Heritability of Upper Airway Dimensions Derived Using Acoustic Pharyngometry

Abstract

Acoustic pharyngometry represents a simple, quick, non-invasive method for measuring upper airway dimensions which are predictive of sleep apnea risk. In this study we sought to assess the genetic basis for upper airway size as obtained by pharyngometry.

Participants over age 14 y in the Cleveland Family Study underwent three acoustic pharyngometry measurements. Variance component models adjusted for age and sex were used to estimate heritability of pharyngometry-derived airway measures.

A total of 568 of 655 subjects (87%) provided quality pharyngometry curves. Although African-Americans tended to have narrower airways compared to Caucasians, heritability patterns were similar in these two groups. Minimum cross-sectional area had a heritability of 0.34 (p=0.004) in Caucasians and 0.39 (p<0.001) in African-Americans, suggesting that 30-40% of the total variance in this measure is explained by shared familial factors. Estimates were unchanged after adjustment for body mass index or neck circumference. In contrast, oropharyngeal length did not have significant heritability in either ethnic group.

The minimum cross-sectional area in the oropharynx is a highly heritable trait suggesting the presence of an underlying genetic basis. These findings demonstrate the potential utility of acoustic pharyngometry in dissecting the genetic basis of sleep apnea.

Introduction

Obstructive sleep apnea (OSA) has been shown to have an important familial component suggesting the presence of a genetic basis for this disorder.^1-3 The pathways by which genetic polymorphisms might influence OSA susceptibility are not completely clear but upper airway anatomy likely represents an important mechanism. Numerous studies have identified specific anatomic features which predispose to OSA.^4-7 Many of the bony craniofacial risk factors have been shown to be strongly heritable.^{8, 9} More recently, using magnetic resonance imaging (MRI), soft tissue structures in the airway have also been shown to demonstrate familial correlation.¹⁰ Unfortunately, such elegant imaging is both time and cost intensive limiting the use of this modality to identify susceptibility genes in large populations and cannot be performed on extremely obese individuals. Acoustic pharyngometry represents a relatively simple and quick method of assessing upper airway dimensions which has been shown to predict OSA status.^{11, 12} In this study, we assessed the heritability of upper airway measurements derived from acoustic pharyngometry in participants in the Cleveland Family Study in order to estimate the potential utility of this tool for use in large-scale phenotyping efforts.

Methods

Subjects

The Cleveland Family Study is a longitudinal family-based epidemiological cohort designed to study the genetics of OSA. Details on recruitment of this cohort have been previously described.^{1, 13} Briefly, index probands with a laboratory confirmed diagnosis of OSA and at least two first-degree relatives available to be studied, were recruited along with family members. A subset of 725 individuals was selected for detailed phenotyping based on expected genetic informativity by choosing pedigrees where siblings had extremes (either high or low) of AHI. A more detailed explanation of the selection scheme has been previously published.² Because of potential confounding effects due to adenotonsillar hypertrophy in young children, only participants over age 14 were included in this analysis. The protocol was approved by the University Hospitals Case Medical Center institutional review committee and all participants provided written informed consent.

Phenotype collection

Measurements of height, weight, and neck circumference were made in duplicate and averaged. Body mass index (BMI) was calculated as the ratio of weight to height squared. Attended overnight laboratory polysomnography (Compumedics, Abbotsford, AU) was performed using both an oronasal thermocouple and nasal pressure cannula to assess airflow. Apneas and hypopneas were defined using Sleep Heart Health Study criteria, modified to include consideration of the nasal pressure signal.¹⁴ The apnea hypopnea index (AHI) was computed by dividing the number of respiratory events by the total sleep time.

Acoustic Pharyngometry

Pharyngometry (Eccovision, Hood Laboratories, Pembroke MA) was performed with the subject seated comfortably and breathing orally with the head in the Frankfort horizontal plane through a rubber mouthpiece that included a midline bridge to stabilize tongue position on the evening prior to polysomnography. Each measurement consists of a plot of cross-sectional area (CSA) as a function of distance from the mouth (Figure 1). An initial plot was performed with the subject breathing nasally. This was followed by three tracings done with the subject breathing orally at functional residual capacity. Tracings were scored as being of poor, adequate, or high quality according to clarity in identifying landmarks. Subjects with at least two adequate or high quality tracings were included in this analysis.

Figure 1. Schematic of Pharyngometry Variables.

Sample acoustic pharyngometry tracing displayed. Mean CSA obtained by averaging results between the proximal and distal minima. Volume is the product of mean CSA and oropharyngeal length. Relative maximum location is the ratio of maximum location divided by oropharyngeal length. CSA: cross-sectional area.

The oropharyngeal segment was defined as the region from the proximal minimum to the distal minimum CSA. These points correspond anatomically to the oropharyngeal junction and epiglottis. Eight dimensions were obtained from each curve and averaged over the 2-3 quality curves: five cross-sectional dimensions (proximal minimum CSA, distal minimum CSA, overall minimum CSA, maximum CSA, mean CSA) and three axial dimensions (oropharyngeal segment length, relative position of the maximum CSA over the segment length, and segment volume). Segment volume was computed as the product of mean CSA and segment length.

Pharyngometry validation

In a subgroup of 10 individuals (6 women, 4 men) with a wide range of AHI (range 2-68/hr), acoustic pharyngometry was assessed immediately prior to MRI of the upper airway. Axial images were obtained with a 1.5T Siemens Espree system (Siemens, Erlangen, Germany) using a 2D spin echo sequence (TR=400ms/TE=12ms) and 5 mm slice thickness with the subject supine, awake, and breathing through the pharyngometry mouthpiece. The air-tissue boundary was manually defined by a technician blinded to pharyngometry measurements and UTHSCSA Image Tool version 2.0 (San Antonio, TX) was used to compute the cross-sectional area in each slice. The proximal minimum CSA obtained by MRI while supine was substantially smaller than that obtained by pharyngometry while seated (1.09 cm² vs. 2.28 cm²). However, the Spearman correlation coefficient between the two measures was strong (r=0.75, p=0.01).

Statistical analysis

Differences between Caucasians and African-Americans were assessed using chi-squared and t-tests. Heritability estimates were computed using the maximum likelihood based variance component approach implemented in the statistical genetics software package, SOLAR version 4.0.7.¹⁵ All models included age and gender as covariates and conditioned on proband data to account for potential ascertainment bias. Heritability is computed as the ratio of the genetic variance to the sum of genetic and environmental variances and represents the proportion of the total variance in a trait (after adjusting for covariate effects) that is explained by additive genetic effects. Additional analyses were performed including BMI and neck circumference as covariates to estimate the heritability of upper airway measures independent of these potential contributing variables. Because of potentially different patterns of genetic transmission across races, all analyses were performed separately in Caucasians and African-Americans.

Results

Subject Characteristics

Out of 655 subjects > 14 y of age who performed pharyngometry, 568 (87%) had curves which met minimum quality criteria to be used in this analysis. Participant characteristics for each racial group are provided in Table 1. In general, the two groups were similar in weight, and OSA severity. However, the African-American cohort tended to have a narrower airway as evidenced by smaller cross-sectional mean and minimal pharyngeal areas.

Table 1.

Participant Characteristics

	Caucasians	African- Americans	P-value
Subjects (N)	229	339
Male (%)	102 (44.5%)	140 (41.3%)	0.44
Age (yrs)	42.1 (19.2)	38.2 (18.9)	0.02
BMI (kg/m2)	32.0 (9.3)	32.4 (9.9)	0.62
Neck Circumference (cm)	38.4 (5.2)	38.3 (5.2)	0.67
AHI(events/hr)	16.6 (24.6)	15.5 (24.1)	0.60
Maximum CSA (cm2)	3.24 (0.94)	2.84 (0.89)	< 0.001
Mean CSA (cm2)	2.65 (0.67)	2.34 (0.68)	< 0.001
Minimum CSA (cm2)	1.93 (0.57)	1.75 (0.55)	< 0.001
Proximal Minimum CSA (cm2)	2.14 (0.71)	1.86 (0.64)	< 0.001
Distal Minimum CSA (cm2)	2.56 (0.81)	2.38 (0.79)	0.008
Relative Maximum Location	0.57 (0.24)	0.63 (0.22)	0.004
Length (cm)	4.93 (1.27)	4.85 (1.30)	0.48
Volume (cm3)	13.31 (5.57)	11.57 (5.21)	< 0.001

Values expressed as number (%) or mean (standard deviation). BMI: body mass index; AHI: apnea hypopnea index; CSA: cross-sectional area.

Heritability Analyses

Six subjects were excluded from the genetic analysis as they had no family members with pharyngometry results. The remaining subjects came from 131 families (224 individuals in 53 Caucasian families and 338 individuals in 78 African-American families). Among Caucasians, the distal minimum CSA and the overall minimum CSA were the most heritable pharyngometry measures with heritabilities (h²) of 0.37 ± 0.19 and 0.34 ± 0.15 respectively (Table 2). The proximal minimum CSA also showed evidence of a genetic basis (h² = 0.24 ± 0.13) while the heritabilities of the mean and maximum CSA were much lower.

Table 2.

Heritability of Pharyngometry Measures in Caucasians

	Miximum CSA	Mean CSA	Minimum CSA	Proximal Minimum CSA	Distal Minimum CSA	Relative Maximum Location	Length	Volume
Age, sex adjusted
Heritability	0.06	0.18	0.34	0.24	0.37	0.06	0.00	0.00
P-value	0.31	0.08	0.004	0.01	0.02	0.36	0.50	0.50
Age, sex, BMI-adjusted
Heritability	0.04	0.15	0.32	0.17	0.38	0.04	0.00	0.00
P-value	0.41	0.19	0.01	0.07	0.02	0.40	0.50	0.50
Age, sex, neck circumference-adjusted
Heritability	0.08	0.19	0.34	0.22	0.35	0.03	0.00	0.00
P-value	0.27	0.08	0.004	0.03	0.02	0.43	0.50	0.50

BMI: body mass index; CSA: cross-sectional area.

In the African-American sample, the distal minimum CSA and overall minimum CSA were also the most heritable pharyngometry measures with heritabilities of 0.37 ± 0.13 and 0.39 ± 0.13 respectively (Table 3). In contrast to the Caucasian cohort, the other cross-sectional measures including mean and maximum oropharyngeal CSA also showed evidence of a genetic basis with heritabilities in the 0.20-0.30 range.

Table 3.

Heritability of Pharyngometry Measures in African-Americans

	Miximum CSA	Mean CSA	Minimum CSA	Proximal Minimum CSA	Distal Minimum CSA	Relative Maximum Location	Length	Volume
Age, sex adjusted
Heritability	0.21	0.26	0.39	0.27	0.37	0.00	0.17	0.08
P-value	0.03	0.01	<0.001	0.01	<0.001	0.50	0.08	0.16
Age, sex, BMI-adjusted
Heritability	0.19	0.24	0.37	0.26	0.36	0.01	0.16	0.06
P-value	0.04	0.01	<0.001	0.01	<0.001	0.46	0.09	0.26
Age, sex, neck circumference-adjusted
Heritability	0.19	0.25	0.39	0.28	0.37	0.02	0.17	0.07
P-value	0.04	0.01	<0.001	0.01	<0.001	0.43	0.08	0.23

BMI: body mass index; CSA: cross-sectional area.

Adjustment for BMI or neck circumference had minimal effect on heritability estimates in either ethnic group. In contrast to the substantial heritability found for cross-sectional airway measures, little evidence was found for a genetic basis for axial measures. Oropharyngeal length, relative location of the maximal cross-sectional area, and oropharyngeal airway volume were not heritable in either Caucasians or African-Americans.

Secondary analyses were performed limited to individuals where at least 2 curves met the highest quality rating. In general, heritabilities were greater in this subgroup of 224 individuals. For example, the heritability of the minimum cross-sectional area was 0.56 ± 0.19 (p=0.002) in Caucasians and 0.44 ± 0.18 (p=0.004) in African-Americans.

Discussion

Our results suggest that upper airway dimensions derived via acoustic pharyngometry demonstrate substantial intra-familial correlation. For the minimum oropharyngeal cross-sectional area, the heritability was .30-.40 implying that minimally 30-40% of the overall variance in this measure may be explained by intra-familial factors such as shared genetic polymorphisms. Higher heritability estimates are obtained when only the highest quality curves are considered. Findings were independent of BMI or neck circumference suggesting the relevant genes act independently of overall obesity. A previous study by Mathur et al demonstrated that the airways of relatives of apneics as assessed by pharyngometry were narrower than the airways of controls, although that work did not quantify the strength of the familial correlation.¹⁶ Since studies have demonstrated that a small minimum oropharyngeal CSA predicts the presence of OSA,^{12, 17} genes which influence the minimum CSA are likely to also influence OSA status.

The heritability of the minimum CSA is of similar magnitude to that of the AHI which has been estimated at 0.32-0.37 in several studies.^{2, 3, 18} An important difference in these two apnea related traits is that there are likely fewer genes responsible for the overall genetic effect on minimum CSA so that the locus-specific heritability for the genes with strongest effect is greater for minimum CSA. In addition to genes influencing upper airway anatomy, the AHI is likely influenced by genes which regulate such varied phenotypes as obesity, ventilatory control, arousal threshold, and loop gain. Genetic analyses of an upper airway phenotype may provide insight into one of the causal pathways patho-etiologically related to OSA. Acoustic pharyngometry, because of its ease of use, is ideally suited for the study of the thousands of subjects required for epidemiologic studies aimed at dissecting the genetics of OSA.

An additional finding in this work is the similar inheritance patterns between Caucasians and African-Americans, despite a smaller airway in African-Americans. In both groups, the distal minimum CSA and overall minimum CSA were the most heritable measures followed by the proximal minimum CSA. Minimal cross sectional area was also the pharyngometry measure found to best discriminate children with and without OSA.¹⁹ These data also suggest that cross-sectional airway dimensions have a greater genetic basis than axial dimensions. This may in part be due to greater measurement error in estimating airway length than cross-sectional area with acoustic pharyngometry. However, similar results were found in the work by Schwab et al using MRI.¹⁰ In that study, the greater heritability for cross-sectional airway measures compared to volumetric measures suggests airway length may have less of a genetic basis than other dimensions. Furthermore, fluid dynamic theory would argue that OSA pathogenesis is much more sensitive to changes in cross-sectional as compared to axial dimensions of the upper airway.

Another notable finding is the increase in heritability associated with limiting analysis to those subjects with high quality curves. The heritability of minimum CSA increased to 0.45-0.55 in this subset, suggesting that measurement error may represent a portion of the non-genetic variance. Of note, these values are similar to the 0.46 heritability reported by Schwab et al for minimum CSA using MRI.¹⁰ Lower quality curves may result from challenges in collecting such measurements in some individuals due to swallowing or tongue placement during the maneuver. These findings highlight the importance of using careful, standardized methodology in obtaining pharyngometry, and the need to further improve methodology for stabilizing the tongue during testing maneuvers.

The overall validity of pharyngometry for assessment of pharyngeal cross sectional area is supported both by prior work showing its ability to discriminate subjects with and without OSA,^{12, 17} as well as the high correlation we observed between minimum CSA obtained by pharyngometry with measurements obtained by MRI. However, a limitation of acoustic pharyngometry is that it does not provide information about specific tissue structures such as the genioglossus muscle or parapharyngeal fat pads. If airway dimensions are defined secondarily by the residual volume remaining after defining the structure of the bones, muscles, fat, and connective tissue in the neck, one would expect heritabilities for the volumes of these structures to be greater than that of the airway lumen. However, the heritabilities of airway measurements in the study by Schwab et al were greater than those for individual soft tissue structures.¹⁰ This suggests the possibility that genetic mechanisms might primarily define airway dimensions and this secondarily limits the size of surrounding structures. Alternatively, the various structures surrounding the airway may be influenced by the same set of genes so that the power to detect the effects of these genes is increased by considering a summary measure such as the joint effects on the airway lumen as opposed to the magnitude of each individual structure.

Several limitations regarding acoustic pharyngometry should be noted. First, it cannot distinguish airway narrowing caused by impingement from surrounding tissues from reduced neuromuscular compensation. Thus, we cannot exclude the possibility that the genetic basis observed for airway caliber is due to genes that act to modulate neural control of upper airway musculature rather than genes that influence anatomic structure. Second, acoustic pharyngometry provides no information about nasopharyngeal dimensions, which may be a relevant region for collapse in many patient with OSA. However, previous studies have demonstrated that oropharyngeal dimensions as measured by acoustic pharyngometry predict OSA status so we believe our findings are relevant to OSA gene discovery. Finally, the pharyngometry measurements used in this study were obtained with subjects seated rather than supine. Our validation study, though small, suggests that seated pharyngometry values do correlate with supine MRI measurements. Previous studies have found the magnitude of decrease in airway lumen going from seated to supine varies by gender. As a result, our results on seated measurements may represent a biased estimate of the heritability of supine airway dimensions.

In summary, this work demonstrates the utility of acoustic pharyngometry in studying the genetic basis for variability in upper airway shape by demonstrating the substantial heritability of pharyngometry-derived airway measures. While clearly pharyngometry cannot provide detail on specific structures that impinge on the airway as can be obtained with MRI or other technologies, pharyngometry is relatively low-cost, minimally burdensome and non-invasive, and thus amenable to use in the large scale studies needed to discover genes for OSA-related traits.