Primary Ciliary Dyskinesia Subjects Research Articles

TAS2R38 Genotype Does Not Affect SARS-CoV-2 Infection in Primary Ciliary Dyskinesia.

Several chronic respiratory diseases could be risk factors for acquiring SARS-CoV-2 infection: among them, Primary Ciliary Dyskinesia (PCD) is a rare (about 1:10.000) inherited ciliopathy (MIM 242650) characterized by recurrent upper and lower respiratory tract infections due to a dysfunction of the respiratory cilia. In this study, we aimed to investigate whether PCD subjects are more susceptible to infection by SARS-CoV-2 and whether some polymorphisms of the TAS2R38 bitter taste receptor correlate with an increased prevalence of SARS-CoV-2 infection and severity of symptoms. Patients answered several questions about possible SARS-CoV-2 infection, experienced symptoms, and vaccinations; in the case of infection, they also filled out a SNOT-22 questionnaire and ARTIQ. Forty PCD adult patients (mean age, 36.6 ± 16.7 years; 23 females, 17 males) participated in this study, out of which 30% had tested positive for COVID-19 during the last four years; most of them reported a mildly symptomatic disease. We found no differences in age or sex, but a statistically significant difference (p = 0.03) was observed in body mass index (BMI), which was higher in the COVID-acquired group (23.2 ± 3.3 vs. 20.1 ± 4.1 kg/m2). Genotyping for TAS2R38 polymorphisms showed a prevalence of 28.6% PAV/PAV, 48.6% PAV/AVI, and 22.8% AVI/AVI individuals in our cohort. In contrast to our hypothesis, we did not observe a protective role of the PAV allele towards SARS-CoV-2 infection. Conclusions: Our findings suggest that subjects with PCD may not be at increased risk of severe outcomes from COVID-19 and the TAS2R38 bitter taste receptor genotype does not affect SARS-CoV-2 infection.

The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8Hz versus control = 15.8Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X.laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results inPCD.

The effect of l-Arginine on Ciliary Beat Frequency in PCD patients, non-PCD respiratory patients and healthy controls

ObjectivesFew studies have examined the potentially therapeutic effect of increasing the production of endogenous nitric oxide (NO) in Primary Ciliary Dyskinesia (PCD) and other chronic respiratory conditions. Nasal NO is low in PCD and has been found to correlate with compromised Ciliary Beat Frequency (CBF). In this study we assessed the effect of increasing l-Arginine, as the substrate of NO synthases, on CBF in biopsies of human respiratory ciliated epithelium. MethodologyA total of 28 suspect cases with chronic respiratory manifestations referred for PCD diagnostic testing and 8 healthy controls underwent nasal brushing. Obtained epithelial cells were divided between three culture medium 199 solutions, containing different levels of l-Arginine (0.33 mM as baseline, 1 mM and 10 Mm as increased levels). CBF measurements were obtained at 37 °C and 25 °C at 1, 3 and 24 h after sample acquisition. ResultsAmong a total of 36 recruited subjects, 8 had PCD confirmed (PCD n = 8), 20 had PCD excluded (non-PCD n = 20) and 8 were healthy controls (Healthy Controls = 8). Among PCD subjects, ciliary motility was characterized by rotational (n = 5) or dyskinetic (n = 3) beating. At 37 °C, compared to baseline, higher levels of l-Arginine resulted in up to 9% CBF increase at 1 h (p = 0.007), up to 9% CBF increase at 3 h (p < 0.001) and up to 12% CBF increase at 24 h (p = 0.002). Similar although smaller scale increases were recorded at 25 °C. The effect of l-Arginine was time dependent (interaction p = 0.002) and was similar in PCD patients, non-PCD chronic respiratory patients and healthy controls (interaction p = 0.800). Conclusionsl-Arginine increases CBF and merits to be evaluated as a potential stimulator of mucociliary clearance in chronic respiratory conditions and congenital ciliary disorders with residual motility. Larger human studies are needed to confirm these findings.

Hypovitaminosis D: a novel finding in primary ciliary dyskinesia.

BackgroundA relationship between low levels of serum vitamin D and respiratory infections has been established. No study has examined the frequency and clinical relevance of vitamin D deficiency in patients with primary ciliary dyskinesia (PCD).MethodsVitamin D levels were measured in 22 PCD patients (7 females, 10.5 years, range, 2–34 years). In PCD, pulmonary function tests (PFTs), sputum microbiology, self-reported physical activity (PA) level, and quality of life (QoL) by means of the Saint George’s Respiratory Questionnaire (SGRQ), were also assessed.ResultsSeventy-two percent of PCD patients were vitamin-D deficient-to-insufficient and 28% were sufficient. No differences in PFTs parameters were found between vitamin D deficiency-to-insufficiency and sufficiency groups. Patients with vitamin D deficiency-to-insufficiency had significantly higher SGRQ total scores, and thus poorer QoL (p = 0.03). Seventy-nine percent of PCD subjects had limitations in performing vigorous activities, and 53% performed less than 3 hours of PA per week. Vitamin D deficiency-to-insufficiency and sufficiency groups did not show any differences in age at PCD diagnosis or at onset of respiratory symptoms, BMI, atopy, current asthma or bronchiectasis. However, 79% of patients with bronchiectasis had vitamin D deficiency-to-insufficiency. No differences were found in the rate of positive sputum cultures and in the number of antibiotic courses between the two groups.ConclusionsHypovitaminosis D is common in PCD patients, and is associated with poorer QoL. We recommend the assessment and treatment of hypovitaminosis D to be included in the routine management of PCD.

Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

BackgroundThe diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns.MethodsWe retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture.ResultsCiliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced.ConclusionsThe change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia

No study has evaluated the correlation between different expression of nitric oxide synthase (NOS) isoforms in nasal epithelial cells and nasal NO (nNO) level in primary ciliary dyskinesia (PCD). Gene expression of endothelial (NOS3) and inducible NOS (NOS2) and their correlation with nNO level, ciliary function and morphology were studied in patients with PCD or secondary ciliary dyskinesia (SCD). NOS3 gene polymorphisms were studied in blood leukocytes. A total of 212 subjects were studied (48 with PCD, 161 with SCD and three normal subjects). nNO level correlated with mean ciliary beat frequency (p = 0.044; r = 0.174). The lower the nNO level the higher was the percentage of immotile cilia (p<0.001; r = -0.375). A significant positive correlation between NOS2 gene expression and nNO levels was demonstrated in all children (p = 0.001; r = 0.428), and this correlation was confirmed in patients with PCD (p = 0.019; r = 0.484). NOS2 gene expression was lower in PCD than in SCD (p = 0.04). The NOS3 isoform correlated with missing central microtubules (p = 0.048; r = 0.447). nNO levels were higher in PCD subjects with the NOS3 thymidine 894 mutation, and this was associated with a higher ciliary beat frequency (p = 0.045). These results demonstrate a relationship between nNO level, NOS mRNA expression and ciliary beat frequency.

Long-term clearance from small airways in subjects with ciliary dysfunction

The objective of this study was to investigate if long-term clearance from small airways is dependent on normal ciliary function.Six young adults with primary ciliary dyskinesia (PCD) inhaled 111 Indium labelled Teflon particles of 4.2 μm geometric and 6.2 μm aerodynamic diameter with an extremely slow inhalation flow, 0.05 L/s. The inhalation method deposits particles mainly in the small conducting airways. Lung retention was measured immediately after inhalation and at four occasions up to 21 days after inhalation. Results were compared with data from ten healthy controls. For additional comparison three of the PCD subjects also inhaled the test particles with normal inhalation flow, 0.5 L/s, providing a more central deposition. The lung retention at 24 h in % of lung deposition (Ret24) was higher (p < 0.001) in the PCD subjects, 79 % (95% Confidence Interval, 67.6;90.6), compared to 49 % (42.3;55.5) in the healthy controls. There was a significant clearance after 24 h both in the PCD subjects and in the healthy controls with equivalent clearance. The mean Ret24 with slow inhalation flow was 73.9 ± 1.9 % compared to 68.9 ± 7.5 % with normal inhalation flow in the three PCD subjects exposed twice. During day 7–21 the three PCD subjects exposed twice cleared 9 % with normal flow, probably representing predominantly alveolar clearance, compared to 19 % with slow inhalation flow, probably representing mainly small airway clearance.This study shows that despite ciliary dysfunction, clearance continues in the small airways beyond 24 h. There are apparently additional clearance mechanisms present in the small airways.

Markers of airway inflammation in primary ciliary dyskinesia studied using exhaled breath condensate

Macroscopically, the airways in primary ciliary dyskinesia (PCD) are inflamed and infected, and the eventual result is bronchiectasis. The measurement of noninvasive markers of inflammation in PCD may allow determination of mechanisms of tissue damage, and even allow monitoring of therapy. The aim of this study was to measure in exhaled breath condensate (EBC) of children with PCD the concentrations of the neutrophil chemoattractants leukotriene (LT) B4 and interleukin (IL)-8 and the marker of oxidative stress 8-isoprostane (8-IP), and to try determining whether these markers can be used to assess mechanisms of airway inflammation in these patients. Concentrations of LTB4, IL-8, and 8-IP in the EBC of 23 PCD and 11 age-matched healthy children were measured using an enzyme immunoassay (EIA). The children also performed spirometry and underwent sputum induction, the latter for differential cell count. The concentrations of 8-IP in EBC of children with stable PCD were significantly increased compared to normal controls (median, 7.8 pg/ml vs. 3.1 pg/ml; P = 0.004). There was no difference in the median concentrations of EBC LTB4 between PCD subjects and healthy controls (28 pg/ml vs. 28 pg/ml; P = 0.5). IL-8 levels were below the detection limit of the assay, and were not analyzed further. There was no correlation between concentrations of either 8-IP or LTB(4) in EBC and forced expired volume in 1 sec in PCD children. Sputum induction was successful in 83% of the subjects; the median induced sputum neutrophil count was 69% (interquartile range, 59.3-73.6). No significant correlation was found between sputum neutrophils and either EBC 8-IP or LTB4 concentrations in PCD children. This study showed that oxidative stress, as reflected by increased exhaled 8-IP concentration, is increased in PCD children. The mechanism of airway neutrophilia is unclear, but is unlikely to be related to increased production of LTB4, at least in stable PCD patients.