The AHI data of seven people were not available.

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表4   rs11615756各基因型特征

Tab 4  Characteristics of each genotype of rs11615756

VariableOverall (n=6 026)CC (n=3 352)TC (n=2 236)TT (n=385)P value
Genotype/n(%)<0.001
CC3 352 (55.6)3 352 (100.0)0 (0)0 (0)
TC2 236 (37.1)0 (0)2 236 (100.0)0 (0)
TT3 85 (6.4)0 (0)0 (0)385 (100.0)
NA53 (0.9)0 (0)0 (0)0 (0)
Gender/n(%)0.027
Male5 325 (88.4)2 965 (88.5)1 956 (87.5)355 (92.2)
Female701 (11.6)387 (11.5)280 (12.5)30 (7.8)
Smoking status/n(%)0.599
Never3 926 (65.2)2 183 (65.1)1 465 (65.5)242 (62.9)
Ever2 100 (34.8)1 169 (34.9)771 (34.5)143 (37.1)
Drinking status/n(%)0.513
Never3 637 (60.4)2 004 (59.8)1 371 (61.3)231 (60.0)
Ever2 389 (39.6)1 348 (40.2)865 (38.7)154 (40.0)
AHI (events/h)①/n(%)0.685
AHI< 5 (Non-OSA)764 (12.7)421 (12.6)293 (13.1)44 (11.4)
5 ≤AHI < 15 (Mild-OSA)318 (5.3)184 (5.5)109 (4.9)22 (5.7)
15 ≤AHI < 30 (Moderate-OSA)1 245 (20.7)700 (20.9)459 (20.6)70 (18.2)
≥30 (Severe-OSA)3 692 (61.3)2 044 (61.0)1 371 (61.4)249 (64.7)
T2DM status/n(%)0.989
No5 580 (92.6)3 104 (92.6)2 072 (92.7)356 (92.5)
Yes446 (7.4)248 (7.4)164 (7.3)29 (7.5)
Hypertension status/n(%)0.568
No5 141 (85.3)2 863 (85.4)1 896 (84.8)334 (86.8)
Yes885 (14.7)489 (14.6)340 (15.2)51 (13.2)

Note:①The AHI data of seven people were not available.

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表5   rs7305526和rs11615756在非OSA和OSA人群中的各基因型比率

Tab 5  Individual genotype ratios of rs7305526 and rs11615756 in non-OSA and OSA populations

SNPGenotypeRatio of individuals of each genotype/%
Non-OSAOSA
rs7305526AA28.4633.66
CA48.3248.78
CC23.2217.55
rs11615756CC55.5456.22
TC38.6537.23
TT5.806.55

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2.4.2 OSA呼吸、血氧和睡眠结构性状关联分析

进一步使用加性模型分析rs7305526和rs11615756与OSA相关呼吸、血氧和睡眠结构性状的关系。结果：rs7305526（C>A）与部分OSA呼吸、血氧和睡眠性状显著相关。具体而言，在呼吸性状方面，rs7305526与总AHI（OR=0.126，95%CI 0.049~0.326，P<0.001）、AHIREM（OR=0.189，95%CI 0.071~0.502，P<0.001）、AHINREM（OR=0.153，95%CI 0.057~0.415，P<0.001）和SSS（OR=0.866，95%CI 0.787~0.954，P=0.004）呈显著负相关；在血氧性状方面，rs7305526同ODI（OR=0.111，95%CI 0.040~0.313，P<0.001）、LT90（OR=0.008，95%CI 0.001~0.188，P=0.003）、LT80（OR=0.160，95%CI 0.027~0.964，P=0.046）和LT70（OR=0.436，95%CI 0.192~0.990，P=0.047）呈显著负相关，与LSpO2（OR=4.455，95%CI 2.548~7.784，P<0.001）呈显著正相关；在睡眠结构性状方面，rs7305526与TIB中WK次数（OR=1.239，95%CI 1.011~1.517，P=0.039）、持续时长（OR=34.022，95%CI 3.300~350.724，P=0.003）和占比（OR=1.848，95%CI 1.136~3.007，P=0.013）呈显著正相关，而与N2占TIB的比例［（N2/TIB）%］（OR=0.482，95%CI 0.258~0.901，P=0.022）呈显著负相关（图4A、C和表6）。而rs11615756（T>C）仅与睡眠结构性状中N2（OR=0.364，95%CI 0.184~0.720，P=0.004）和N3（OR=0.440，95%CI 0.240~0.807，P=0.008）次数呈显著负相关（图4B、C和表6），与其他呼吸、血氧及睡眠结构性状均无相关性。

图4

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图4   rs7305526和rs11615756与OSA临床数量性状的关联

Note： A. Forest plot illustrating the association of genetic variant rs7305526 with clinical quantitative traits of OSA. B. Forest plot illustrating the associations of genetic variant rs11615756 with clinical quantitative traits of OSA. C. Forest plot illustrating the associations of rs7305526 and rs11615756 with additional clinical quantitative traits of OSA.

Fig 4   Associations of genetic variants rs7305526 and rs11615756 with clinical quantitative traits of OSA


表6   rs7305526和rs11615756与OSA临床数量性状的关联

Tab 6  Associations of genetic variants rs7305526 and rs11615756 with clinical quantitative traits of OSA

SNPAlleleTraitOR95%CIP value
LowerUpper
rs7305526C>AAHItotal0.1260.0490.326<0.001
AHIREM0.1890.0710.502<0.001
AHINREM0.1530.0570.415<0.001
ODI0.1110.0400.313<0.001
LT900.0080.0010.1880.003
LT800.1600.0270.9640.046
LT700.4360.1920.9900.047
LSpO24.4552.5487.784<0.001
SSS0.8660.7870.9540.004
WK(TIB) frequency1.2391.0111.5170.039
WK(TIB) duration34.0223.300350.7240.003
WK/TIB1.8481.1363.0070.013
N2 frequency0.8180.4461.5000.515
N2/TIB0.4820.2580.9010.022
N3 frequency0.8610.5011.4770.586
rs11615756T>CAHItotal1.3520.4643.9390.581
AHIREM1.5780.5214.7830.420
AHINREM0.8620.2802.6580.796
ODI1.2250.3813.9390.733
LT900.0320.0011.1650.061
LT800.5120.0673.9080.519
LT700.9110.3612.3010.844
LSpO20.9460.5011.7850.864
SSS1.0250.9191.1430.661
WK(TIB) frequency0.8800.7001.1070.275
WK(TIB) duration1.6900.12023.7120.697
WK/TIB1.2040.6932.0910.510
N2 frequency0.3640.1840.7200.004
N2/TIB0.7370.3551.5330.414
N3 frequency0.4400.2400.8070.008

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2.4.3 OSA临床数量性状关联亚组分析

在女性中，NOS1 rs7305526和rs11615756与OSA临床数量性状均无明显相关性；而男性中，rs7305526与部分呼吸、血氧和睡眠结构性状的相关性同整体人群的趋势一致（表7）。根据AHI将入组患者划分为非OSA组，以及轻度、中度和重度OSA组。rs7305526与非OSA组（OR=2.039，95%CI 1.124~3.699，P=0.019）、中度OSA组（OR=2.280，95%CI 1.129~4.605，P=0.022）和重度OSA组（OR=2.217，95%CI 1.226~4.011，P=0.008）的LSpO2均呈显著正相关；然而在轻度OSA组，该位点仅与LT90（OR=23.117，95%CI 1.232~433.679，P=0.036）显著正相关。rs11615756与总人群的呼吸、血氧性状均无显著关联，而与非OSA组REM的AHI（OR=0.251，95%CI 0.074~0.856，P=0.027）和NREM的AHI（OR=0.270，95%CI 0.083~0.875，P=0.029）、轻度OSA组SSS（OR=0.626，95%CI 0.423~0.926，P=0.019）显著负相关，与中度OSA组LT70（OR=1.166，95%CI 1.011~1.344，P=0.035）显著正相关，与重度OSA组LT90（OR=0.002，95%CI 0.001~0.148，P=0.006）显著负相关（表8）。

表7   男性与女性rs7305526和rs11615756与OSA临床数量性状的关联

Tab 7  Associations of genetic variants rs7305526 and rs11615756 with clinical quantitative traits of OSA in male and female groups

GenderSNPAlleleTraitOR95%CIP value
LowerUpper
Malers7305526C>AAHItotal0.1030.0370.285<0.001
AHIREM0.1890.0660.5350.002
AHINREM0.1110.0380.322<0.001
ODI0.0930.0310.281<0.001
LT900.0040.0010.1160.001
LT800.1770.0291.0870.061
LT700.5400.2521.1540.112
LSpO24.7822.8218.108<0.001
SSS0.8650.7810.9580.006
WK (TIB) frequency1.2150.9791.5080.078
WK (TIB) duration36.6963.154426.9910.004
WK/TIB2.0021.2083.3180.007
N2 frequency0.7810.4121.4820.450
N2/TIB0.1290.0062.8670.195
N3 frequency2.8810.26830.9580.382
rs11615756T>CAHItotal1.2510.3983.929<0.001
AHIREM1.2460.3854.0390.002
AHINREM0.8900.2682.962<0.001
ODI1.0490.3033.638<0.001
LT900.0490.0012.0510.001
LT800.3690.0472.8680.061
LT700.7480.3171.7640.112
LSpO21.1590.6382.105<0.001
SSS1.0200.9091.1440.006
WK (TIB) frequency0.8620.6761.1000.078
WK (TIB) duration4.2120.26467.1220.004
WK/TIB1.4300.8082.5310.007
N2 frequency0.3560.1740.7260.450
N2/TIB0.8230.02527.4240.195
N3 frequency0.1970.0142.8760.382
Femalers7305526C>AAHItotal0.7260.0608.7860.801
AHIREM0.3710.0255.4990.471
AHINREM1.5860.10623.7350.738
ODI0.7610.04512.9260.850
LT900.2380.0025 373.9210.779
LT800.1280.001195.2860.581
LT700.1630.0064.5160.283
LSpO21.1370.2664.8540.862
SSS0.8460.6351.1270.253
WK (TIB) frequency1.3520.7662.3840.298
WK (TIB) duration26.2860.01741 622.2300.384
WK/TIB1.8570.3948.7600.434
N2 frequency1.0200.1526.8410.984
N2/TIB0.0020.0012.3740.078
N3 frequency11.9130.0178 142.9300.456
rs11615756T>CAHItotal3.2740.17461.7380.428
AHIREM19.3440.799468.3190.068
AHINREM0.8080.03419.3830.895
ODI5.7000.202161.0960.307
LT900.0040.001716.6650.375
LT8014.0170.00365 377.3710.539
LT7020.6630.444960.8110.122
LSpO20.3970.0732.1620.285
SSS1.0920.7811.5290.606
WK (TIB) frequency1.0470.5372.0400.893
WK (TIB) duration0.0020.0013.1070.088
WK/TIB0.3030.0491.8710.198
N2 frequency0.5460.0575.2290.599
N2/TIB0.0930.0012 235.4850.644
N3 frequency0.0960.001201.3060.547

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表8   非OSA组，以及轻度、中度和重度OSA组rs7305526和rs11615756与OSA临床数量性状的关联

Tab 8  Associations between genetic variants rs7305526 and rs11615756 with clinical quantitative traits of OSA in non-OSA， mild-OSA， moderate-OSA and severe-OSA groups

SNPTraitNon-OSA (n=764)Mild-OSA (n=318)Moderate-OSA (n=1 245)Severe-OSA (n=3 692)

P

value

OR

(95%CI)

P

value

OR

(95%CI)

P

value

OR

(95%CI)

P value

OR

(95%CI)

rs7305526AHItotal0.530

0.955

(0.826‒1.104)

0.290

0.818

(0.563‒1.188)

0.619

1.090

(0.777‒1.528)

0.206

0.593

(0.264‒1.334)

AHIREM0.763

1.176

(0.410‒3.380)

0.965

1.070

(0.054‒21.232)

0.890

0.894

(0.182‒4.383)

0.577

0.749

(0.271‒2.070)

AHINREM0.464

1.461

(0.529‒4.0332)

0.143

4.276

(0.611‒29.910)

0.846

1.076

(0.517‒2.239)

0.206

0.538

(0.205‒1.408)

ODI0.821

0.847

(0.199‒3.602)

0.562

1.330

(0.505‒3.501)

0.124

0.516

(0.222‒1.200)

0.464

0.684

(0.247‒1.892)

LT900.633

0.536

(0.041‒6.958)

0.036

23.117

(1.232‒433.679)

0.632

1.864

(0.146‒23.881)

0.122

0.032

(0.001‒2.517)

LT800.982

0.999

(0.907‒1.100)

0.097

1.397

(0.941‒2.074)

0.678

0.878

(0.475‒1.624)

0.392

0.290

(0.017‒4.948)

LT700.278

0.998

(0.993‒1.002)

0.613

0.990

(0.951‒1.030)

0.331

1.064

(0.939‒1.205)

0.179

0.424

(0.121‒1.481)

LSpO20.019

2.039

(1.124‒3.699)

0.958

1.028

(0.372‒2.841)

0.022

2.280

(1.129‒4.605)

0.008

2.217

(1.226‒4.011)

SSS0.421

0.888

(0.666‒1.186)

0.333

1.193

(0.834‒1.706)

0.995

1.001

(0.817‒1.226)

0.153

0.923

(0.826‒1.030)

WK (TIB) frequency0.215

1.498

(0.791‒2.838)

0.016

0.369

(0.163‒0.832)

0.130

1.432

(0.900‒2.281)

0.244

1.158

(0.905‒1.4811)

WK (TIB) duration0.119

406.376

(0.213‒775 370.391)

0.606

0.060

(0.001‒2674.448)

0.046

269.283

(1.095‒66 255.145)

0.230

5.352

(0.345‒83.043)

WK/TIB0.097

3.846

(0.784‒18.871)

0.714

0.661

(0.072‒6.081)

0.142

2.251

(0.763‒6.641)

0.240

1.411

(0.794‒2.508)

N2 frequency0.788

0.857

(0.277‒2.650 6)

0.432

0.454

(0.063‒3.278)

0.921

0.942

(0.289‒3.071)

0.892

0.942

(0.398‒2.230)

N2/TIB0.214

0.009

(0.001‒15.720)

0.035

0.002

(0.001‒0.405)

0.678

0.272

(0.001‒128.335)

0.695

0.462

(0.010‒21.825)

N3 frequency0.295

0.045

(0.001‒14.873)

0.021

10 267.538

(3.963‒26 600 123.192)

0.528

4.297

(0.046‒400.757)

0.648

2.015

(0.099‒40.930)

rs11615756AHItotal0.777

0.976

(0.823‒1.157)

0.875

0.968

(0.639‒1.464)

0.626

1.104

(0.742‒1.642)

0.626

0.801

(0.328‒1.957)

AHIREM0.027

0.251

(0.074‒0.856)

0.791

1.551

(0.060‒40.217)

0.085

5.121

(0.800‒32.802)

0.750

1.202

(0.389‒3.711)

AHINREM0.029

0.270

(0.083‒0.875)

0.504

0.500

(0.065‒3.838)

0.392

1.457

(0.616‒3.448)

0.377

0.620

(0.214‒1.793)

ODI0.123

0.266

(0.050‒1.429)

0.846

1.112

(0.381‒3.249)

0.840

1.108

(0.411‒2.98)

0.994

1.004

(0.327‒3.080)

LT900.511

2.705

(0.139‒52.722)

0.076

0.053

(0.002‒1.364)

0.324

4.571

(0.222‒93.989)

0.006

0.002

(0.001‒0.148)

LT800.407

0.952

(0.848‒1.069)

0.757

1.074

(0.682‒1.690)

0.077

1.885

(0.933‒3.807)

0.290

0.184

(0.008‒4.251)

LT700.314

0.997

(0.992‒1.003)

0.565

0.987

(0.943‒1.033)

0.035

1.166

(1.011‒1.344)

0.857

0.881

(0.221‒3.504)

LSpO20.951

1.022

(0.511‒2.046)

0.314

1.780

(0.577‒5.489)

0.480

0.743

(0.326‒1.696)

0.357

1.362

(0.706‒2.627)

SSS0.697

1.070

(0.762‒1.501)

0.019

0.626

(0.423‒0.926)

0.949

0.992

(0.782‒1.259)

0.435

1.050

(0.929‒1.186)

WK (TIB) frequency0.238

0.636

(0.300‒1.349)

0.835

1.101

(0.444‒2.731)

0.917

0.971

(0.561‒1.682)

0.470

0.905

(0.689‒1.188)

WK (TIB) duration0.397

46.583

(0.006‒338 729.248)

0.531

43.721

(0.001‒6 081 353.502)

0.772

0.381

(0.001‒256.719)

0.879

1.266

(0.061‒26.225)

WK/TIB0.456

2.041

(0.313‒13.327)

0.661

1.729

(0.148‒20.150)

0.845

0.881

(0.245‒3.167)

0.517

1.234

(0.653‒2.331)

N2 frequency0.135

0.366

(0.098‒1.370)

0.934

0.911

(0.099‒8.373)

0.918

0.930

(0.234‒3.703)

0.005

0.261

(0.101‒0.671)

N2/TIB0.091

2 007.782

(0.296‒13 627 828.272)

0.832

3.998

(0.001‒1 516 906.783)

0.407

0.046

(0.001‒66.068)

0.498

0.228

(0.003‒16.421)

N3 frequency0.979

1.094

(0.001‒976.421)

0.665

0.145

(0.001‒929.697)

0.778

0.464

(0.002‒96.210)

0.173

0.098

(0.004‒2.775)

Note: AHI< 5 (Non-OSA); 5 ≤AHI < 15 (Mild-OSA); 15 ≤AHI < 30 (Moderate-OSA); AHI≥30 (Severe-OSA).

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3 讨 论

NO已被证实是一种促眠的气体信号分子。NOS1的表达水平和活性会直接影响中枢神经系统NO介导的生理功能，然而NOS1在人类睡眠调节以及OSA临床数量性状中的作用尚不清楚。我们研究发现，NOS1基因在人类睡眠和呼吸调控相关脑区、核团中高度表达，其遗传变异rs11615756与人类睡眠时长性状显著相关，另一个遗传变异rs7305526与OSA呼吸、血氧和睡眠结构等性状均呈显著相关，提示NOS1在人类睡眠时长以及OSA临床数量性状中起到重要的调节作用。

保护性位点NOS1 rs7305526（C>A）对AHI和SpO2产生了协同影响，这表明rs7305526遗传变异对血氧的调控并非独立存在，而是基于其引起的AHI的变化。尽管rs7305526（C>A）有助于改善OSA的呼吸和血氧性状，但会增加觉醒并缩短N2。这与我们所知道的OSA患者在通气改善后觉醒次数减少、NREM延长的情况相反。因此，rs7305526（C>A）对睡眠性状的调节独立于对呼吸性状的调节。

NOS1 rs7305526和rs11615756与OSA临床数量性状的相关性存在性别差异。女性组中无显著相关性，而男性组与整体人群的趋势一致，这可能与女性样本量较少有关。rs11615756与整体人群OSA血氧、呼吸性状无显著相关性，但在根据OSA严重程度分层后，rs11615756和rs7305526与部分呼吸、血氧性状存在显著相关性。先前的研究［19-20］已经确定NOS1在孤束核存在表达，本研究基于AHBA人脑图谱分析发现NOS1在臂旁外侧核、舌下神经核、杏仁核等控制呼吸的核团中高表达，提示NOS1可能在呼吸中枢对呼吸、血氧的调节中发挥重要作用。

既往研究［21-23］表明，NO信号与脑衰老及相关的神经退行性疾病，如阿尔茨海默病、帕金森病和亨廷顿病密切相关。然而，具体的神经生物学机制仍待阐明。我们的研究发现NOS1遗传变异与睡眠时长性状显著相关，且NOS1定位于睡眠、呼吸相关脑区和认知记忆相关脑区——海马，而在大脑、小脑皮层中NOS1表达水平相对较低甚至不表达，提示NOS1可能更广泛地参与了睡眠和呼吸调节的过程，而并不深度参与躯体感觉、运动协调以及语言等神经生物学功能。海马区域负责清醒阶段记忆编码、睡眠阶段记忆重组，然而NOS1在海马以及睡眠调控区域均有高度表达，提示它可能通过调控睡眠和认知记忆形成过程进而影响阿尔茨海默病的发生［24-25］。

此前基于UKB的队列研究［9-10］发现NOS1 rs11615756遗传变异与日间小睡密切相关，而本研究同样基于UKB队列GWAS数据，发现该遗传变异与人类睡眠时长性状显著相关，然其在SSHS中仅与部分睡眠结构性状相关（N2和N3次数）；此前基于SSHS发现的与OSA的LSpO2显著相关的位点rs7305526遗传变异与UKB队列的失眠和睡眠时长2种睡眠性状无显著相关性，但在SSHS中，该遗传变异不仅影响呼吸及血氧性状，还影响睡眠结构性状。NOS1不同遗传变异存在着不同的生物学效应，这种现象可能是由于NOS1的不同位点在调控性状时可能存在高度的人群特异性，也可能是由于不同位点受到不同信号的调节或表观遗传状态的影响。

NO供体药物已被广泛应用于不同的临床疾病治疗中。例如硝化甘油和硝普钠可通过稳定释放NO来松弛血管平滑肌，分别用于治疗高血压和心绞痛［26］。OSA患者常因慢性间歇性缺氧导致血氧分压降低，易引起全身多脏器的缺氧损伤，甚至导致高血压、冠心病等多系统的并发症。NOS1作为睡眠和呼吸中枢的双重调节因子，已被证实与OSA患者呼吸、血氧以及睡眠结构等性状存在不同程度的相关性，可作为治疗靶点，用于纠正患者睡眠中的通气不足和睡眠结构紊乱。

本研究尚存在以下不足之处。虽然本研究收集自SSHS的中国汉族人群中最大的OSA遗传样本库，然而前来就诊的多为重度OSA患者，且男性患者占比较大。同时本研究分析的NOS1的脑区表达模式源自未知种族的健康捐赠者的正常人脑，可能与中国汉族OSA患者脑中NOS1的表达情况存在差异。此外，在人类睡眠性状分析中，本研究仅选取了失眠和睡眠时长2个性状，仍需更多类别的睡眠障碍数据集以评估NOS1在特定睡眠阶段中的作用。

NOS1基因rs7305526和rs11615756遗传变异与人类睡眠性状及OSA呼吸、血氧事件和睡眠结构等临床数量性状密切相关，该基因有望成为纠正OSA患者通气障碍和睡眠结构紊乱的重要治疗靶点。

作者贡献声明

袁灏琳、李念念和刘峰参与数据分析，袁灏琳、李念念、胡珺晖、沈锦虹、高振飞、关建、刘峰和殷善开参与论文写作和修改。

AUTHOR's CONTRIBUTIONS

YUAN Haolin, LI Niannian and LIU Feng participated in data analysis. YUAN Haolin, LI Niannian, HU Junhui, SHEN Jinhong, GAO Zhenfei, GUAN Jian, LIU Feng and YIN Shankai participated in manuscript drafting and revision.

利益冲突声明

所有作者声明不存在利益冲突。

COMPETING INTERESTS

All authors disclose no relevant conflict of interests.

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