Identification of Craniofacial Risk Factors for Obstructive Sleep Apnea Using Three-

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1 Identification of Craniofacial Risk Factors for Obstructive Sleep Apnea Using Three Dimensional MRI Luqi Chi, MD; FrancoisLouis Comyn, DMD; Nandita Mitra, PhD; Muredach P. Reilly, MD; Fei Wan, BS; Greg Maislin MS; Lauren Chmiewski, BS; Matthew D. ThorneFitzGerald, BS;; Uduak N. Victor, Allan I. Pack, MD, PhD; Richard J. Schwab, MD Online Data Supplement 1

2 METHODS Subject Selection Control (normal) subjects were recruited through local advertisements in the neighborhood (same school district) of the cases (patients with obstructive sleep apnea). Controls were of the same sex and ethnic background as the case subjects. To qualify as controls, individuals needed to be free of sleep apnea. This was confirmed in a sleep study and almost all controls had an apnea hypopnea index (AHI) less than 5 episodes/hour. Four controls had an AHI slightly greater than 5 (5.3, 5.4, 5.7 and 6.3). Cases were recruited primarily from the Penn Center for Sleep Disorders (Philadelphia, PA) outpatient practice and were newly diagnosed as having sleep apnea. They had symptoms of sleep apnea. To qualify as a "case" such patients had to have an apnea hypopnea index (AHI) greater than 15 episodes/hour. Upper airway imaging was performed prior to the patient being started on nightly nasal continuously positive airway pressure (CPAP). Patients with sleep apnea already receiving chronic CPAP therapy were excluded from the study since such therapy might alter upper airway tissue properties (1). Potential controls with an AHI greater than 5 but less than 15 episodes/hour were considered indeterminate and were not studied further. They did not undergo MRI. Exclusionary criteria include: 1) age under 18; 2) subjects chronically taking medications that affected upper airway tone (i.e., benzodiazepines, or sedatives, etc.); 3) exclusions related to use of MRI, specifically: a) body weight more than 136 kg (table limit of the magnetic resonance scanner); b) presence of metallic implants (pacemaker, ferromagnetic clips, etc.); or c) severe claustrophobia. No subjects used steroids and there was no clinical evidence for hypothyroidism in any of the subjects. 2

3 Subjects were paid $ for the polysomnography and $ for the MRI. The patients received MRI and sleep study reports at the same time so weight loss secondary to knowing that they had OSA could not have occurred. The MRI was performed within one week of the sleep study. The study was approved by the University of Pennsylvania Institutional Review Board, and written informed consent was obtained from all subjects. Polysomnography Standard polysomnography procedures and scoring were performed, as previously described from our laboratory (25), using a computerized polysomnography system (Sandman, Mellville Diagnostics, Ottawa, Ontario). Controls underwent full night polysomnography. Cases (defined by an AHI greater than 15 episodes/hour) initially had a clinical sleep study (split night protocol diagnostic study in the first half of the night and CPAP in the second half of the night). Once recruited to this study, all cases had an upper airway MRI and the majority of cases (39 out of 55) had a repeat diagnostic study before starting CPAP, i.e. a full night study without CPAP to determine the AHI over the full night analogous to the controls. Polysomnograms were scored by registered sleep technologists and reviewed by certified sleep physicians using the standard criteria of Rechtschaffen and Kales (25) and the criteria proposed by the American Academy of Sleep Medicine (6). Obstructive apneas were defined as airflow cessation for more than 10 seconds; hypopneas were defined as 50% or more reductions in airflow that exceeded 10 seconds and were associated with 3% or greater decrements in blood oxygen saturation and/or an arousal (6). Nasal pressure monitors were utilized to determine airflow abnormalities in all subjects. In addition to AHI, we examined sleep efficiency, total sleep time, total test time, (time in bed), arousal index, minutes in NREM (stages 14) and REM sleep, and latency to REM sleep. 3

4 We did not record the duration of the apneic events themselves on the sleep studies. Snoring was noted but not quantified. Upper Airway Magnetic Resonance Imaging Upper airway magnetic resonance imaging was performed identically in normals and apneics using a 1.5 Tesla magnetic resonance imaging scanner (Signa Advantage: General Electric, Milwaukee, WI). Since head and neck position (flexion and extension) have been shown to alter upper airway anatomy (3, 4), each subject s head was placed in a neutral position by aligning in the Frankfurt plane perpendicular to the floor before initiating scanning. Foam padding aided in the stabilization of the subject s head and insulated the head from the anterior neck coil (General Electric Medical Systems). Subjects were instructed to breathe normally through their nose, not to move their head and were encouraged to refrain from swallowing during the scanning. The subjects were also instructed to keep their mouth closed and assume a normal relaxed bite (with the tongue touching the front teeth) during the scanning. Verbal communication was used to insure that the entire study was performed during wakefulness. We communicated with the subjects after each MR sequence to make sure that they did not fall asleep during the scans. If the initial MR sagittal scan demonstrated neck flexion/extension or mouth opening the scans were repeated. The initial sagittal localizer scan used the following parameters: TR (repetition time) = ms; TE (echo time) = 16.0 ms; 256 x 128 matrix; 1.0 NEX (number of signal averages); CF (center frequency) automated to water, flip angle = 90 ; FOV (field of view) = 24.0 cm, slice thickness of 3.0 mm and 1.5 mm skip; scan range from ear to ear. These localizing scans were used to identify the boundaries of the nasopharynx and the larynx, which comprised the range of the subsequent axial scans. Contiguous axial T1weighted spin echo data sets (TR = ms, 4

5 TE = 16.0 ms, 0.5 NEX, flip angle = 90, FOV = 24.0 cm, slice thickness of 5.0 mm and 0.0 mm skip) were utilized to acquire images from the most superior aspect of the mandibular rami to the false vocal cords. The upper airway volumetric reconstructions were performed from the axial data set. Total MR scanning lasted for approximately 10 minutes (3.5minute sagittal localizer run, 5.5minute contiguous axial data set acquisition). Anatomic Definitions, Measurements and Analysis The technicians who performed the MR analysis were blinded to case or control status. Axial Measurements Characterization of the Mandible (see Figures E1E3) The mandible has been separated into two sections for this craniofacial analysis, upper and lower portions. The upper portion is the rami of the mandible and the lower portion is the jaw. Figure E1 describes the locations and labels to all points used to characterize the mandible. Figure E2 shows axial MRI measurements that characterize the mandible. Figure E1. Definition of mandibular measurements and MR correlates Point Definition on Figure E1 (MRI correlates see Figure E2) Rami Centroid of rami (left and right) (MR correlates see figure E2A) 5

6 Gonion (Go): The left and right Go are the most posterior and inferior points of the mandible (MR correlates see figure E2B) 1 Centroid of lower central tooth (MR correlates see figure E2B) 2 Centroid of lower lateral central tooth (MR correlates see figure E2B) 3 Centroid of lower cuspid teeth (MR correlates see figure E2B) 4 6 Centroid of 1 st, 2nd and 3 rd molars (MR correlates see figure E2B) 7 (outside) 7 (inside) Mandibular body thickness (MR correlates see figure E2B) Gnathion (Gn) The most anterior inferior point of the mandible (MR correlates see Figure E2C) Distances in Figure E1A The length of mandibular rami (left/right): the distance measured from Go (left/right) to rami (left/right). The four principle points are the centroid of rami (left, right) and Go (left, right). Mandibular body thickness: the distance measured from 7 (inside) to 7 (outside). It is the maximum lateral thickness of the lower mandible. Typically measured just posterior to the 3 rd molar or in place of the 3 rd molar (it is not unusual to find a patient without the 3 rd molars). It is measured on the same slice as the mandibular unit width measurements. It is also recorded for the left and right side. Distances in Figure E1B Mandibular unit width: the multiple measurement description for the space enclosed laterally by the mandible s lower portion, or the jaw of the mandible. It is characterized by the linear distances between the centroids of specific teeth (see Figure E1B): lateral distance between the centroids of left and right centrals (1), lateral centrals (2), cuspids (3), 1 st molars (4), 6

7 2nd molars (5), and 3rd molars (6). All measurements initiate with a specific point on one side of the mandible and connect with its sister point on the other side. Figure E2. MRI mandible dimensional measurements (refer to figure E1 for craniofacial correlates) Figure E2A: Left and right mandibular rami The centroid of rami has been calculated on this slice for coordinate analysis (refer to Figure E1 for craniofacial correlate). Figure E2B: Lower centrals (1), cuspids (3), 1st, 2nd, 3rd molars (4, 5, 6, respectively), maximal mandibular thickness (7 inside 7 outside) 7

8 Most of the measurements used to characterize the mandible are obtained on this MR slice (Figure E2B). Figure E2C: Anteriorinferior mandibular point (Gn). The last slice fully containing the anterior portion of the mandible (Figure E2C). The measurement for mandibular length is shown in Figure 1 and Figure E3. When axial MR slices are stacked on top of each other, the linear distance between the most anterior inferior point of the mandible (Gnathion) and the most posterior and inferior points of the mandible (left and right Gonion) is the linear distance traversing axial planes, interpolated from threedimensional coordinates (x,y,z). The linear distance between any two points in 3dimensions can be determined mathematically using the following formulas. 8

9 Figure E3: Demonstration of the craniofacial coordinate analysis. The five points (Gn, Go, and centroids of the mandible rami) are demonstrated on 3 MR slice levels. The left and right mandibular body length (Gn to left and right Go, the green lines) and the length of mandible rami (black lines) were calculated from coordinates (x, y, slice number and slice increment). Slice 19 is the axial plane containing Gn; slice 14 is the axial plane containing left and right Go; and slice 3 is the axial plane containing the centroids of the mandible rami. Mandibular depth is the blue dashed line from Gn to H, where H is the midpoint between the left to the right Go. Formulas 1, 2 and 3 were used to calculate the 3dimentional left mandibular body length, right mandibular body length and mandibular depth, respectively. Twodimensional coordinate analysis (Formula 4) 9

10 was used to calculate mandibular width (the red dotted line between the left and the right Go). Formulas 5 and 6 were used to calculate the length of mandible rami. Twodimensional coordinate analysis (Formula 7) was used to calculate the width of rami (the distance between centroids of mandible rami). Formula 1. Mandibular body Length, left = (x 1 x 3 ) 2 + (y 1 y 3 ) 2 + (z 1 z 3 ) 2 Formula 2. Mandibular body Length, right = (x 2 x 3 ) 2 + (y 2 y 3 ) 2 + (z 2 z 3 ) 2 Coordinate values on individual 2dimensional MRI slices determine the X and Y values, for each point. Slice number and slice thickness (5 mm) determine the Z value. The distance from z 1 to z 3 (z 1 z 3 ) is calculated as the thickness of the slices multiplied by the number of slices separating the two slices of interest (Figure E3). The mandibular body length is the average of the two green GnGo line segments (left and right). Mandibular depth (the blue dashed line) is a 3dimensional distance from the line segment drawn from point H (x 4, y 4, and z 4 ) to Gn, where H is the midpoint of the red dotted line between the left and right Go (Formula 3). The distance from z 4 to z 1 (z 4 z 1 ) is equal to the distance from z 1 to z 2 (z 1 z 2 ). Formula3. Mandibular Depth = (x 4 x 3 ) 2 + (y 4 y 3 ) 2 + (z 4 z 3 ) 2 Where x 4 =(x 1 +x 2 )/2, and y 4 =(y 1 +y 2 )/2 Mandibular width is a 2dimensional linear distance (Formula 4). Formula 4. 10

11 Mandibular Width = (x 1 x 2 ) 2 + (y 1 y 2 ) 2 The linear distance between the left and right Go to the respective centroids of each ramus can be determined mathematically using formula 5 and formula 6. Formula 5. Length of Ramus, right = (x 5 x 1 ) 2 + (y 5 y 1 ) 2 + (z 5 z 1 ) 2 Formula 6. Length of Ramus, left = (x 6 x 2 ) 2 + (y 6 y 2 ) 2 + (z 6 z 2 ) 2 Coordinate values on individual 2dimensional MRI slices determine the X and Y values, for each point. Slice number and slice thickness (5 mm) determine the Z value. The distance from z 1 to z 3 (z 1 z 3 ) or from z 2 to z 4 (z 2 z 4 ) can be calculated as the thickness of the slices multiplied by the number of slices separating the two slices of interest (Figure E3). The reported length of rami in the present study is the average of the line segments from the left and right Go to the respective centroids of each ramus. The width of rami is a twodimensional linear distance between the centroids of the left and right rami. It was determined using formula 7. Formula 7. Width of Rami = (x 5 x 6 ) 2 + (y 5 y 6 ) 2 Characterization of the Maxilla (see Figures E4E5): 11

12 The maxilla is shaped like a horseshoe. Specific points have been chosen for analysis. Figure E4 has the locations and labels to all points used to characterize the maxilla. These points were selected from a specific MR image. Threedimensional coordinates were measured and a variety of linear distance measurements were derived from them. All measurements except for Anterior Arch of the Atlas were made on one MR slice, the slice containing the roots of the teeth and the incisive canal (Figure E5A). The Anterior Arch of the Atlas is usually clearly visible on only one axial slice above, below or the same slice as the principle slice used for maxilla measurements. 12

13 Figure E4. Description of dimensional measurement variables of maxilla (MR correlates see Figure E5) Point definition on Figure E4 (MR correlates see Figure E5A and Figure E5B) 89 Centroid of upper central and lateral central teeth 10 Centroid of cuspid tooth 13

14 1113 Centroid of 1 st, 2 nd and 3 rd molar 14 Posterior points of the maxilla (left/right) 15 Anterior Arch of the Atlas M The midpoint between the left and right centroid of the upper central teeth (near 8). Distances Figure E4A: Maxillary unit length (Vshape) This is the simplest method of characterizing the maxilla s length and general shape. Three principle points were chosen for analysis. The linear distances from point M to the posterior points (14 left/right) are recorded as maxilla unit length (Vshape). The reported maxillary unit length (Vshape) is the average of the left and the right. Figure E4B: Maxillary unit length (Ushape) UShape is more descriptive in its characterization of the maxilla s length and shape. U shape1(left/right) are the distances measured from point M to the centroid of the cuspid (10 left/right). Ushape2 (left/right) are the distances measured from the centroid of the cuspid (10 left/right) to the posterior point of the maxilla (14 left/right). Figure E4C: Maxillary unit width measurements Maxillary unit width is comprised of seven principle measurements. Like mandibular unit width, it is characterized by the linear distances between the centroids of specific teeth. Each measurement is taken from a specific point on one side of the maxilla and correlated with its sisterpoint on the other side of the maxilla. Lateral distances between left and right centrals (8), lateral centrals (9), cuspids (10), 1 st molar (11), 2 nd molar (12), 3 rd molar (13), and the linear distance between the left and right posterior points of maxilla (14 left/right) were measured. Figure E4D: Maximum maxillary depth 14

15 Maximum maxillary depth is the linear distance from the point M to the midpoint of the posterior line, drawn from specific points (14 left/right). Figure E4E: Relations of maxillary specific points with the anterior arch of the atlas Linear distances measured from the centroid of the anterior arch of the atlas (15) to the point M, the left and right posterior maxilla (14 left/right) (the anterior arch of the atlas has been drawn into the diagram for this analysis) Figure E5. MRI maxilla dimensional measurements (see Figure E4 for craniofacial correlates) Figure E5A: Maxillary measurements: upper centrals (8), cuspids (10), 1st, 2nd, and 3rd molars (11,12,13, respectively), posterior points of the maxilla (14 left/right) The MR slice contains incisive canal is the main slice for maxillary analysis. This patient does not have 3rd molars. Figure E5B: Anterior arch of the atlas 15

16 This MR slice is one slice above the one used for maxillary unit measurements. The centroid of the anterior arch of the atlas has been calculated for coordinate analysis. The linear distance between any two points in 3dimensions can be determined mathematically as in formula 1 3. List of secondary measurements: Mandible (see Figures E1E3): Mandibular Rami Length (average left and right) Mandibular Body Thickness (average left and right) Width Rami to Rami Width 3 rd molar to 3 rd molar Width 2 nd molar to 2 nd molar Width 1 st molar to 1 st molar Width Cuspid to Cuspid Width Central to Central Maxilla (see Figures E4E5): Maxillary Unit Length (V shape) average left and right Maxillary Unit Length (Ushape1) average left and right Maxillary Unit Length (Ushape2) average left and right Maxillary Unit Width (Posterior to Posterior) Maxillary Unit Width (3 rd molar to 3 rd molar) Maxillary Unit Width (2 nd molar to 2 nd molar) Maxillary Unit Width (1 st molar to 1 st molar) 16

17 Maxillary Unit Width Cuspid to Cuspid Maxillary Unit Width Central to Central Anterior Arch of Atlas to centre of Maxilla Centrals Average of Anterior Arch of Atlas to Left and Right Posterior Maxilla MRI Cephalometry (see Figure E6) Figure E6: Standard cephalometric measurements (angles and lines): SNA Angle (sellanasionsubspinale) SNB Angle (sellanasionsupramentale) ANB Angle: SNA Angle minus SNB Angle SN: Distance from Sella (S) to Nasion (N) SA: Distance from Sella (S) to Subspinale (A) NA: Distance from Nasion (N) to Subspinale (A) SB: Distance from Sella (S) to Supramentale (B) NB: Distance from Nasion (N) to Supramentale (B) AB: Distance from Supramentale (B) to Subspinale (A) 17

18 Statistical Analysis (exploratory) We examined secondary craniofacial variables in association with sleep apnea, seeking to provide a more complete assessment of craniofacial risk factors in increasing risk for sleep apnea. However, these analyses were likely to lack the necessary power to find significant differences because of multiple testing and interactions especially with small effects. Exploratory analyses were performed as the same manner as the primary analysis, described in the main manuscript. RESULTS Demographics of Case Subjects and Control Subjects Cases and controls did not differ significantly with respect to marital status (p = 0.12) and education (p = 0.32) (Table E1). Although the entire distribution of gender and race was balanced between apneics and controls, we had more Caucasian males (32.7%) than African American males (12.7%) and fewer Caucasian female apneics (12.7%) than African American Females (36.4%) (Table E1). Table E1: Description of Case and Control Subjects Online Repository Factor Cases (n) (%) Controls (n) % p value* Race (male) 1.00 White % %. African American % %. Asian 1 1.8% 1 1.8%. Hispanic 1 1.8% 1 1.8%. Race (female) White % %. African American % %. Asian 1 1.8% 1 1.8%. Hispanic 0 0.0% 0 0.0%. Marital Status Married 27 54% 16 32% 0.12 Single 14 28% 23 46%. 18

19 Separated/Divorced 8 16% 10 20%. Widow (er) 1 2% 1 2%. Education <High School 2 4% 0 0% 0.32 High School 12 42% 15 29%. 2 year college 10 20% 12 23%. 4 year college 11 22% 18 35%. Graduate school 6 12% 7 13%. * Fisher's Exact Test, comparing apneic and normal subjects Polysomnography As shown in Table E2, sleep efficiency was not significantly different between cases and controls (p = 0.91). Cases had significantly less rapid eye movement (REM) sleep than did controls (p = 0.01). In contrast, there was no significant difference between cases and controls for mean times spent in stage 1 sleep (p = 0.21) and stage 2 sleep (p = 0.61). Cases tended to have less delta sleep but this was not quite significant (p = 0.06). Cases had a reduction in REM sleep time (p = 0.01). There were also significantly more nocturnal arousals (46.6 ± 31.5 arousals/hour) among the cases than among the controls (17.5 ± 7.2 arousals/hour, p < ). Presumably the reduced REM sleep time and increased arousals among the subjects with sleep apnea were related to their sleepdisordered breathing. Table E2. Summary of Polysomnography Results for Case and Controls Subjects Cases Controls p value Sleep Study Variable Mean SD Mean SD (ttest) Sleep Efficiency% Arousal Index* < Time in Stage 1, minutes Time in Stage 2 (minutes) Time in Stage 3/4 (minutes) Time in REM Stage (minutes) Latency to REM Stage (minutes)

20 Total Sleep Time (minutes) Total Test Time (minutes) Significant differences (p < 0.05) are presented in bold. * Number of EEG arousals/hour. Reliability and Validity Analysis Comparison Between MRI Cephalometry versus Conventional Cephalometric Radiographs (Table E3a, E3b and E3c) Very high inter and intrareader reliability for mandibular and hyoid measurements are shown in Table E3a and E3b. We have also performed standard cephalometric measurements by MRI and conventional cephalometric radiographs in 10 normal subjects. There were no significant differences between these measurements using conventional cephalometric radiographs or MR cephalometrics (Table E3c). Table E3a. Intra reader reliability analysis Variable Intraclass Correlation (ICC) Mandible Mandibular Length 0.99 Mandibular Width 0.99 Mandibular Depth 0.98 Hyoid Hyoid to Sella 0.99 Hyoid to Nasion 0.99 Hyoid to Supramentale 0.98 Table E3b. Inter reader reliability analysis Variable Variance Total Component Variance % of Total Variance Mandible Mandibular Length Mandibular Width Mandibular Depth Hyoid Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale

21 Table E3c. Validity Analysis Comparison of Lateral Cephalogram to MRI Cephalometry n = 10 MRI Lateral Cephalogram Mean SE p SNA Angle ( o ) SNB Angle ( o ) ANB Angle ( o ) Saddle Angle ( o ) Upper Facial Height (cm)

22 Lower Facial Height (cm) Anterior Facial Height (cm) Upper Facial Height / Anterior Facial Height Additional Primary Mandibular and Hyoid Measurements (Figure E4E7) Primary analysis for mandible and hyoid was repeated with total upper airway soft tissue volume in place of tongue volume. This did not change the magnitude of the results (E4a, E5a, 22

23 E6a and E7a). We used neck fat since it is the relevant fat for the upper airway. Repeated analysis including BMI in place of fat pad as a covariate did not alter the results (Table E4b, E5b, E6b and E7b). TABLE E4a. Comparison of Adjusted Mean Differences in Mandibular Measurements between Apneic and Normal Subjects, Stratified for Gender Mandibular Measurements (mm) Age, Height, Volume of Fat Pad, Volume of Total Soft Tissue Age, Height, Volume of Fat Pad, Volume of Total Soft Tissue, ANB Angle Diff. Diff. SE p Diff. SE p Men Mandibular Body Length <0.01* <0.01* Mandibular Width Mandibular Depth <0.01* <0.001* Women Mandibular Body Length Mandibular Width Mandibular Depth Significant differences (p < 0.05) presented in bold. Definition of abbreviations: Diff. = adjusted difference (apneic normal); SE = standard error. * Significant after Bonferonni correction for multiple testing. TABLE E4b. Comparison of Adjusted Mean Differences in Mandibular Measurements between Apneic and Normal Subjects, Stratified for Gender Mandibular Measurements (mm) Age, Height, BMI, Volume of Tongue Age, Height, BMI, Volume of Tongue, ANB Angle Diff. Diff. SE p Diff. SE p Men Mandibular Body Length Mandibular Width Mandibular Depth Women Mandibular Body Length Mandibular Width Mandibular Depth

24 Significant differences (p < 0.05) presented in bold. Definition of abbreviations: Diff. = adjusted difference (apneic normal); SE = standard error. BMI = body mass index. TABLE E5a. Comparison of Odds Ratios in Mandibular Measurements between Apneic and Normal Subjects, Stratified by Gender Mandibular Measurements (mm) SD Unadjusted Age, Height, Volume of Fat Pad, Volume of Total Soft Tissue Age, Height, Volume of Fat Pad, Volume of Total Soft Tissue, ANB Angle OR 95% CI OR* 95% CI OR* 95% CI Men Mandibular Body Length Mandibular Width Mandibular Depth Women Mandibular Body Length Mandibular Width Mandibular Depth Definition of abbreviations: OR = odds ratio for 1SD increase; OR* = adjusted OR; 95% CI = 95% confidence interval. Significant differences (95% CI not including 1.0) are presented in bold. TABLE E5b. Comparison of Odds Ratios in Mandibular Measurements between Apneic and Normal Subjects, Stratified by Gender Mandibular Measurements (mm) SD Unadjusted Age, Height, BMI, Volume of Tongue Age, Height, BMI, Volume of Tongue, ANB Angle OR 95% CI OR* 95% CI OR* 95% CI Men Mandibular Body Length Mandibular Width Mandibular Depth Women Mandibular Body Length Mandibular Width Mandibular Depth Definition of abbreviations: OR = odds ratio for 1SD increase; OR* = adjusted OR; 95% CI = 95% confidence interval. Significant differences (95% CI not including 1.0) are presented in bold. BMI = body mass index 24

25 TABLE E6a. Comparison of Adjusted Mean differences in Hyoid Measurements between Apneic and Normal Subjects, Stratified by Gender Men Hyoid Measurements (mm) Age, Height, Volume of Fat Pad, Volume of Total Soft Tissue Age, Height, Volume of Fat Pad, Volume of Total Soft Tissue, ANB Angle Diff. Diff. SE p Diff. SE p Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Women Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Significant differences (p < 0.05) presented in bold. Definition of abbreviations: Diff. = adjusted difference (apneic normal); SE = standard error. n = number of Subjects. TABLE E6b. Comparison of Adjusted Mean differences in Hyoid Measurements between Apneic and Normal Subjects, Stratified by Gender Hyoid Measurements (mm) Age, Height, BMI, Volume of Tongue Age, Height, BMI, Volume of Tongue, ANB Angle Diff. Diff. SE p Diff. SE p Men Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Women Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Significant differences (p < 0.05) presented in bold. Definition of abbreviations: Diff. = adjusted difference (apneic normal); SE = standard error. BMI = Body Mass Index TABLE E7a. Comparison of Odds Ratios in Hyoid Measurements between Apneic and Normal Subjects, Stratified by Gender Hyoid Measurements (mm) SD Unadjusted Age, Height Age, Height, Volume of Total 25

26 Soft Tissue OR 95% CI OR* 95% CI OR* 95% CI Men Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Women Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Definition of abbreviations: OR = odds ratio for 1SD increase; OR* = adjusted OR; 95% CI = 95% confidence interval. Significant differences (95% CI not including 1.0) are presented in bold. TABLE E7b. Comparison of Odds Ratios in Hyoid Measurements between Apneic and Normal Subjects, Stratified by Gender Hyoid Measurements (mm) SD Unadjusted Age, Height Age, Height, BMI OR 95% CI OR* 95% CI OR* 95% CI Men Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Women Hyoid to Sella Hyoid to Nasion Hyoid to Supramentale Definition of abbreviations: OR = odds ratio for 1SD increase; OR* = adjusted OR; 95% CI = 95% confidence interval. BMI = body mass index. Significant differences (95% CI not including 1.0) are presented in bold. Secondary Mandibular Measurements (Table E8E11) Unadjusted mean comparisons in secondary mandibular measurements between apneics and controls are shown in Table E8. As shown in Table E8, the mandibular unit width between the left to right second molar and the unit width between the left to the right first molar were significantly larger in cases than in controls (p = 0.04, p = , respectively). After stratification for gender (Table E9), the differences between male apneics and their normal 26

27 controls in the same measures was insignificant (p = 0.47, p = 0.05, respectively), but the differences remained significant in women (p = 0.02 and p = 0.001, respectively). The width from the left to right third molar was not different between apneics and controls before gender stratification but became significant in men after stratifying for gender. However, the sample size was small (male n = 6 and female n = 4) since many subjects were missing their third molar. There were no significant differences between apneic and normal subjects in other mandibular measurements before and after gender stratification. Table E8. Crude Comparisons of Mandibular Measurements Between Apneic and Normal Subjects in Men and Women Mandibular Measurement (mm) Apneic Subjects Normal Subjects N Mean SD N Mean SD % Diff. p* Mandibular Rami Length Average Left and Right Width Rami to Rami Width 3rd molar to 3rd molar Width 2nd molar to 2nd molar Width 1st molar to 1st molar Width Cuspid to Cuspid Width Central to Central Maximum MandibThickness Average Left and Right Significant differences (p < 0.05) presented in bold; % Diff. = % Difference = [(apneic normal)/apneic]*100. N = number of subjects; * p value for Student's ttest. smaller sample size due to missing molars. measurements were unable to obtained in 5 subjects due to MRI artifacts. Table E9. Crude Comparisons of Mandibular Measurements Between Apneic and Normal Subjects, Stratified by Gender Maxillary Measurements (mm) Apneic Men Normal Men Apneic Women Normal Women % p* Diff. N Mean SD N Mean SD N Mean SD N Mean SD Mandibular Rami Length Average Left and Right Mandibular Unit Width Rami to Rami % Diff. p* 27

28 Mandibular Unit Width 3rd molar to 3rd molar Mandibular Unit Width 2nd molar to 2nd molar Mandibular Unit Width 1st molar to 1st molar Mandibular Unit Width Cuspid to Cuspid Mandibular Unit Width Central to Central Maximum Mandibular Thickness Average Left and Right Significant differences (p < 0.05) presented in bold; % Diff. = % Difference = [(apneic normal)/apneic]*100. N = number of subjects; * p value for Student's ttest. smaller sample size due to missing molars. measurements were unable to obtained in 5 subjects due to MRI artifacts. Table E10 shows differences in means of secondary mandibular measurements between apneics and controls, adjusting for important factors that may affect the outcome and stratifying for gender. In men, when adjusted for age, race height, only a larger mandibular unit width between left to right first molar was found in apneics compared to normal controls (p = 0.04). After controlling for parapharyngeal fat pad tongue volume or total soft tissue volume in addition to above confounders, the difference became insignificant. Other variables were insignificant before and after gender stratification. It was noted that apneic men had a smaller mandibular unit width from the left to the right rami, and smaller width from left to right cuspid compared in normal controls, but these were not significant. In women, the apneics had significantly larger mandibular unit widths between the left to right second molars after adjusting for age, race and height (p = 0.04 and p = respectively). After controlling for tongue or soft tissue volume, the difference in unit width between the second molars was insignificant, but the difference in unit width between the first molars 28

29 remained significant (p = 0.01). However, the smaller sample size, due to missing molars, should be noted (Table E8, E12 and E16). Other secondary mandibular measurements were not statistically significant between female apneic and normal subjects either before or after above adjustments. Table E10. Comparison of Adjusted Mean Differences in Mandibular Measurements Between Apneic and Normal Subjects, Stratified by Gender Mandibular Measurements (mm) Age, Height Age, Height, Volume of Fad Pad, Volume of Tongue Age, Height, Volume of Total Soft Tissue Age, Height, ANB Angle, Volume of Fad Pad, Volume of Tongue Age, Height, ANB Angle, Volume of Total Soft Tissue Diff. SE p Diff. SE p Diff. SE p Diff. SE p Diff. SE p Men Mandibular Rami Length Average Left and Right Width Rami to Rami Width 3rd molar to 3rd molar Width 2nd molar to 2nd molar Width 1st molar to 1st molar Width Cuspid to Cuspid Width Central to Central Maximum MandibThickness Average Left and Right Women Mandibular Rami Length Average Left and Right Width Rami to Rami Width 3rd molar to 3rd molar Width 2nd molar to 2nd molar

30 Width 1st molar to 1st molar Width Cuspid to Cuspid Width Central to Central Maximum Mandibular Thickness Average Left and Right Significant differences (p < 0.05) presented in bold. Definition of abbreviations: Diff. = adjusted difference (apneic normal); SE = standard error. Table E11 summarizes the multiple logistic regression analyses for secondary mandibular measurements. In men, the odds ratios for 1SD increase in mandibular unit width between left first molar to right first molar was significantly larger than one, indicating a wider width between lower left to right first molar is associated with increased risk of sleep apnea in men (OR: 2.84, 95% CI: ) after adjusting for race, age, and height. However, adjusting for the total soft tissue volume abolished the difference. Replacing the volume of the parapharyngeal fat pad and tongue with total soft tissue volume demonstrated the same results. Adding skeletal type using ANB angle into the models did not change the altitude of the results. In women, mandibular unit widths between the left to right second molars and the left to right first molars were associated with increased risk for OSA after controlling for age, race and height. However, these differences were insignificant after adjusting for total soft tissue volume or tongue volume, ANB angle and parapharyngeal fat pad in addition to above adjustments (Table E11). Table E11. Comparison of Odds Ratios in Mandible Measurements Between Apneic and Normal Subjects, Stratified by Gender Mandibular Measurements (mm) SD Unadjusted Adjusted for Race, Age, Height Adjusted for Race, Age, Height, Volume of Total Soft Tissue Adjusted for Race, Age, Height, ANB Angle, Volume of Fat Pad, Volume of Tongue Adjusted for Race, Age, Height, ANB Angle, Volume of Total Soft Tissue OR 95% CI OR* 95% CI OR* 95% CI OR* 95% CI OR* 95% CI Men 30

31 Mandibular Rami Length Average Left and Right Width Rami to Rami Width 3rd molar to 3rd molar Width 2nd molar to 2nd molar Width 1st molar to 1st molar Width Cuspid to Cuspid Width Central to Central Maximum MandibThickness Average Left and Right Women Mandibular Rami Length Average Left and Right Width Rami to Rami Width 3rd molar to 3rd molar Width 2nd molar to 2nd molar Width 1st molar to 1st molar Width Cuspid to Cuspid Width Central to Central Maximum MandibThickness Average Left and Right Definition of abbreviations: OR = odds ratio for 1SD increase; OR* = adjusted OR; 95% CI = 95% confidence interval. Significant differences (95% CI not including 1.0) are presented in bold. : not estimable due to small sample size (majority of subjects had missing molars) Secondary maxillary measurements (Table E12E15) Unadjusted mean comparisons in maxillary measurements between apneics and controls are shown in Table E12. Before stratification for gender, the average maxillary unit length 1 (U 31

32 shape1) and maxillary unit width (the upper left cuspid to right cuspid) were significantly larger in cases than in controls (p = 0.01 and p = 0.01, respectively). After stratification for gender (shown in Table E13), there were no significant differences in means of maxillary measurements between apneics and in their normal controls. Apneic women had bigger maxillary unit widths (Vshape, Ushape1, the distance between the left to right cuspids, and the distance between the left to right first molars), deeper maxillary depth and the longer distance from anterior Arch of Atlas to maxilla centrals. Table E12. Crude Comparisons of Maxillary Measurements Between Apneic and Normal Subjects in Men and Women Maxillary Measurement (mm) Apneic Subjects Normal Subjects N Mean SD N Mean SD % Diff. p* Maxilla Unit Length (Vshape) Average Left and Right Maxilla Unit Length 1 (Ushape1) Average Left and Right Maxilla Unit Length 2 (Ushape2) Average Left and Right Maxilla Unit Width Posterior to Posterior Maxilla Unit Width 3rd molar to 3rd molar Maxilla Unit Width 2nd molar to 2nd molar Maxilla Unit Width 1st molar to 1st molar Maxilla Unit Width Cuspid to Cuspid Maxilla Unit Width Central to Central Maximum Maxilla Depth Anterior Arch of Atlas (AAA) to Maxilla Centrals Distance AAA to Posterior Maxilla Distance Average Left and Right Significant differences (p < 0.05) presented in bold; % Diff. = % Difference = [(apneic normal)/apneic]*100. N = number of subjects; * p value for Student's ttest. smaller sample size due to missing molars. measurements were unable to obtained in 5 subjects due to MRI artifacts. Table E13. Crude Comparisons of Maxillary Measurements Between Apneic and Normal Subjects, Stratified by Gender Maxillary Measurements (mm) Apneic Men Normal Men Apneic Women Normal Women % p* Diff. N Mean SD N Mean SD N Mean SD N Mean SD % Diff. p* 32

33 Maxilla Unit Length (V) Average L and R Maxilla Unit Length 1 (UShape1) Average L and R Maxilla Unit Length 2 (USahpe2) Average L and R Maxilla Unit Width Posterior to Posterior Maxilla Unit Width 3rd molar to 3rd molar Maxilla Unit Width 2nd molar to 2nd molar Maxilla Unit Width 1st molar to 1st molar Maxilla Unit Width Cuspid to Cuspid Maxilla Unit Width Central to Central Maximum Maxilla Depth Anterior Arch of Atlas (AAA) to Maxilla Centrals Distance AAA to Posterior Maxilla Distance Average L and R Significant differences (p < 0.05) presented in bold; % Diff. = % Difference = [(apneic normal)/apneic]*100. N = number of subjects; * p value for Student's ttest. smaller sample size due to missing molars. measurements were unable to obtained in 5 subjects due to MRI artifacts. L=left, R=right. Table E14 shows differences in means of maxillary measurements between cases and controls, after adjustments. No significant differences were found in all measurements between male apneics and their normal controls. Apneic women had significantly larger maxillary unit lengths (Vshape and Ushape1) and the unit width between the left to right first molar, and the unit width between the left to right cuspids) after adjusting demographic factors. After controlling for total soft tissue volume, ANB angle and parapharyngeal fat fad in addition to above confounders, difference in maxillary unit length (Vshape) was insignificant, whereas the difference in Ushape1 and the distance between the left to right cuspids remained significant. 33

34 Table E14. Comparison of Adjusted Mean Differences in Maxillary Measurements Between Apneic and Normal Subjects, Stratified by Gender Maxillary Measurements (mm) Age, Height, Volume of Fad Pad, Volume of Tongue Age, Height, Volume of Total Soft Tissue Age, Height, ANB Angle, Volume of Fad Pad, Volume of Tongue Age, Height, ANB Angle, Volume of Total Soft Tissue Diff. SE p Diff. SE p Diff. SE p Diff. SE p Men Maxilla Unit Length (V) Average Left and Right Maxilla Unit Length 1 (U Shape1) Average Left and Right Maxilla Unit Length 2 (U Sahpe2) Average Left and Right Maxilla Unit Width Posterior to Posterior Maxilla Unit Width 3rd molar to 3rd molar Maxilla Unit Width 2nd molar to 2nd molar Maxilla Unit Width 1st molar to 1st molar Maxilla Unit Width Cuspid to Cuspid Maxilla Unit Width Central to Central Maximum Maxilla Depth Anterior Arch of Atlas (AAA) to Maxilla Centrals Distance AAA to Posterior Maxilla Distance Average Left and Right Women Maxilla Unit Length (V) Average Left and Right Maxilla Unit Length 1 (U Shape1) Average Left and Right Maxilla Unit Length 2 (U Shape2) Average Left and Right Maxilla Unit Width Posterior to Posterior

35 Maxilla Unit Width3rd molar to 3rd molar Maxilla Unit Width 2nd molar to 2nd molar Maxilla Unit Width1st molar to 1st molar Maxilla Unit WidthCuspid to Cuspid Maxilla Unit WidthCentral to Central Maximum Maxilla Depth Anterior Arch of Atlas (AAA) to Maxilla Centrals Distance AAA to Posterior Maxilla Distance Average Left and Right Significant differences (p < 0.05) presented in bold. Definition of abbreviations: Diff. = adjusted difference (apneic normal); SE = standard error. not estimable due to small sample size (majority of subjects had missing molars). Table E15 summarizes the multiple logistic regression analyses for maxillary measurements. In men, there were no significant maxillary structures associated with increased risk of OSA before and after adjustment. In women, with 1SD increase in maxillary unit length (Vshape1 and Ushape1), widths between the left to right 1 st molar and between the left and right cuspid and mandibular depth (maximum maxilla depth and the distance from Anterior Arch of Atlas to Maxillary centrals), the 95% CIs were significantly larger than one. However, these structures were not associated with significantly increased odds ratios for OSA after adjusting for soft tissue volume or tongue volume. Table E15. Comparison of Odds Ratios in Maxillary Measurements Between Apneic and Normal Subjects, Stratified by Gender Maxillary Measurements (mm) SD Unadjusted Age, Height, Volume of Total Soft Tissue Age, Height, ANB Angle, Volume of Fat Pad, Volume of Tongue Age, Height, ANB Angle, Volume of Total Soft Tissue OR 95% CI OR* 95% CI OR* 95% CI OR* 95% CI Men Maxilla Unit Length (V) Average Left and Right