Abstract
Background
Primary ciliary dyskinesia (PCD) is a rare disease caused by mutations in >50 genes that impair the function of motile cilia. The clinical phenotype is heterogeneous and recent studies have begun to investigate genotype–phenotype relationships to better understand disease pathogenesis and develop improved treatments. The major cause of morbidity and mortality among individuals with PCD is the lack of mucociliary clearance (MCC) that results in chronic respiratory infections and leads to bronchiectasis. Here we examine the relationship between MCC and genotype in two groups of PCD individuals; one with mutations in a gene (DNAH5) that causes PCD with mostly immotile cilia and one with mutations in a gene (RSPH1) that cause PCD with cilia that beat with a near-normal frequency, but an abnormal, sometimes circular waveform.
Methods
Patients with known pathogenic variants in DNAH5 (n=8) or RSPH1 (n=7), along with healthy controls (n=8), were assessed for clearance of an inhaled radioactive tracer by mucociliary and cough clearance as measured by gamma scintigraphy.
Results
Neither DNAH5 nor RSPH1 subjects showed clear evidence of MCC under either baseline or albuterol stimulated conditions. Unexpectedly, subjects with RSPH1 mutations demonstrated cough clearance (median 9.7%, IQR 6.2–17%) that was significantly higher than subjects with DNAH5 mutations (4.2% (0.94–5.1%); p=0.015) and was not significantly different from healthy control subjects (8.3% (4.2–16%); p=0.88).
Conclusions
The results confirm impaired MCC in people with PCD of both genotypes. However, in this small cohort, the results suggest cough clearance may differ between these two genotypes.
Shareable abstract
PCD patients with DNAH5 mutations had low levels of both mucociliary and cough clearance; those with mutations in RSPH1 had low levels of mucociliary clearance but, on average, exhibited near-normal levels of cough clearance https://bit.ly/4580LyZ
Introduction
Primary ciliary dyskinesia (PCD) is a rare heterogenous, genetic disorder that results from mutations that impair the function of motile cilia. PCD occurs with an estimated global prevalence of 1:10 000 to 1:30 000, although a recent study suggests the prevalence may be as high as 1:7500 individuals [1, 2]. Motile cilia play a key role in multiple organ systems, including the upper and lower respiratory tract, the middle ear, the ventricles of the brain and the reproductive system [3, 4]. Consequently, individuals with PCD suffer from chronic bronchitis, sinusitis, otitis media and reduced fertility, particularly in male subjects. During development, motile cilia present at the embryonic node play a critical role in left–right patterning. As a result, mutations that impair nodal cilia function also cause situs inversus totalis and other laterality defects, including congenital heart defects [5]. The clinical phenotype of PCD is variable, with individuals experiencing a range of disease severity [6, 7]. Although PCD affects multiple organs, the major cause of morbidity and mortality is due to impaired MCC that causes recurring and chronic respiratory infection leading to bronchiectasis and in severe cases, lung failure [1].
PCD is usually inherited in an autosomal recessive manner, and is heterogenous at the genetic level, with causative mutations identified in over 50 different genes [1, 8, 9]. However, the relationship between genotype and phenotype in PCD has not yet been well defined. Recent studies have reported associations between specific genotypes and more or less severe phenotypes. For example, PCD patients with disease-causing mutations in CCDC39 or CCDC40 exhibit a more severe pulmonary phenotype with lower forced expiratory volume in 1 s (FEV1) and a higher incidence of bronchiectasis [3, 6, 10, 11]. Individuals with mutations in CCNO have also been reported to have a lower FEV1 [10]. In contrast, patients with mutations in DNAH11 or ODAD1 have a less severe pulmonary phenotype [10]. Interestingly, patients with mutations in RSPH1 have also been reported to have a less severe pulmonary phenotype, along with a lower incidence of neonatal respiratory distress, later onset of daily cough, and higher nasal nitric oxide (nNO) levels, compared with PCD patients with mutations in other genes [12, 13]. Cilia from individuals with mutations of RSPH1 and other radial spoke proteins display a variety of waveforms, including a circular pattern when viewed from above [12, 14–16] and this aberrant waveform is replicated in mouse models [17, 18]. Interestingly, Rsph1−/− mice demonstrated evidence of mucociliary clearance (MCC) in the nasal cavity up to ∼1 month of age, compared with Dnaic1−/− mice that exhibited no MCC [18].
MCC is an innate protective airway mechanism essential to maintain airway health, and impaired MCC underlies much of the disease pathogenesis of PCD [19, 20]. A number of laboratories have established procedures for measuring pulmonary MCC. In most studies, subjects inhale an aerosol containing radioactively labelled particles of a defined size (e.g. Tc99m-labelled sulfur colloid). The deposition of particles in the lung is then quantified by use of a gamma camera, and the disappearance of the tracer is followed over time. Typically, images are collected every few minutes for a period of 1–24 h. As the tracer particles are too large to be absorbed, the loss of radioactivity from the lungs is taken as a measure of MCC. Although the basic procedure for measuring pulmonary MCC is straightforward, the results can be influenced by many factors, including the initial deposition pattern of the particles (large airways clear faster than smaller airways) and the occurrence of cough, whether spontaneous or voluntary [21]. Thus, methods for taking these variables into account have been described [22, 23].
The direct measurement of MCC has been utilised primarily in research centres to understand the pathophysiology and the effect of treatments for several respiratory diseases including cystic fibrosis (CF), COPD and PCD [22, 24, 25]. Furthermore, MCC has also been studied as a potential diagnostic tool by observing diminished MCC in patients with PCD [26, 27], and will likely be an important measure of efficacy in clinical trials of PCD therapeutics.
Previous MCC studies have described severely impaired MCC in PCD subjects compared with healthy controls; however, these studies have not extensively investigated the effect of genotype [26–29]. A recent retrospective study by Marthin et al. [29] reported the absence of MCC in 49 PCD subjects with 23 different genotypes, except for one subject with homozygous p.His154Pro mutations in CCDC103. CCDC103 is expressed at relatively low levels in the axoneme [30], and the p.His154Pro mutation results in a variable ciliary phenotype, often resulting in near-normal ciliary activity [31]. We hypothesise that there is a range of MCC that may be seen in PCD subjects, dependent on the particular gene mutated and on the specific type of mutation (e.g. loss of function compared with hypomorph). Here we investigated whether PCD subjects with mutations in RSPH1 may exhibit residual MCC as a result of the high levels of ciliary activity often observed in these subjects [12, 32]. We compared MCC in PCD subjects with RSPH1 mutations to PCD subjects with mutations in DNAH5 and healthy controls. We also investigated the effect of a short-acting β agonist (SABA) on clearance, as stimulation of ciliary beat frequency in PCD subjects with residual motility may be beneficial [33, 34]. In addition, many PCD individuals are routinely treated with SABAs in an attempt to improve lung function [35, 36] but with the potential for enhancing MCC or cough clearance (CC) as observed in COPD [33, 34]. Finally, we measured the effect of controlled CC, as this is an important clearance mechanism for PCD subjects.
Methods
We performed a cross-sectional study to measure MCC and CC in two cohorts of PCD subjects and a group of healthy controls (HCs). An experimental sub-study to determine the effect of β agonist treatment on MCC was also conducted.
Confirmed PCD subjects with mutations in RSPH1 or DNAH5, along with age-matched HCs between 12 and 90 years old were recruited under protocols approved by UNC IRB (Clinical Trial NCT04901715). All PCD subjects met the diagnostic criteria for PCD published by the American Thoracic Society [37], having bi-allelic pathogenic or likely pathogenic variants in a PCD-associated gene with a compatible clinical phenotype. Informed consent was obtained from each subject before enrolment. Exclusion criteria included an FEV1 below 30% of per cent predicted (ppFEV1) or a sino-nasal pulmonary exacerbation in the past 4 weeks. Complete eligibility criteria are provided in supplementary data. The screening included a basic physical examination, vital signs and baseline spirometry. For safety and confounding rationale (i.e. potential effect of agonists on MCC) subjects were asked to stop inhaled bronchodilators 12 h prior to the MCC studies; hypertonic saline and pulmozyme were stopped 24 h prior to the start of the study. Eleven subjects withheld the use of bronchodilators, 10 withheld hypertonic saline, two withheld pulmozyme and three did not report using these medications. A full listing can be found in supplementary table S1. A total of 23 subjects were studied.
MCC and CC were measured by a well-established procedure in which subjects inhaled a tracer compound (Tc99m-labelled sulfur colloid) that deposits in the bronchial airways [21, 23]. In brief, subjects inhale tracer particles using a “slow inhalation-large particle” technique designed to increase deposition in the bronchial airways. Before each measurement of clearance, a Co57 transmission scan was performed to define lung regions (central and peripheral) which were used for subsequent analyses of regional deposition and clearance. The clearance of the tracer was measured using a planar gamma camera. Serial images (2 min) were captured and used to determine retention at 10-min intervals. Figure 1a illustrates the measured order of clearance after deposition of tracer. All subjects were instructed to suppress voluntary cough throughout the initial measurements of MCC. Baseline MCC was measured over a 60-min period. The average per cent clearance from 0–60 min (Ave60Clr) was determined by averaging the 10-min clearance values from 0–60 min for each subject as previously described in detail [23]. Thus, our primary outcomes have a single value per subject over a given period, e.g. 60 min for Ave60Clr. A SABA (albuterol) was then administered (four puffs, 90 μg per puff via metered dose inhaler with spacer) and FEV1 was measured. Clearance was again measured over the next 60 min as described above. Controlled coughs were then performed by each subject for a total of 30 coughs (10 coughs×3). Mean peak flow rate was measured during the controlled cough period [38]. Finally, a 15-min scan was obtained at 6 h from the start of the acquisition to measure long-term retention of labelled colloid.
FIGURE 1.
a) Schematic of the lung clearance study. After obtaining pulmonary function testing (PFT) and a transmission scan of the lung, subjects inhaled the radioactive tracer at t=0. Clearance of radioactive particles was monitored at different time periods over the next 6 h. b) Median whole-lung retention in primary ciliary dyskinesia (PCD) subjects and healthy controls. Graph shows overall retention data for healthy controls, RSPH1 and DNAH5 subjects. Administration of albuterol at 60 min (short-acting β agonist; SABA) and initiation of controlled cough (cough) at 120 min are indicated. Details can be found in the text. MCC: mucociliary clearance.
Statistical analysis
The predefined primary analysis was a comparison of whole-lung Ave60Clr (MCC), Ave 60-120Clr (post-bronchodilator MCC) and Ave120-150Clr (CC) between the three cohorts. Results were analysed using the nonparametric Kruskal–Wallis test. Results are presented as median and interquartile range except where noted. Post hoc tests of comparisons between groups were performed without p-value adjustment given the small cohort of subjects using Dunn's multiple comparisons test. A Wilcoxon matched-pairs signed-rank test was used to compare differences within groups (e.g. before and after albuterol). Correlations between continuous measures were calculated and the Spearman correlation coefficient and associated p-value are reported. Statistical analysis was performed using Excel and GraphPad Prism.
Results
Patient demographics
The study was designed to compare lung clearance parameters in subjects with disease-causing mutations in RSPH1 to subjects with mutations in DNAH5. For comparison, we also included a separate group of HCs. After screening, 23 subjects were included in the study: seven subjects with RSPH1 variants, eight with DNAH5 variants and eight HCs. Patient characteristics including age, sex, situs, nNO, ppFEV1 and z-score are presented in table 1. The specific mutations identified in these subjects are detailed in supplementary table S1. All patients were diagnosed according to the Official American Thoracic Society Clinical Practice Guidelines [37].
TABLE 1.
Clinical characteristics and demographics of study subjects
| Study ID | Gene | Age at visit, years | Sex | Situs | nNO# | ppFEV1 | FEV1 Z-score |
|---|---|---|---|---|---|---|---|
| PCD-001 | RSPH1 | 29 | F | SS+ | 69 | 63 | −3.25 |
| PCD-004 | RSPH1 | 18 | F | SS | 171 | 67 | −3.13 |
| PCD-006 | RSPH1 | 73 | M | SS | 434 | 38 | −4.05 |
| PCD-008 | RSPH1 | 13 | F | SS | 161 | 92 | −0.64 |
| PCD-015 | RSPH1 | 15 | F | SS | 30 | 82 | −1.33 |
| PCD-016 | RSPH1 | 15 | M | SS | NA | 90 | −0.76 |
| PCD-018 | RSPH1 | 24 | M | SS | (low normal) | 46 | −3.92 |
| PCD-002 | DNAH5 | 27 | M | SI | 12 | 79 | −1.76 |
| PCD-003 | DNAH5 | 17 | M | SI | 7 | 76 | −2.2 |
| PCD-010 | DNAH5 | 20 | F | SI | 20 | 114 | 1.33 |
| PCD-011 | DNAH5 | 14 | M | SS | 21 | 70 | −2.53 |
| PCD-012 | DNAH5 | 17 | M | SS | 27 | 103 | 0.28 |
| PCD-009 | DNAH5 | 20 | M | SI | 202 | 74 | −2.57 |
| PCD-013 | DNAH5 | 23 | M | SS§ | 183 | 96 | −0.35 |
| PCD-014 | DNAH5 | 21 | M | SS | 191 | 95 | −0.45 |
| PCD-02C | Control | 29 | M | SS | NA | 112 | 1.13 |
| PCD-03C | Control | 19 | F | SS | NA | 83 | −1.61 |
| PCD-04C | Control | 23 | F | SS | NA | 122 | 2.07 |
| PCD-05C | Control | 24 | M | SS | NA | 116 | 1.53 |
| PCD-06C | Control | 20 | M | SS | NA | 112 | 1.2 |
| PCD-07C | Control | 26 | F | SS | NA | 112 | 1.12 |
| PCD-08C | Control | 25 | M | SS | NA | 100 | 0 |
| PCD-09C | Control | 57 | M | SS | NA | 110 | 0.77 |
nNO: nasal nitric oxide; ppFEV1: forced expiratory volume in 1 s % predicted; F: female; M: male; SS: situs solitus; SI: situs inversus totalis; NA: not available; #: nL·min−1. +: Right middle and lower lobectomy; §: Right middle lobectomy.
There was no significant difference in average age between groups (RSPH1=26.7, DNAH5 19.9, HC=27.9; p=0.18) (table 1). As expected, average ppFEV1 of both PCD groups was significantly lower than the HC group (RSPH1, 68%; DNAH5, 88%; HC, 108%; p<0.05 for both groups) but was not significantly different between RSPH1 and DNAH5 subjects (p=0.13). nNO levels, on average, were higher in the RSPH1 cohort (172 nL·min−1) compared with the DNAH5 group (83 nL·min−1) as previously reported [12], but levels were highly variable among the individual subjects. Three of six RSPH1 subjects had values well above the value of 77 nL·min−1 recommended as a cut-off value for PCD screening [39], whereas two other subjects (PCD-001 and PCD-018) had values near this cut-off. In contrast, five of eight DNAH5 subjects had uniformly low nNO values (mean 17 nL·min−1), whereas three subjects with predicted missense mutations (PCD-009, −013, −014; supplementary table S1) had levels of nNO well above the value of 77 nL·min−1 (mean 191 nL·min−1).
Whole-lung MCC clearance
Figure 1b shows the median overall retention for the three cohorts. As expected, the baseline whole-lung MCC (where % clearance=(1−retention)×100) was higher in the HC group compared with the PCD groups (table 2 and figure 2a; Ave60Clr). The HC group had a median Ave60Clr of 7.3%, compared with the DNAH5 group (Ave60Clr=−1.1%; p<0.002) and the RSPH1 group (Ave60Clr=1.4%; p=0.054). Baseline MCC in the RSPH1 group was not significantly different from the DNAH5 group (p=0.27; table 2).
TABLE 2.
Whole-lung clearance data in healthy controls, DNAH5 and RSPH1 subjects
| Percent clearance | p-value | ||||
|---|---|---|---|---|---|
| Genotype | Median (interquartile range) | HC versus DNAH5 | HC versus RSPH1 | RSPH1 versus DNAH5 | |
| Baseline MCC Ave60Clr | Control | 7.3 (4.9–9.1) | 0.002 | 0.054 | 0.27 |
| DNAH5 | −1.1 (−1.3–2.7) | ||||
| RSPH1 | 1.4 (−0.42–2.6) | ||||
| MCC with albuterol Ave60-120Clr | Control | 9.7 (1.8–12) | 0.014 | 0.066 | 0.58 |
| DNAH5 | −0.42 (−1.4–2.5) | ||||
| RSPH1 | 0.82 (−0.53–1.9) | ||||
| Cough clearance Ave120-150Clr | Control | 8.3 (4.2–16) | 0.018 | 0.88 | 0.015 |
| DNAH5 | 4.2 (0.94–5.1) | ||||
| RSPH1 | 9.7 (6.2–17) | ||||
HC: healthy control; MCC: mucociliary clearance. A negative clearance value can result from labelled tracer moving toward or away from the gamma camera, the subject shifting position in relation to the camera, or potentially from labelled tracer moving caudally.
FIGURE 2.
Whole-lung clearance in healthy controls (HCs), DNAH5 and RSPH1 cohorts at a) baseline, b) following albuterol stimulation and c) during controlled cough. DNAH5 and RSPH1 subjects show little mucociliary clearance (a,b), but RSPH1 subjects show a significant level of cough clearance that is similar to HCs (c). Box-and-whisker plot show median and interquartile range; bars show maximum and minimum values. *: p<0.05; **: p<0.005.
Whole-lung MCC in HCs increased after administration of albuterol from 7.3% at baseline to 9.7% after albuterol, but this increase was not significant (p=0.46) (table 2 and figure 2b). The median MCC in the RSPH1 and DNAH5 groups did not change significantly following administration of albuterol. Again, there was no significant difference in whole-lung MCC when comparing DNAH5 to RSPH1 subjects (p =0.58).
Subjects were instructed to suppress coughing during the above measurements of MCC. Somewhat surprisingly, the PCD subjects spontaneously coughed little before the controlled cough period (average coughs during the first 120 min was 0.67; range 0–3). During the measurement of CC, 11 of 15 PCD subjects coughed 30 times. Additional spontaneous coughs were recorded for two DNAH5 subjects (31 and 32) and two RSPH1 subjects (34 and 46). Interestingly, during the controlled cough period, median whole-lung CC (Ave120-150Clr) was almost identical in the healthy control and RSPH1 subjects (8.3% versus 9.7%; p=0.88) (table 2 and figure 2c). In contrast, the median whole-lung CC was substantially lower in the DNAH5 subjects when compared with HCs (4.2%; p=0.018). Importantly, whole-lung CC in the RSPH1 cohort was also substantially higher than in the DNAH5 group (9.7% versus 4.2%; p=0.015). Although most of the subjects were able to limit coughing to 30 voluntary coughs during the measurement period, one of the RSPH1 subjects (PCD-006) spontaneously coughed an additional 16 times during the CC measurement and exhibited the highest CC (20.7%). Removing this subject from the analysis reduced the median CC (Ave120-150Clr) in the RSPH1 group from 9.7% to 8.2%, which was not significantly different from HC CC (p=0.89), but remained significantly higher than the DNAH5 group (p=0.036). Spearman correlation coefficients were calculated between CC and peak flow. The results (r=0.11, p-value=0.609) indicate that there was no correlation between CC and peak flow in these subjects (supplementary figure S1). Further, there was no obvious relationship between CC and FEV1 (Spearman correlation, r=0.082) and only a weak correlation between CC and age (Spearman, r=0.43).
Regional lung clearance
To analyse clearance more specifically from the central and larger airways, the Co57 transmission scan was used to define central and peripheral regions of the lung [23]. The central to peripheral deposition (C/P) was not significantly different between the three groups (mean C/P; HC=2.0±0.5; DNAH5=1.7±0.2; RSPH1=2.0±0.3; p=0.17).
Central lung clearance
Clearance from the central region followed the same pattern as whole lung clearance (table 3 and figure 3). In HCs, clearance was the same or higher in the central region compared with whole lung clearance during all three study intervals (baseline 9.8% versus 7.3%; p=0.016; albuterol stimulated 9.4% versus 9.47%; p=0.25; CC, 14% versus 8.3%; p=0.02). Neither the RSPH1 nor DNAH5 subjects showed an increase in baseline or albuterol stimulated clearance in the central region compared with whole lung clearance (table 3 and figure 3a,b). However, similar to the HC group, CC in the RSPH1 subjects was increased when measured in the central lung region compared with the whole-lung measurement (18% versus 9.7%; p=0.16). CC in the RSPH1 cohort was similar in magnitude to the HCs (18% versus 14%, table 3 and figure 3c) and was not significantly different (p=0.82). In contrast, both the HCs (14%; p=0.011) and the RSPH1 (18%; p=0.026) cohort had significantly higher CC compared with the DNAH5 (0.86%) subjects.
TABLE 3.
Central lung clearance data in healthy controls, DNAH5 and RSPH1 subjects
| Percent clearance | p-value | ||||
|---|---|---|---|---|---|
| Genotype | Median (interquartile range) | HC versus DNAH5 | HC versus RSPH1 | RSPH1 versus DNAH5 | |
| Baseline MCC Ave60Clr | Control | 9.8 (7.4–14) | 0.0006 | 0.001 | 0.94 |
| DNAH5 | −0.15 (−3.1–2.6) | ||||
| RSPH1 | 0.35 (−3.8–2.9) | ||||
| MCC with albuterol Ave60-120Clr | Control | 9.4 (4.2–17) | 0.007 | 0.029 | 0.67 |
| DNAH5 | −4.6 (−7.9–(−0.31)) | ||||
| RSPH1 | −0.22 (−5.6–4.7) | ||||
| Cough clearance Ave120-150Clr | Control | 14 (8.3–29) | 0.011 | 0.815 | 0.026 |
| DNAH5 | 0.86 (−3.9–6.5) | ||||
| RSPH1 | 18 (−1.1–32) | ||||
HC: healthy control; MCC: mucociliary clearance. A negative clearance value can result from labelled tracer moving toward or away from the gamma camera, the subject shifting position in relation to the camera, or potentially from labelled tracer moving caudally.
FIGURE 3.
Central lung clearance in healthy controls (HCs), DNAH5 and RSPH1 cohorts at a) baseline, b) following albuterol stimulation and c) during controlled cough. DNAH5 and RSPH1 subjects show little mucociliary clearance (a,b), but RSPH1 subjects show a significant level of cough clearance that is similar to HCs (c). Box-and-whisker plot show median and interquartile range; bars show maximum and minimum values. *: p<0.05;**: p<0.005; ***: p<0.001.
Peripheral lung clearance
Clearance from the peripheral region was generally lower compared with clearance measured from the whole or central lung regions, likely reflecting the deposition of labelled tracer into alveolar and small airway regions with little MCC and less effective CC. There was no significant difference in the average baseline peripheral MCC between the three groups, although clearance trended higher in the HC group (table 4 and figure 4a). Similar to whole lung and central clearance, albuterol slightly increased MCC clearance in the HC group (9.3% versus 5.6%), and again, trended higher compared with the RSPH1 group (−0.07%; p=0.047) and the DNAH5 group (1.7%; p=0.47) (table 4 and figure 4b). CC from the peripheral region was similar between the three groups, with no significant differences (table 4 and figure 4c).
TABLE 4.
Peripheral lung clearance data in healthy controls, DNAH5 and RSPH1 subjects
| Percent clearance | p-value | ||||
|---|---|---|---|---|---|
| Genotype | Median (interquartile range) | HC versus DNAH5 | HC versus RSPH1 | RSPH1 versus DNAH5 | |
| Baseline MCC Ave60Clr | Control | 5.6 (3.1–6.9) | 0.08 | 0.53 | 0.28 |
| DNAH5 | 0.77 (−2.6–4.4) | ||||
| RSPH1 | 2.9 (−2.1–13) | ||||
| MCC with albuterol Ave60-120Clr | Control | 9.3 (−1.1–13) | 0.24 | 0.06 | 0.47 |
| DNAH5 | 1.7 (0.99–4.0) | ||||
| RSPH1 | −0.07 (−1.8–3.8) | ||||
| Cough clearance Ave120-150Clr | Control | 3.5 (1.7–13) | >0.99 | 0.72 | 0.72 |
| DNAH5 | 4.2 (3.0–8.6) | ||||
| RSPH1 | 4.7 (0.37–7.4) | ||||
HC: healthy control; MCC: mucociliary clearance. A negative clearance value can result from labelled tracer moving toward or away from the gamma camera, the subject shifting position in relation to the camera, or potentially from labelled tracer moving caudally.
FIGURE 4.
Peripheral lung clearance in healthy controls (HCs), DNAH5 and RSPH1 cohorts at a) baseline, b) following albuterol stimulation and c) during controlled cough. Peripheral clearance was reduced compared with whole lung or central clearance and was not significantly different between the groups. Box-and-whisker plot show median and interquartile range; bars show maximum and minimum values.
Discussion
PCD is a heterogeneous disease at the level of both genotype and phenotype. Thousands of pathogenic mutations, including missense, nonsense, splicing variants and deletions, have been identified in over 50 different genes, and additional mutations will undoubtedly be discovered [2, 8–10]. The clinical phenotype is also variable, with most individuals suffering from chronic upper and lower respiratory infections, persistent middle ear effusions and otitis media, and subfertility [1]. Pulmonary disease is responsible for the majority of the morbidity and mortality among individuals with PCD, and this is presumed to be due to the lack of effective MCC. Several previous studies of subjects with PCD have reported essentially no MCC, and measurement of MCC has been proposed as a useful diagnostic tool [26, 27]. Strategies to improve or restore MCC are the goal of several therapeutic approaches, and it is important to determine the level of MCC required to improve clinical outcomes [40].
Recent studies have begun to explore the relationships between genotype and phenotype to further our understanding of disease pathogenesis and develop improved treatments for this rare disease. We previously provided evidence that subjects with mutations in RSPH1 may, on average, have a less severe phenotype, as evidenced by a lower incidence of neonatal respiratory distress, a later onset of daily wet cough, better lung function and higher nNO values [12]. However, this previous study compared subjects with RSPH1 mutations with all other PCD subjects, including those with CCDC39/CCDC40 mutations, which are now known to have more severe disease, and may have accounted for the observed difference in this study. Other studies have not found a significant difference in lung function comparing RSPH1 subjects with PCD subjects with other genotypes [10, 11]. However, it is worth noting that the median age and ppFEV1 of RSPH1 subjects in the study by Raidt et al. [10] were higher (23.4 versus 14.7 years old; 87% versus 79.8%, respectively), compared with the overall cohort. RSPH1 mutant cilia often have a beat frequency that is near normal [12, 14, 15] and display a heterogeneous waveform, sometimes beating in a circular pattern, similar to nodal cilia. It is worth noting that nodal cilia, which lack a central pair and radial spokes, generate a leftward flow that determines laterality. Thus, it is possible that the RSPH1 mutant cilia maintain a low level of MCC sufficient to modify the disease phenotype. Indeed, neonatal Rsph1−/− mice demonstrated a low level of MCC in the nasopharynx [18]. However, this low level of MCC was lost by ∼3 weeks of age.
To test the hypothesis that PCD subjects with RSPH1 mutations may have a residual level of MCC that could be responsible for their apparently less severe phenotype, we measured MCC directly in a group of RSPH1 subjects. For comparison, we also measured MCC in a group of PCD subjects with DNAH5 mutations, along with HCs.
Our results show that both the RSPH1 and DNAH5 subjects exhibit little to no MCC, either at baseline or following albuterol stimulation. These results agree with a retrospective study by Marthin et al. [29] who reported extremely low or unmeasurable MCC in a group of PCD subjects, with no significant differences between genotypes (including one subject with RSPH1 mutations). As expected, the HC group showed a significantly higher level of baseline MCC that increased following albuterol stimulation. These results agree with previous studies of MCC in PCD patients and controls [26–29, 35]. Although the absence of MCC differs from the finding in the neonatal mouse model, it is not yet practical to measure MCC in infants. Intriguingly, the youngest RSPH1 subject in our study (table 1; PCD-008), had a baseline whole-lung MCC of 8.9%, which was higher than the median clearance of the HC group (7.3%). As noted above, RSPH1 subjects were reported to have a lower incidence of neonatal respiratory distress and a later onset of daily wet cough. These data may suggest that a low level of MCC is capable of modifying the early disease phenotype but is not sufficient to maintain clearance and alter the overall progression of disease.
While impaired MCC is a well-established feature of PCD, we also observed a substantial reduction of CC in DNAH5 subjects, compared with HCs. This reduction was observed in subjects with two loss-of-function mutations, as well as three subjects with missense mutations and relatively high levels of nNO and preserved lung function (table 1; PCD-009, PCD-013 and PCD-014). To our knowledge, this is the first observation of impaired CC in PCD subjects. The concentration of mucus in sputum samples from people with PCD has been reported to reach values predicted to impede both MCC and CC [41]. Surprisingly, the RSPH1 subjects demonstrated substantially higher CC compared with the DNAH5 subjects. CC in the RSPH1 group was not different from the HC group, when measured from the whole lung or when measured in the central region. While the mechanisms regulating the integrated process of cough and MCC are complex, our observation of preserved CC in RSPH1 subjects may be explained by several possibilities. First, based in part on the demonstration of low levels of MCC in the mouse model, it is possible that the ciliary activity in RSPH1 patients more actively moves deposited material from the distal airways into the larger central airways, where they are more easily cleared by cough. Second, based on the observation that normal ciliary activity can sense and respond to the hydration state of the mucus layer [42], it is possible that the ciliary activity in RSPH1 subjects maintains the ability to properly hydrate the mucus layer, thus improving CC. These two mechanisms are not mutually exclusive, and the improved CC observed may be the result of both. CC is an essential “back-up” mechanism of lung clearance and is especially important to PCD patients who have little or no MCC. In the absence of specific treatments that correct the underlying genetic defect, improving the clearance of airway secretions by cough may improve the clinical outcome of individuals with PCD. In a previous study, Noone et al. [25], demonstrated that aerosolised uridine-5′-triphosphate (UTP) was able to significantly improve CC in a group of PCD subjects, likely through its actions to stimulate Cl− secretion and mucin release by goblet cells. More recently, inhaled hypertonic saline co-administered with the sodium channel inhibitor (ENaC) idrevloride was shown to improve pulmonary function, likely by hydrating mucus in the PCD airway and enhancing CC [43]. Thus, further studies of CC and ways to improve CC in this study population are clearly warranted.
The major limitation of the current study is the small sample size. PCD is a rare disease, and because our study was focused on a specific genotype (i.e. RSPH1), the number of subjects available for study was further limited. Because of the limited size of our cohorts, post hoc tests of comparisons between groups were performed without p-value adjustment to prioritise power over adjustments of the p-value. In addition, the measurement of mucociliary and CC is dependent on a number of factors, including the deposition pattern of the radioactive tracer, the effort exerted during the controlled cough period and the existence and extent of lung disease. We observed no significant difference between the groups in the deposition pattern, no correlation between CC and average peak flow of controlled coughs, and all subjects performed the cough manoeuvre with no complications. However, the findings reported here require validation in additional studies.
In summary, our findings suggest that PCD patients with causative mutations in both DNAH5 and RSPH1 have little MCC. However, taking into account the limitations detailed above, we observed impaired CC in DNAH5 subjects, whereas CC appeared to be relatively preserved in subjects with RSPH1 mutations. In the absence of treatments to restore ciliary function in people with PCD, our results highlight the need for the development of effective mucolytic and/or hydrating agents to improve CC in PCD subjects and to include measurements of CC in future clinical trials.
Acknowledgements
The authors would like to acknowledge K. Sullivan and N. Capps (Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, NC, USA) for assistance with patient recruitment, and J. Wu (Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA) for technical assistance with the studies. We would also like to thank all the subjects who volunteered to be part of this study.
Footnotes
Provenance: Submitted article, peer reviewed.
This clinical trial is prospectively registered with ClinicalTrials.gov as NCT04901715.
Ethics statement: All study protocols were approved by the University of North Carolina Institutional Review Board.
Conflict of interest: All authors have confirmed that they have no conflicts of interest to declare.
Support statement: Funding for this work was provided by R01 2-R01-HL117836-06 and by US National Institutes of Health/Office of Rare Diseases Research (ORDR)/National Center for Advancing Translational Sciences (NCATS)/National Heart, Lung, and Blood Institute (NHLBI) grant U54HL096458. The Genetic Disorders of Mucociliary Clearance Consortium (U54HL096458) is part of the NCATS Rare Diseases Clinical Research Network (RDCRN), and supported by the RDCRN Data Management and Coordinating Center (U2CTR002818). RDCRN is an initiative of the ORDR funded through a collaboration between NCATS and NHLBI. Funding information for this article has been deposited with the Open Funder Registry.
Supplementary material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material
00681-2025.SUPPLEMENT
Data availability
We will share individual participant data that underlie the results reported in this article after deidentification (text, tables, figures and appendices). The study protocol, statistical analysis plan and analytic code will also be made available. They will be provided to anyone who wishes to access the data for any purpose beginning 6 months and ending 5 years following article publication. Requests for data should be directed to either L.E. Ostrowski (ostro@med.unc.edu) or W.D. Bennett (william_bennett@med.unc.edu).
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Supplementary Materials
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
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00681-2025.SUPPLEMENT
Data Availability Statement
We will share individual participant data that underlie the results reported in this article after deidentification (text, tables, figures and appendices). The study protocol, statistical analysis plan and analytic code will also be made available. They will be provided to anyone who wishes to access the data for any purpose beginning 6 months and ending 5 years following article publication. Requests for data should be directed to either L.E. Ostrowski (ostro@med.unc.edu) or W.D. Bennett (william_bennett@med.unc.edu).