A candidate gene study of obstructive sleep apnea in European Americans and African Americans.
ABSTRACT Obstructive sleep apnea (OSA) is hypothesized to be influenced by genes within pathways involved with obesity, craniofacial development, inflammation, and ventilatory control.
We conducted the first candidate gene study of OSA using family data from European Americans and African Americans, selecting biologically plausible genes from within these pathways.
A total of 1,080 single nucleotide polymorphisms (SNPs) were genotyped in 729 African Americans and 505 SNPs were genotyped in 694 European Americans. Coding for SNPs additively, association testing on the apnea-hypopnea index (AHI) as a continuous trait, and OSA as a dichotomous trait (AHI ≥15) was conducted using methods that account for familial correlations in models adjusted for age, age-squared, and sex, with and without body mass index.
In European Americans, variants within C-reactive protein (CRP) and glial cell line-derived neurotrophic factor (GDNF) were associated with AHI (CRP: β = 4.6; SE = 1.1; P = 0.0000402) (GDNF: β = 4.3; SE = 1; P = 0.0000201) and with the dichotomous OSA trait (CRP: odds ratio = 2.4; 95% confidence interval, 1.5-3.9; P = 0.000170) (GDNF: odds ratio = 2; 95% confidence interval, 1.4-2.89; P = 0.0000433). In African Americans, rs9526240 within serotonin receptor 2a (HTR2A: odds ratio = 2.1; 95% confidence interval, 1.5-2.9; P = 0.00005233) was associated with OSA.
This candidate gene analysis identified the potential role of genes operating through intermediate disease pathways to influence sleep apnea phenotypes, providing a framework for focusing future replication studies.
- [show abstract] [hide abstract]
ABSTRACT: Patients with obstructive sleep apnoea-hypopnoea syndrome (OSAHS) have elevated circulating levels of tumour necrosis factor (TNF)-alpha. The hypothesis in this study was that OSAHS might be associated with the TNF-alpha (-308A) gene polymorphism, which results in increased TNF-alpha production. This hypothesis was examined in OSAHS patients, their siblings and population controls. A total of 206 subjects were recruited. All underwent sleep studies and clinical review, and were subsequently classified as having OSAHS or not depending on apnoea-hypopnoea frequency, sex, age and symptoms. All subjects had blood collected and genotyping was performed on DNA extracted from peripheral leukocytes. Some 192 random UK blood donors were used as population controls. The results demonstrated a significant association for TNF-alpha (-308A) allele carriage with OSAHS (OR=1.8; 95% Confidence interval: 1.18-2.75) when compared with population controls. Siblings with OSAHS were significantly more likely to carry the TNF-alpha (-308A) allele. In addition, 21 pairs of male siblings discordant for carriage of the -308A allele showed a significant level of discordance for the OSAHS phenotype. In conclusion, this study demonstrates an association of tumour necrosis factor-alpha (-308A) carriage with obstructive sleep apnoea-hypopnoea syndrome, suggesting that inflammation may be implicated in the pathogenesis of this condition.European Respiratory Journal 11/2005; 26(4):673-8. · 6.36 Impact Factor
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ABSTRACT: Obstructive sleep apnea (OSA) is a common, chronic, complex disease associated with serious cardiovascular and neuropsychological sequelae and with substantial social and economic costs. Along with male gender, obesity is the most characteristic feature of OSA in adults. To identify susceptibility loci for OSA, we undertook a 9-cM genome scan in 66 white pedigrees (n=349 subjects) ascertained on the basis of either an affected individual with laboratory-confirmed OSA or a proband who was a neighborhood control individual. Multipoint variance-component linkage analysis was performed for the OSA-associated quantitative phenotypes apnea-hypopnea index (AHI) and body mass index (BMI). Candidate regions on chromosomes 1p (LOD score 1.39), 2p (LOD score 1.64), 12p (LOD score 1.43), and 19p (LOD score 1.40) gave the most evidence for linkage to AHI. BMI was also linked to multiple regions, most significantly to markers on chromosomes 2p (LOD score 3.08), 7p (LOD score 2.53), and 12p (LOD score 3.41). Extended modeling indicated that the evidence for linkage to AHI was effectively removed after adjustment for BMI, with the exception of the candidate regions on chromosomes 2p (adjusted LOD score 1.33) and 19p (adjusted LOD score 1.45). After adjustment for AHI, the primary linkages to BMI remained suggestive but were roughly halved. Our results suggest that there are both shared and unshared genetic factors underlying susceptibility to OSA and obesity and that the interrelationship of OSA and obesity in white individuals may be partially explained by a common causal pathway involving one or more genes regulating both AHI and BMI levels.The American Journal of Human Genetics 03/2003; 72(2):340-50. · 11.20 Impact Factor
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ABSTRACT: We have previously shown that plasma levels of orexin-A, a neuropeptide with an arousal-stimulating action, were decreased in parallel with the severity of the disease in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). To clarify the effects of nasal continuous positive airway pressure (nCPAP) treatment on plasma orexin-A levels in patients with this syndrome. Sleep tests and blood sample collections were conducted at the sleep-related respiratory disorders clinic and the sleep laboratory of the Iwate Medical University Hospital. We studied 27 patients with OSAHS (apnea-hypopnea index [AHI], >/= 20 by polysomnography) who were treated with nCPAP for 3 to 6 months. These patients were divided into the following two groups according to the arousal index (AI): group A (n = 11), >/= 60; group B (n = 16), < 60. Plasma samples were obtained before and after the nCPAP treatment for 3 to 6 months. Plasma immunoreactive (IR)-orexin-A concentrations were measured by radioimmunoassay after the extraction using cartridges. Plasma IR-orexin-A concentrations were inversely correlated with the AI (r = -0.807; p < 0.0001) and AHI (r = -0.661; p < 0.0001) in 27 patients before the nCPAP treatment. Mean (+/- SEM) plasma IR-orexin-A concentrations were significantly lower in group A (1.0 +/- 0.3 pmol/L) than in group B (4.6 +/- 0.4 pmol/L). Mean plasma IR-orexin-A concentrations were significantly increased after the nCPAP treatment in group A (to 3.4 +/- 1.2 pmol/L; p = 0.0069), whereas they were not significantly changed in group B. The increases in plasma IR-orexin-A concentrations after the nCPAP treatment were in parallel with the improvements in AI and Epworth sleepiness scale (a marker of severity of daytime excessive sleepiness) score in group A. The low plasma orexin-A levels were increased by the nCPAP treatment in patients with severe OSAHS, suggesting that orexin-A is a plasma marker that reflects the severity of OSAHS and the response to treatment.Chest 03/2005; 127(3):731-7. · 5.85 Impact Factor
A Candidate Gene Study of Obstructive Sleep Apnea
in European Americans and African Americans
Emma K. Larkin1,2,3*, Sanjay R. Patel2,4*, Robert J. Goodloe2, Yali Li3, Xiaofeng Zhu3, Courtney Gray-McGuire3,5,
Mark D. Adams6, and Susan Redline2,4
1Division of Allergy, Pulmonary and Critical Care, Vanderbilt University Medical Center, Nashville, Tennessee;2Center for Clinical Investigation,
4Division of Pulmonary, Critical Care and Sleep Medicine, and6Department of Genetics, Case Western Reserve University, Cleveland;3Department
of Epidemiology and Biostatistics, Case Western Reserve University School of Medicine, Cleveland, Ohio;5Arthritis and Immunology Program,
Oklahoma City Medical Research Foundation, Oklahoma City, Oklahoma
Rationale: Obstructive sleep apnea (OSA) is hypothesized to be
influenced by genes within pathways involved with obesity, cranio-
facial development, inflammation, and ventilatory control.
family data from European Americans and African Americans,
selecting biologically plausible genes from within these pathways.
Methods: A total of 1,080 single nucleotide polymorphisms (SNPs)
were genotyped in 729 African Americans and 505 SNPs were
genotyped in 694 European Americans. Coding for SNPs additively,
association testing on the apnea-hypopnea index (AHI) as a contin-
using methods that account for familial correlations in models
Measurements and Main Results: In European Americans, variants
within C-reactive protein (CRP) and glial cell line–derived neuro-
trophic factor (GDNF) were associated with AHI (CRP: b 5 4.6; SE 5
the dichotomous OSA trait (CRP: odds ratio 5 2.4; 95% confidence
interval, 1.5–3.9; P 5 0.000170) (GDNF: odds ratio 5 2; 95%
confidenceinterval, 1.4–2.89; P 50.0000433). In AfricanAmericans,
rs9526240 within serotonin receptor 2a (HTR2A: odds ratio 5 2.1;
95% confidence interval, 1.5–2.9; P 5 0.00005233) was associated
Conclusions: This candidate gene analysis identified the potential
role of genes operating through intermediate disease pathways to
influence sleep apnea phenotypes, providing a framework for
focusing future replication studies.
Keywords: sleep apnea; body mass index; genetics; candidate gene
Sleep-disordered breathing (SDB) or its clinical counterpart,
obstructive sleep apnea (OSA), is a common disorder charac-
terized by intermittent collapse of the upper airway during
sleep-disrupting breathing, and associated with significant
morbidity including daytime sleepiness, hypertension, and
elevated cardiovascular risk. It has been recognized for more
than three decades that SDB aggregates within families,
suggesting the presence of a genetic basis for this disease
(1). Approximately 30–40% of the variance in the most
common disease-defining metric for SDB, the apnea-hypopnea
index (AHI), has been estimated to be explained by genetic
factors (2). Having a first-degree relative with SDB increases
one’s risk of the disorder by more than 1.5-fold (2). Several
genome-wide linkage and association studies have been con-
ducted for SDB-related traits (3–5) (summarized in Riha ).
The latter have been limited by considering only a handful of
polymorphisms in one gene or by consideration of more than
a few genes in each given population, and by limited statistical
power. To date, no genetic variants have been shown to
associate strongly with SDB.
We postulated that identifying genetic variants for SDB
would be facilitated by studying polymorphisms in genes in
pathways intermediate to SDB and previously established by
human or animal work to have a genetic basis, such as obesity,
ventilatory control, inflammation, and craniofacial morphol-
ogy (7). Using knowledge of underlying biology and prior
genetic studies, we selected a set of candidate genes and then
densely genotyped, in a systematic fashion, each candidate to
identify polymorphisms associated with SDB in a family-
based study, enriched with SDB. Some of the results of these
studies have been previously reported in the form of an
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Obstructive sleep apnea has a strong heritable component,
although its genetic basis is poorly understood. Existing
studies to date have focused on single genes, have been of
relatively small samples, or have had limited genetic
coverage (i.e., examining known functional variants).
What This Study Adds to the Field
This large-scale candidate gene study of sleep apnea
involves systematic coverage of 53 genes representing
intermediate disease pathways implicated in this disorder.
The results from this study identify significant associations
between sleep-disordered breathing and genetic variants
for C-reactive protein, glial-derived growth factor, and
serotonin 2A receptor genes. Our analyses show that the
genetic associations with sleep apnea persist after adjusting
for body mass index, suggesting that the underlying genetic
mechanism of sleep apnea is not dependent on obesity,
a common comorbidity.
(Received in original form February 8, 2010; accepted in final form June 10, 2010)
* These authors contributed equally and are listed alphabetically.
Supported by National Institutes of Health grants HL07567, HL081385,
HL046380, and RR024990. In addition, some of the results of this paper were
obtained by using the software package S.A.G.E., which is supported by a US
Public Health Service Resource Grant (RR03655) from the National Center for
Correspondence and requests for reprints should be addressed to Susan Redline,
M.D., M.P.H., Case Western Reserve University, Center for Clinical Investigation,
Iris S. & Bert L. Wolstein Building, 2103 Cornell Road, Room 6129, Cleveland, OH
44106–7291. E-mail: email@example.com
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.201002-0192OC on June 10, 2010
Internet address: www.atsjournals.org
Vol 182. pp 947–953, 2010
See the online supplement for detailed methods. The Cleveland Family
Study (CFS) has assembled a collection of 2,534 individuals (43%
African American) from 356 families, details of which have been
previouslypublished(2). Inbrief, indexcaseswith laboratory-diagnosed
sleep apnea, their family members, and neighborhood control families
have been followed longitudinally on as many as four occasions over
a period of 16 years and have had measurements made of sleep apnea,
anthropometry, and other related phenotypes. DNA samples were
available for 711 European Americans and 759 African Americans.
The main metric of analyses was the AHI, defined as the average
number of apneas and hypopneas, each associated with a 3% desatu-
ration, per hour of sleep. The AHI was derived from either portable
sleep apnea monitoring (9) (with oxygen saturation, body position,
airflow by thermistry, chest wall effort, and heart rate sensors)
(Edentrace, Eden Prairie, MN) performed in participants studied
before 2000 or by 14-channel attended overnight polysomnography
(Compumedics E series, Abottsford, AU) obtained in a clinical re-
search unit after 2000. The details about comparability of the AHI
determined from the two recording approaches and how the longitu-
dinal data were used are described in the online data supplement. Body
mass index (BMI) was defined as weight divided by the square of
height derived from the same examination as the AHI.
DNA was isolated from blood and buccal swabs using Puregene kits
(Qiagen, Valencia CA). A total of 52 candidate genes for OSA were
TABLE 1. CANDIDATE GENES
PathwaysGene DescriptionGene RefSeq Chr
Craniofacial morphology Fibroblast growth factor receptor 1
Fibroblast growth factor receptor 2
Fibroblast growth factor receptor 3
Muscle segment homeobox 1
Muscle segment homeobox 2
Paired-like homeobox 2B
Transforming growth factor-b receptor 1
Cholecystokinin A receptor
Hypocretin receptor 2
Insulin-induced gene 2
Melanocortin 3 receptor
Melanocortin 4 receptor
Plasminogen activator inhibitor 1
Tumor necrosis factor
Uncoupling protein 2
Uncoupling protein 3
Achaete-scute complex 1
Brain-derived neurotrophic factor
Endothelin-coverting enzyme 1
Endothelin receptor A
Glial cell line–derived neurotrophic factor
Hypoxia-inducible factor 1 alpha
Nitric oxide synthase 3
Rearranged during transfection protooncogene
GABA B receptor 1
Guanine nucleotide-binding protein 3
Serotonin receptor 1B
Serotonin receptor 2A
Serotonin receptor 2C
Serotonin receptor 3A
Serotonin receptor 3B
Serotonin receptor 3C
Serotonin receptor 3D
Serotonin receptor 3E
Insulin receptor substrate 1
Peripheral myelin protein 22
Ventilatory control 17
Definition of Abbreviations: Chr 5 chromosome; RefSeq 5 reference sequence accession number; SNP 5 single nucleotide polymorphism.
948 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1822010
selected based on putative roles in intermediate pathways (craniofacial
morphology, ventilatory control, obesity, and inflammation) hypothe-
sized to be relevant to sleep apnea pathogenesis (Table 1). A total of
522 tagged single nucleotide polymorphisms (SNPs) from European
Americans and 1,095 SNPs from African Americans were genotyped
using the Illumina Golden Gate Assay.
Quality control procedures included (1) elimination of individuals
with call rates less than 90%, (2) visual examination of individual
cluster plots and elimination of SNPs with poor call signals or GenTran
scores less than 0.20, (3) resolution of mendelian inconsistencies, and
(4) evaluation of SNPs for a departure from Hardy-Weinberg equilib-
rium. A total of 505 SNPs in 694 European Americans and 1,080 SNPs
in 729 African Americans from 52 genes met quality control criteria
and are included in this analysis (see online supplement).
The phenotypes modeled were AHI as a continuous variable and OSA
defined as an AHI greater than or equal to 15. Because AHI thresholds
that define abnormality differ in children, the dichotomous OSA
analysis was limited to individuals over the age of 18 years. For
continuous traits, linear mixed models were used to account for familial
correlations as implemented in the ASSOC procedure of the Statistical
Analysis of Genetic Epidemiology program (10). For dichotomous
traits, logistic regression was performed by SAS v9.1 (SAS Institute,
Research Triangle Park, NC) using generalized estimating equations
with an exchangeable correlation structure to account for family
relatedness. SNPs were coded additively based on the number of
minor alleles present. All models were adjusted for age; age-squared;
sex; and an index of racial admixture (percentage European or
percentage African ancestry, as described in the online supplement).
Secondary models were generated additionally adjusting for BMI
(which may be an intermediate trait) as a covariate. To account for
multiple comparisons and balance the type I and type II error rates,
a Benjamini-Hochberg false discovery rate (FDR) was calculated (11).
Thus, in addition to P values, we report q-values, which can be
interpreted as the lowest FDR for which a hypothesis test would be
Haplotype analysis was performed for genes with multiple signif-
icant SNPs from the association analysis (see online supplement).
Demographic characteristics of the sample are shown in Table
2. The sample included a slight predominance of women, on
average was overweight, and included a wide age range. Among
the 553 European Americans and 539 African Americans over
the age of 18, the prevalence of OSA was 26% and 35%,
Among the European Americans, three SNPs were associ-
ated with the age, age-squared, sex, and ancestry-adjusted
dichotomous OSA trait (Table 3), with the strongest association
(q 5 0.02) to rs2808630, a SNP in the 39 untranslated region of
the C-reactive peptide (CRP) gene. Each additional minor allele
at this SNP was associated with a 2.04-fold increased odds of
OSA. The other two SNPs associated with OSA were in the glial
cell–derived neurotrophic factor (GDNF) gene. As shown in
Figure 1, SNPs within GDNF were in strong linkage disequilib-
rium with each other. For the two most significant SNPs
(rs2910705 and rs2975100), the pairwise D’5 0.977 and r25 0.93.
In analyses of AHI as a continuous trait in European
Americans, two SNPs met a 5% FDR threshold and a third
met a 10% FDR (Table 4). Each additional risk allele for these
SNPs increased the mean AHI by four to five events per hour.
The CRP SNP was the same SNP observed with analysis of the
dichotomous phenotype. The other two SNPs were both in
GDNF with a pairwise D’5 1 and r25 0.46. Although these
SNPs did not overlap with the two GDNF SNPs that had a q-
value less than 10% from the OSA analysis, the pairwise D’
between SNPs ranged from 0.935 to 1.00. Furthermore, as
shown in the online supplement, the nominal P values for these
four SNPs support consistent results between these traits.
Of the five SNPs associated with either OSA or AHI in
European Americans, one (rs2808630 in CRP) was associated
with SDB in African Americans at a nominal P less than 0.05.
Each G allele at this SNP increased the risk of OSA by 1.48-fold
(95% confidence interval, 1.04–2.11; P 5 0.029) in African
Analysis in the African American cohort identified a SNP in
the serotonin 2A receptor (HTR2A) to be associated with OSA
(Table 5) at a FDR less than 10% (i.e., q 5 0.05). Each ad-
ditional minor allele was associated with doubling the risk of
OSA. This SNP was not associated with OSA at a P less than
0.05 threshold in the European American cohort, although
other SNPs within HTR2A were associated with SDB at a
nominal 0.05 level (rs6561332, rs1923885, and rs7322347). None
of the genotyped SNPs in the African Americans were associ-
ated with AHI at a 10% FDR threshold.
In secondary analyses, adjusting for BMI, AHI remained
associated with rs2808630 in CRP and rs2973042 in GDNF in
European Americans. For every additional G allele in rs2808630,
the AHI was increased by 4.09 6 1.02 (q 5 0.03) and for every
additional C allele in rs2973042, the AHI was increased by 3.52 6
0.90 (q 5 0.03). In contrast, after adjusting for BMI in African
Americans, no SNPs were associated with either OSA or AHI at
a 10% FDR threshold.
Haplotype analyses were performed on GDNF where mul-
tiple SNPs were associated with AHI or OSA. In general,
haplotypes were not more predictive of OSA status than single
SNPs. Haplotype models tested are presented in the online
supplement. It should be noted that because of the tag SNP
approach used in selecting SNPs for genotyping, few haplo-
blocks contained more than one SNP, limiting this approach.
In addition to the significant results with q-values less than or
equal to 0.10, we also identified several noteworthy SNPs that
TABLE 2. DEMOGRAPHIC CHARACTERISTICS
European Americans (n 5 694) African Americans (n 5 729)
Number of pedigrees
Body mass index, kg/m2
Apnea hypopnea index*
Apnea hypopnea index >15†
7.2 6 4.9
38.8 6 19.6, range 3–81
28.8 6 8.5, range 13–80
2.5 (0.9, 11.6)
6.1 6 4.4
36.4 6 19.1, range 3–82
30.8 6 9.6, range 12–85
3.8 (1.1, 16.7)
Values expressed as n and percentage or mean and SD.
* Median and interquartile range.
†Limited to the 553 European Americans and 539 African Americans over 18 yr of age.
Larkin, Patel, Goodloe, et al.: A Candidate Gene Study of Obstructive Sleep Apnea 949
did not meet our strict threshold. In European Americans we
report an increased risk of AHI at 15 or greater associated with
rs5370 of endothelin 1 (odds ratio 5 1.77; 95% confidence
interval, 1.26–2.48; P , 0.001; q 5 0.12) and rs2071943 in
linkage disequilibrium with rs5370 with a similar odds ratio
estimate, P value, and q-value. In African Americans we found
a haploblock of nine SNPs in strong linkage disequilibrium
(LD) within the leptin receptor with q-values 5 0.16 and P
values ranging from 0.0005 to 0.002 (rs11208674, rs10493379,
rs6676495, and rs12038998). We also note a SNP within
hypocretin receptor 2 (rs7768760) was modestly associated
with OSA in African Americans (odds ratio 5 1.76; 95%
confidence interval, 1.28–2.43; P 5 0.0005; q 5 0.16).
Although we restricted reporting of our primary findings to
associations with an FDR less than 10%, we have provided
regression coefficients, standard errors, P values, and q-values
for all SNPs in the online supplement to assist researchers in
identifying potential opportunities for future replication.
SDB is known to aggregate strongly within families. In the
Cleveland Family Study, individuals with an affected relative
have been shown to have a 1.5-fold greater risk of having OSA
themselves (2). The heritability for AHI is 32–36% in both
European Americans and African Americans (4, 5). In this
study, we sought to evaluate the role of polymorphisms in 52
candidate genes in explaining the familial aggregation of this
disorder. The set of candidate genes was selected based on
biologic knowledge of relevant pathways, similarity in pheno-
type to monogenic diseases, and linkage data from our cohort.
Our results support a potential pathogenic role for polymor-
phisms in GDNF and CRP in European Americans and for
a polymorphism in HTR2A in African Americans. The persis-
tence of associations between SDB with CRP and GDNF after
BMI adjustment suggests that these genetic variants influence
SDB susceptibility through obesity-independent pathways. In
contrast, the attenuation of the association between HTR2A
and SDB suggests that this association is likely through the
influence of HTR2A on weight.
The finding that GDNF variants are associated with SDB
phenotypes is especially interesting in light of the likely in-
fluences of ventilatory control abnormalities in the pathogenesis
of SDB. Ventilatory control abnormalities, such as those that
influence the sensing of oxygen or CO2or ventilation at sleep
state transitions, may predispose to OSA by promoting venti-
latory instability (and periodic breathing) (12), impairing the
arousal response to airway obstruction (13), or contributing to
imbalanced activation of upper airway muscles compared with
chest wall muscles (14). GDNF plays a particularly important
role in the development of neural pathways vital for normal
TABLE 3. ADDITIVE MODELS FOR SINGLE NUCLEOTIDE POLYMORPHISMS AND THE RISK OF OBSTRUCTIVE SLEEP APNEA
IN EUROPEAN AMERICANS ADJUSTED FOR AGE, SEX, AND EUROPEAN ANCESTRY
Unadjusted for BMIAdjusted for BMI
ChrGene SNPLocation Risk Allele Risk Allele Frequency OR (95% CI)P Value Q-Value OR (95% CI)P Value Q-Value
Definition of abbreviations: BMI 5 body mass index; Chr 5 chromosome; CI 5 confidence interval; CRP 5 C-reactive protein; GDNF 5 glial cell line–derived
neurotrophic factor; OR 5 odds ratio; SNP 5 single nucleotide polymorphism; UTR 5 untranslated region.
rium patterns in glial cell–derived
neurotrophic factor in European
Americans, derived from Haplo-
view v. 4.1 Red squares indicate
pairs of single nucleotide poly-
morphisms (SNPs) with high
by a high D’ and few recombi-
nation events (LOD >2). White
squares indicate pairs of SNPs for
which there is strong evidence
for linkage equilibrium. Areas of
pink and blue indicate pairs of
SNP where D’,1 and LOD >2
or D’ 5 1 and LOD ,2, respec-
tively. Haplotype blocks are out-
lined in heavy black triangular
950AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1822010
respiration, specifically influencing the development and differ-
entiation of noradrenergic neurons, including those in the A5
nucleus of the ventrolateral pons, which plays a critical role in
respiratory pattern generation (15). The knockout of the GDNF
gene results in abnormal central respiratory output. In addition,
GDNF seems to play a trophic role for sensory afferent neurons
in the carotid body (16), and thus may be important in the
development of hypoxic responses. The importance of GDNF
in ventilatory control is highlighted by the fact that severe
mutations in GDNF are associated with the central congenital
hypoventilation syndrome (17). Although reductions in respi-
ratory responses to hypoxia and to inspiratory load challenges
among family members of OSA probands have been demon-
strated (18–20), this is the first evidence that variants in a gene
in a ventilatory control pathway are associated with SDB.
The finding that variants in CRP were associated with SDB
provides further evidence that pathways that mediate inflam-
mation may be important in the pathogenesis of SDB. Although
most research of inflammation and OSA has considered in-
flammation as a response to OSA-related stresses (21–23), it is
also plausible that abnormalities in inflammatory pathways may
contribute to OSA severity by influencing pharyngeal patency
through effects on mucosal edema, by contributing to pharyn-
geal neuropathic changes (24), or possibly through effects on
central ventilatory control. Prior small candidate genes studies
have suggested that SDB is associated with other inflammatory
cytokines, such as tumor necrosis factor-a (25, 26) and IL-6
(27). If replicated, such findings may suggest a role for antiin-
flammatory therapy in the management of OSA.
Because serotonin plays an important role not only in sleep-
wake and appetite regulation but also in upper airway dilator
muscle activity through modulation of hypoglossal motor out-
put, serotonergic receptors have gained consideration as candi-
date genes for SDB. In particular, the serotonin 2A receptor has
been found to be the predominant excitatory serotonin recep-
tor subtype at hypoglossal motor neurons (28), and administra-
tion of serotonin 2A receptor agonist improves upper airway
stability in animal models (29). Although HTR2A may in-
fluence SDB through pleiotropic pathways (influencing both
airway function and obesity), the association we observed was
attenuated with BMI adjustment. It was also stronger in our
African American sample, which tended to be more obese.
Thus, further disentangling the influence of this genetic variant
on intermediate pathways may require a larger sample with
a greater BMI range. In both Chinese and Turkish populations,
the A allele in a SNP in the HTR2A promoter region (A-
1438G; rs2070040) has been associated with greater OSA
severity (30, 31). This SNP is in weak LD (D’ 5 0.12; r25
0.01) with the strongest OSA SNP identified for the African
American cohort in this study.
In addition to our primary findings (q values < 0.10), we
observed suggestive associations between the SDB traits with
genetic variants in the leptin receptor (LEPR) in African Amer-
icans, hypocretin 2 receptor (HCRTR2) in African Americans,
and endothelin-1 (EDN1) in European Americans (lowest q-
values of 16, 16, and 12%, respectively) highlighted in the online
supplement. Leptin plays a key role in weight homeostasis and
has been implicated in influencing hypercapnic ventilatory drive
(32). Hypocretin/orexin is a neurotransmitter known to have
important effects on sleep/wake regulation, with more recent
work implicating hypocretins in influencing upper airway neuro-
muscular activity (33). Circulating hypocretin levels have been
reported to be inversely correlated with OSA severity (34). Prior
analysis from European Americans in the CFS found evidence
for linkage to AHI at 6p12, the location of the hypocretin
receptor 2 (HCRTR2) (3). Endothelin 1 (EDN1), a potent va-
soconstrictor implicated in hypertension, is associated with
respiratory failure at birth in EDN1 knockout mice and hypo-
ventilation and blunted ventilatory response to hypoxemia and
hypercapnia in mice with one functional EDN1 gene (35). EDN1
knockout mice also show marked craniofacial morphologic
abnormalities characteristic of disturbed pharyngeal arch de-
velopment, as seen in humans with OSA caused by Treacher
Collins syndromeandPierre Robin syndrome(36).Ina European
population, evidence for an increase in the AHI was observed for
homozygote carriers of the minor allele for a missense coding
SNP (rs5370) in EDN1 (37). The same SNP allele showed an
increased risk of AHI greater than or equal to 15 in the current
sample of European Americans and can be considered replica-
tion at a nominal 0.05 threshold.
It is of interest that modeling the dichotomous and contin-
uously measured traits revealed both overlapping and unique
associations. Whereas genetic variants for the OSA trait are
most likely to identify susceptibility loci influencing clinical
TABLE 4. ADDITIVE MODELS FOR SINGLE NUCLEOTIDE POLYMORPHISMS AND THE APNEA HYPOPNEA INDEX IN EUROPEAN
AMERICANS ADJUSTED FOR AGE, SEX, AND EUROPEAN ANCESTRY
Unadjusted for BMIAdjusted for BMI
Chr GeneSNP Location Risk AlleleRisk Allele Frequency Beta (SE)P Value (empirical P value*) Q-Value Beta (SE)P Value Q-Value
Definition of abbreviations: BMI 5 body mass index; Chr 5 chromosome; CRP 5 C-reactive protein; GDNF 5 glial cell line–derived neurotrophic factor; SNP 5 single
nucleotide polymorphism; UTR 5 untranslated region.
* See online supplement for calculation of empirical P values.
TABLE 5. ADDITIVE MULTIVARIATE LOGISTIC REGRESSION MODELS FOR SINGLE NUCLEOTIDE POLYMORPHISMS AND THE RISK
OF OBSTRUCTIVE SLEEP APNEA IN AFRICAN AMERICANS ADJUSTED FOR AGE, SEX, AND AFRICAN ANCESTRY
Unadjusted for BMIAdjusted for BMI
ChrGene SNPLocation Risk AlleleRisk Allele Frequency OR (95% CI)P Value Q-ValueOR (95% CI)P ValueQ-Value
13HTR2Ars9526240intronA 0.172.06 (1.45–2.91)0.00005230.051.75 (1.13–2.71) 0.01260.49
Definition of abbreviations: BMI 5 body mass index; Chr 5 chromosome; CI 5 confidence interval; HTR2A 5 serotonin receptor 2A; OR 5 odds ratio; SNP 5 single
Larkin, Patel, Goodloe, et al.: A Candidate Gene Study of Obstructive Sleep Apnea 951
disease, those that are associated with AHI are likely variants
that are associated with incremental increases in the AHI across
a wide range.
As has been reported for other traits, many of the identified
genetic regions associated with OSA differed between the two
races. This may reflect differences in allele frequencies for the
causal variants or differing linkage disequilibrium patterns. In
addition, this may reflect a difference in the relevant mecha-
nisms for OSA pathogenesis between the two groups. For
example, the importance of various craniofacial morphologies
on OSA risk differs by race (38). Whether relative differences
exist in other intermediate phenotypes, such as ventilatory
control, is unknown. The fewer number of positive SNPs
identified for the African American cohort may also reflect
the stricter threshold for significance used in this group.
Because of the greater genetic complexity in this population,
nearly double the numbers of SNPs were required for genotyp-
ing to obtain the same level of coverage for the genes of
interest, and so double the number of comparisons for which to
Two areas of overlap should be noted across the two races.
First, rs2808630 found in the 39 untranslated region of CRP was
associated with both OSA and AHI in European Americans
after adjustment for multiple comparisons and also met nominal
significance criteria for association to OSA in African Ameri-
cans. In addition, two separate SNPs in EDN1 were suggestive
of an association with OSA in European Americans (rs2071943)
and African Americans (rs9296344).
Given that the genes studied were selected based on biologic
plausibility, we highlighted findings that met a threshold FDR
of 10%. This threshold corresponds to a nominal P values of
0.0002 and 0.00009 in the European American and African
American cohorts, respectively, and is much stricter than prior
genetic work in OSA and more conservative than a suggested
nominal P value of 0.00005 for candidate gene studies (39).
However, even with this approach, the q-value results demon-
strate that three of the four primary findings would meet
a stricter 5% FDR threshold.
To our knowledge, this is the largest candidate gene study
for sleep apnea to date. We were able to assess simultaneously
the association between SDB phenotypes and 52 genes that
have strong a priori evidence for being involved in sleep apnea
pathogenesis, and were able to examine associations in two
racial groups. Of note, this study includes the only African
American cohort to date to have undergone a genetic associa-
tion study for SDB, despite the fact that this group is at risk for
OSA at an earlier age (40). Strengths include the relatively large
sample size compared with prior studies in this field and the
extensive coverage obtained for each of the genes considered.
Although genome-wide association has gained extensive popu-
larity for identifying causal variants for complex diseases,
because of the large number of hypotheses being simulta-
neously tested in such a study design, the sample size required
to obtain statistical significance with genome-wide association is
substantially larger than with candidate gene studies. This is
a particular problem with regards to SDB research given the
substantial expense of overnight sleep studies required for
The findings in this study await replication in independent
cohorts to confirm the generalizability of the identified
associations. Furthermore, more detailed sequencing is
needed along with functional assays to identify the causal
variants, if any, at each of the regions associated with the
SDB phenotypes. Nevertheless, this study identified genetic
regions in both European American and African American
populations worthy of further investigation as containing risk
alleles. Given the increasingly recognized adverse impact of
SDB on diabetes, heart disease, and stroke risk, these variants
may prove also to be important in determining cardiovascular
Author Disclosure: E.K.L. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. S.R.P. received
$1,001–$5,000 from Health Right Products in industry-sponsored grants, and
more than $100,001 from the National Institutes of Health (NIH) and $50,001–
$100,000 from the American Thoracic Society in sponsored grants. R.J.G. does
not have a financial relationship with a commercial entity that has an interest in
the subject of this manuscript. Y.L. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. X.Z.
does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. C.G-M. does not have a financial
relationship with a commercial entity that has an interest in the subject of this
manuscript. M.D.A. received $50,001–$100,000 from the NIH as a research pilot
grant under CTSC. S.R. received more than $100,001 from Dymedix Inc. in
industry-sponsored grants and more than $100,001 from the NIH in sponsored
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