Respiratory muscle weakness in facioscapulohumeral muscular dystrophy

2.1 Experimental study design

This cross-sectional case–control study was conducted from November 2017 to October 2018. Ethical approval was obtained from the local ethics committee (Ethikkommission der Ärztekammer Westfalen-Lippe und der WWU Münster, AZ 2016-072-f-S). It was part of a wider project investigating the nature of respiratory muscle strength and function in neuromuscular disorders and chronic obstructive pulmonary disease (ClinicalTrials.gov Identifier: NCT03032562).

2.2 Study population

Twenty consecutive patients with genetically proven FSHD from the neuromuscular outpatient clinic at Münster University Hospital were consecutively invited to participate in the study. Consent was given by 14 individuals. All patients carried a chromosomal rearrangement within the subtelomeric region of chromosome 4q35 referred to as the D4Z4 locus.15 Neurological impairment was assessed using the Clinical Severity Scale (CSS), which reflects weakness in various muscle groups, including the spread of symptoms to pelvic and proximal leg muscles, which unequivocally indicates disease progression.16 The CSS score ranges from 0.5 (“facial weakness”) to 5 (“wheelchair-bound”) (see Table S1 online). Clinical symptoms indicative of respiratory muscle weakness or sleep-disordered breathing were assessed using a validated German language questionnaire (Boentert et al, manuscript submitted for publication). Healthy control subjects were consecutively recruited and matched 1:1 for age (±5 years), gender, and body mass index (BMI; ±3 kg/m²). All participants gave written informed consent to participate in the study.

2.3 Spirometry, maximum inspiratory, and expiratory pressures

Lung function tests were performed according to standard recommendations using an electronic spirometer (Vitalograph 3000, Vitalograph, Hamburg, Germany).10 Participants were encouraged to provide a maximum effort during assessment of their FVC and forced expiratory volume in the first second (FEV₁) in the upright position. At least five consecutive tests were performed until the best result was achieved and showed less than 10% variation from the preceding test. FVC was expressed as percentage of the predicted value based on gender, height, and age.17 Maximum inspiratory and expiratory pressures (MIP and MEP, respectively) were obtained using a handheld electronic manometer equipped with a large-diameter flanged mouthpiece (MicroRPM, Care Fusion, Baesweiler, Germany). All participants performed at least three tests (with a 1-second plateau in 3- to 4-second efforts and a 1-minute pause between efforts). Subjects who failed to achieve less than 10% variability from the preceding test within the first 3 runs repeated the test until they were able to achieve more consistent results. MIP was measured first (from residual volume), and MEP was measured second (from total lung capacity), both with the subject sitting upright. All measurements were performed using a nasal clip to prevent air leakage.18

2.4 Diaphragm ultrasound

A portable ultrasound machine (Logiq S8-XD Clear, GE Healthcare, London, UK) with a 3.5-MHz convex transducer was used for assessment of diaphragm excursions in the subcostal view, and a 10-MHz linear transducer was used for evaluation of diaphragm thickness in the zone of apposition. Measurements were performed on the right hemidiaphragm in the supine position. All sonographic recordings were saved for later analysis. All measurements were performed three times and the average value for each parameter was calculated. For real-time evaluation of diaphragm excursions, the probe was positioned in the subcostal area between the midclavicular and anterior axillary line and directed cranially (Figure S1A online). Diaphragm excursion amplitude was assessed during tidal breathing (Figure S1A online), at total lung capacity (TLC), and after a voluntary sniff. Assessment of diaphragm excursion velocity was performed during tidal breathing and after a voluntary sniff maneuver only (Figure S1B online). Excursion amplitude was defined as the upright perpendicular distance from the minimum to the maximum point of diaphragm displacement, and excursion velocity was defined as the upright diagonal distance from the minimum to the maximum point.

Diaphragm thickness was measured as the vertical distance between the pleural and peritoneal layer at both TLC and functional residual capacity (FRC) (Figure S1C,D online). The 10-MHz probe was positioned in the posterior axillary line between the eighth and tenth intercostal space (Figure S1C online). Diaphragm thickness was defined as the distance from the inner part of the pleural layer to the inner part of the peritoneal layer, measured at its thickest portion adjacent to the lung. Diaphragm thickening ratio (DTR) was calculated as thickness at TLC divided by thickness at FRC.

2.5 Phrenic nerve conduction studies after posterior cervical magnetic stimulation

To exclude phrenic nerve dysfunction, diaphragm electrical activity was bilaterally recorded after posterior cervical magnetic stimulation. Surface electromyogram was recorded using a electromyography device (Dantec 2000, Dantec Medical, Skovlunde, Denmark) and surface electrodes were filled with electrode paste. Electrodes were placed in the seventh intercostal space approximately on the anterior axillary line with the reference electrode positioned cranially to the xiphoid process (16-cm interelectrode distance). The ground electrode was placed around the right wrist. Magnetic stimulation was performed with the subject in the seated position. After a 20-minute resting period with quiet breathing and no speaking, stimuli were delivered using a magnetic stimulator equipped with a 12-cm circular coil (C-100 MagPro Compact, MagVenture, Willich, Germany). Square-pulse stimulus duration was 0.1 milliseconds. Equal stimulus intensity was achieved by using maximum magnetic flux density (2 Tesla) for all stimulations. The coil was first placed at C7 and then moved up toward C6 until the highest reproducible diaphragm compound muscle action potential (CMAP) was obtained. For each run of tests, at least five stimuli were delivered to achieve the highest possible diaphragm CMAP amplitude showing less than 10% variation from the preceding two stimulations. To avoid twitch potentiation, stimuli were separated by at least 40 seconds. Stimulation at FRC was determined by visual observation of abdominal movements after instructing subjects to breathe out and hold their breath until the magnetic stimulus was delivered (the stimulus was then deployed within 1 second after FRC had been reached). Figure S2 (online) displays representative diaphragm CMAP values after CMS at FRC.

2.6 Invasive inspiratory muscle strength measurements

Twitch esophageal pressure (twPes) and twPgas were simultaneously obtained using balloon catheters (Cooper Surgical, Trumbull, Connecticut) transnasally inserted into the stomach and the distal esophagus.19 The esophageal and gastric balloons were filled with 1.0 mL and 2.5 mL of air, respectively. To ensure constant filling, both balloons were deflated and refilled after every run of tests. Balloon catheters were connected to a differential pressure transducer (DPT-100, Utah Medical Products, Athlone, Ireland) and a carrier amplifier (ADInstruments, Oxford, UK). Pressure data for twPgas, twPes, and twPdi (defined as twPes minus twPgas) were continuously displayed using LabChart software (ADInstruments) on a personal computer. Figure S3A (online) shows representative tracings of twPdi after CMS at FRC.

In addition to nonvolitional tests, subjects were instructed to perform consecutive maximum sniff maneuvers with a resting period of 5 minutes in between. Subjects were encouraged to achieve maximum deflection of the Pdi curve. After participants had learned and practiced the maneuver several times five measurements were recorded for each, and the highest value was taken for analysis. Figure S3B (online) shows representative pressure tracings after maximum voluntary sniff.

2.7 Invasive expiratory muscle strength measurements

As proposed by Polkey and coworkers,11 the abdominal nerve roots were magnetically stimulated at the 10th vertebrae with a clear instruction to go up and down the vertebral column (by no more than 2 vertebrae) to determine the optimal position where the highest reproducible diaphragm CMAP could be achieved. Abdominal muscle CMAPs were recorded bilaterally using surface electrodes placed in the anterior axillary line close to the lower costal margin. Stimulus duration was 0.1 millisecond. Stimulation intensity was 100% of the maximum magnetic output, as described above. Again, stimulation at FRC was determined by visual observation of abdominal movements. In addition, subjects were instructed to perform a voluntary maximum cough with a resting period of 5 minutes between different maneuvers. Gastric pressure during the cough maneuver and twPgas in response to magnetic stimulation of the abdominal muscles were recorded using a gastric balloon catheter and the same technical setup as described earlier. Representative pressure tracings are shown in Figure S3C and S3D (online).

2.8 Statistical analysis

All analyses were performed using SigmaPlot software version 13.0 (Systat Software, Erkrath, Germany). The data distribution was tested using the Kolmogorov–Smirnov test. Results are expressed as mean and standard deviation for continuous variables, and the t test for independent samples was used for between-group comparisons. For nonparametric data, the Mann–Whitney U test was used for comparison between groups as appropriate. For all analyses, P < .05 was considered statistically significant. GraphPad Prism 7 (GraphPad Software, San Diego, California) was used for graphical illustrations.

3.1 Subjects

Fourteen patients were enrolled in the study. Diagnosis of FSHD was genetically confirmed in all subjects, and exact contraction length of the D4Z4 repeat was known in 10 individuals. Fourteen matched control subjects (were also recruited (Table 1, and Table S2 online). Patients had a moderate to severe disease manifestation based on the CSS score (range, 1.0–5.0; median, 2.75; interquartile range [IQR], 1.50–3.38). Two patients required a wheelchair for ambulation. These two patients also had kyphoscoliosis. Four patients were receiving long-term non-invasive ventilation (NIV) during the night for known sleep-related hypoventilation, as reflected by nocturnal hypercapnia (transcutaneous carbon dioxide tension >50 mmHg or increase from awake baseline >10 mm Hg). Weakness of the lateral abdominal wall was observable in five individuals. According to the respiratory symptom questionnaire, exertional dyspnea was the most common complaint among FSHD patients (n = 9/14), followed by speech dyspnea (n = 6/14), non-restorative sleep (n = 5/14), sleep disturbances (n = 4/14), and daytime sleepiness (n = 3/14). The sum score derived from this questionnaire did not correlate with FVC and MIP, respectively (data not shown).

3.2 Spirometry and maximum inspiratory and expiratory pressures

Compared with controls, FSHD patients showed significantly lower FVC, MIP, MEP, and PCF (Table 1 and Figure 1). FVC correlated inversely with clinical disease severity (as reflected by the CSS score) (Figure 2). When patients receiving nocturnal NIV were compared with patients without home ventilatory support, no differences were found between the two groups. Among FSHD patients, bedside tests of respiratory muscle function (FVC, MIP, MEP and PCF) did not correlate with the length of the D4Z4 contraction (data not shown).

3.3 Diaphragm ultrasound

Diaphragm excursion amplitude during deep breathing and DTR were reduced in FSHD patients compared with controls (Table 2). Reduction of either DTR or diaphragm excursion amplitude during deep breathing was found in FSHD patients with and without NIV (Figure 3). MIP showed significant negative correlation with DTR and diaphragm excursion amplitude derived from ultrasound (Figure 3B,C). No correlation was found between clinical disease severity (CSS score) and diaphragm excursion amplitude or DTR, respectively.

3.4 Phrenic nerve conduction studies

Reproducible diaphragm CMAP could be obtained in all study participants, and no side-to-side difference was found with regard to latency or amplitude in those with FSHD or healthy controls (Table 3). Analysis of CMAP latencies and amplitudes revealed no abnormalities (Table 3).

3.5 Invasive inspiratory muscle strength measurements

Transnasal insertion of balloon catheters was declined by 4 patients with FSHD, leaving 10 patients and 10 control subjects for group comparison of invasive pressure recordings. The twPdi was lower in the FSHD patients compared with controls (Table S3 online, and Figure 4A). Both twPdi and sniffPdi did not differ between patients with (n = 4) and without (n = 6) NIV (Table S2 online). No correlation was found between clinical disease severity (CSS score) and invasively obtained inspiratory muscle strength measures, and neither MIP nor FVC were significantly correlated with twPdi (data not shown). SniffPdi and twPdi showed a statistical correlation in control subjects (r = 0.77; P < .05), but not in FSHD patients. CoughPgas and twPgas after stimulation at Th10 were not associated in both groups (data not shown). FSHD patients were dichotomized for the median MIP and sniffPdi value, and individual patients with more severe inspiratory muscle weakness reported sleep disturbances, exertional dyspnea, and weakness of cough more frequently than patients with better inspiratory muscle function (data not shown).

3.6 Invasive expiratory muscle strength measurements

After magnetic stimulation of the abdominal muscles, mean twPgas was lower in FSHD patients compared with healthy controls (Table S2 online, and Figure 4B). Pgas values after maximum voluntary cough (coughPgas) showed no differences between groups. Again, twitch Pgas did not differ between patients with and without nocturnal NIV. CoughPgas, twPgas, and MEP were not related to the CSS score (data not shown).