The challenges of diagnosing primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare genetic disorder of ciliary structure and function. The diagnosis can be challenging, particularly when using nongenetic assays. The "gold standard" diagnostic test is ultrastructural analysis of respiratory cilia obtained by nasal scrape or brush biopsy. A few specialized centers use high-speed videomicroscopy to examine ciliary beat. Certain beat patterns correlate with ultrastructural defects, and, in some cases, subtle alterations in beat pattern can be seen when ultrastructure is normal. Recent studies have shown that nasal nitric oxide (NO) is very low in patients with PCD compared with healthy control subjects; therefore, this assay may be a useful screening or adjunctive test for PCD. Because acute respiratory illnesses may yield alterations in ciliary ultrastructure, ciliary beat, and nasal NO values, these tests should be performed during a stable baseline period. Identification of an array of PCD genes has provided the opportunity for making a definitive genetic diagnosis for PCD in some cases. All of these approaches have a role in diagnosing PCD. For example, PCD has been confirmed by identifying disease-causing mutations in a heavy dynein chain gene in individuals with normal ciliary ultrastructure but subtle defects in ciliary beat and low nasal NO. Priorities to improve nongenetic diagnostic capability include standardization of nasal NO as a screening test and the development of specialized centers using uniform approaches for the analysis of ciliary ultrastructure and ciliary beat pattern. Another chapter in this issue (see Zariwala and colleagues, pp. 430) addresses the progress toward improved capabilities for definitive genetic testing.     

Ultrastructural ciliary defects in children with recurrent infections of the lower respiratory tract

One hundred fifty-four children with recurrent or chronic infections of the lower respiratory tract compatible with the diagnosis of primary ciliary dyskinesia (PCD) were evaluated for the presence of ultrastructural ciliary abnormalities. Studies were performed on multiple samples of respiratory mucosa obtained by nasal and bronchial brushing. Twenty-eight children showed ultrastructural ciliary defects compatible with the diagnosis of PCD: Twenty-four presented dynein arm deficiency (either as isolated defect or in association with microtubular abnormalities), two had ciliary aplasia, and two showed microtubular abnormalities. Eleven patients with PCD had situs viscerum inversus, bronchiectasis, and chronic sinusitis (Kartagener's syndrome); one child with Kartagener's syndrome had normal ciliary structure. The appearance of respiratory symptoms within the first month of life, the colonization by Haemophilus influenzae, and a history of recurrent rhinitis and otitis were characteristically present in children with PCD. The clinical status of those patients who reached adolescence was, in our experience, remarkably good. An early diagnosis with adequate prevention and therapy of respiratory infections may have an important role in minimizing irreversible lung damage.     

Primary ciliary dyskinesia syndrome associated with abnormal ciliary orientation in infants.

Primary ciliary dyskinesia (PCD) syndrome associated with abnormal ciliary orientation but with normal ciliary ultrastructure has been described in adults, but there are no normal ranges for orientation in infants, despite the fact that half of all patients with PCD present in the new-born period. Nasal brush biopsies were obtained from eight infants (three males), mean age 13.1 months, range 7-23, in order to determine ciliary orientation. They had no upper or lower airway disease and normal organ arrangement and were undergoing general anaesthesia for other reasons. Two infants with typical PCD syndrome but normal ultrastructure of individual cilia also had orientation studies. In the eight normal subjects, a mean of 254 central pairs was examined, range 82-453. The mean ciliary orientation was 14.9 degrees, range 12.9-17.5. The two infants with PCD syndrome but normal ultrastructure of individual cilia had ciliary orientation of (Case 1) 44.5 degrees (range 10.6-64.5) in 218 central pairs; and on a second occasion, 28.9 degrees, (range 9.0-47.5) in 259 central pairs; for Case 2, 24.4 degrees, (range 13.1-38.4) in 196 central pairs. The normal range for ciliary orientation is similar in infants to that described in other work in adults. The two cases of phenotypic primary ciliary dyskinesia in the presence of normal ciliary ultrastructure but abnormal ciliary orientation in infants supports the contention that measurement of ciliary orientation should be part of the assessment of ciliary structure and function in cases of possible primary ciliary dyskinesia, in particular when the ultrastructure of individual cilia appear to be normal.     

Primary ciliary dyskinesia: diagnostic and phenotypic features

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary structure/function. We hypothesized that the major clinical and biologic phenotypic markers of the disease could be evaluated by studying a cohort of subjects suspected of having PCD. Of 110 subjects evaluated, PCD was diagnosed in 78 subjects using a combination of compatible clinical features coupled with tests of ciliary ultrastructure and function. Chronic rhinitis/sinusitis (n = 78; 100%), recurrent otitis media (n = 74; 95%), neonatal respiratory symptoms (n = 57; 73%), and situs inversus (n = 43; 55%) are strong phenotypic markers of the disease. Mucoid Pseudomonas aeruginosa (n = 12; 15%) and nontuberculous mycobacteria (n = 8; 10%) were present in older (> 30 years) patients with PCD. All subjects had defects in ciliary structure, 66% in the outer dynein arm. Nasal nitric oxide production was very low in PCD (nl/minute; 19 +/- 17 vs. 376 +/- 124 in normal control subjects). Rigorous clinical and ciliary phenotyping and measures of nasal nitric oxide are useful for the diagnosis of PCD. An increased awareness of the clinical presentation and diagnostic criteria for PCD will help lead to better diagnosis and care for this orphan disease.     

The saccharin method for testing mucociliary function in patients suspected of having primary ciliary dyskinesia

In order to evaluate the clinical value of the saccharin test as a practical and simple measure of mucociliary clearance, nasal mucociliary clearance (NMCC) and ciliary ultrastructure were studied in 22 patients suspected of having primary ciliary dyskinesia (PCD) based on the saccharin test. Ten patients fulfilling the diagnostic criteria of PCD had a pathological response to the saccharin test (transport time greater than 60 minutes), and this was consistently associated with ultrastructural defects, specific for PCD. These results validate the suitability of the clinical use of the saccharin test as a screening procedure for NMCC. The false-negative results obtained in three cases of PCD, all with borderline values, cannot be ascribed to ineffectiveness of the test, but rather to the persistence of some motility by certain defective cilia, detectable by microphoto-oscillographic investigation of specimens obtained by nasal biopsy or brushing.     

气道纤毛功能及相关呼吸系统疾病研究进展

正常的黏液纤毛清除功能要求黏液具有最佳的黏弹性和厚度,而足够数量、结构完整和一定摆动频率的纤毛则更重要.由于慢性阻塞性肺疾病,原发性纤毛运动障碍、囊性纤维化等多种疾病均存在纤毛结构和功能异常或纤毛周围的黏液流变学特征改变,因此气道黏液纤毛清除功能成为探索相关治疗的新领域之一.本文就影响纤毛结构及运动的因素及相关的呼吸系统疾病的研究进展作综述.     

Dyskinésie ciliaire primitive

Primary ciliary dyskinesia (PCD, MIM 242650) is an inherited respiratory disease caused by functional and ultrastructural abnormalities of respiratory cilia. This disorder, which affects 1 in 16,000 individuals, is usually transmitted as an autosomal recessive trait. In half the cases, PCD is associated with situs inversus (Kartagener syndrome). PCD is characterized by impaired mucociliary clearance resulting from a lack of ciliary motion, which is responsible for recurrent respiratory infections. The most frequent and first identified ciliary defect involves the dynein arms. The genetic heterogeneity underlying PCD is extremely important, and only three genes have as yet been identified in a few PCD patients with absence of outer dynein arms. The main clinical symptoms, at pulmonary and ENT levels, the abnormalities of ciliary structure and function, and the molecular basis of PCD will be reported in this review.     

Acquired ciliary defects in bronchial epithelium of patients with chronic bronchitis

In order to quantify and classify ciliary changes of bronchial epithelium in chronic bronchitis, an ultrastructural study on biopsy specimens of 30 patients submitted to bronchoscopy was performed. Fourteen patients were affected by endoscopically and clinically confirmed chronic bronchitis, 8 showed an endoscopic picture of chronic bronchitis but without the clinical features of this pathology, 8 were free of chronic bronchitis. The mean percentage of abnormal cilia in control subjects (1.9 +/- 1.0%) was significantly lower than in patients either with only 'endoscopic' chronic bronchitis (5.7 +/- 2.6%; p less than 0.001) or with 'clinical' chronic bronchitis (14.8 +/- 15.9%; p less than 0.05). Like the ciliary body, the ciliary tip can also show some abnormalities. It is possible to subdivide them in two types: (1) ciliary membrane blebs; (2) ciliary membrane extrusions, variously shaped and sized, with homogeneous content. Bronchial cilia abnormalities become more serious as soon as the clinical picture worsens and this is likely to contribute to the impairment of the mucociliary clearance in chronic bronchitis patients.     

Respiratory ciliary function in bone marrow recipients

Bone marrow transplantation (BMT) recipients, particularly those with chronic graft-versus-host disease (GVHD), suffer from respiratory tract problems, including bronchiolitis obliterans (BO) and recurrent lower respiratory tract infections. Minute cilia beat continuously on the surface of respiratory mucosa, and this beating maintains the sterility of the lower respiratory tract. Dysfunction of respiratory cilia could lead to development of recurrent respiratory tract infections, which are also features of BMT recipients, although ciliary function has not been systematically studied among these subjects. We have, therefore, investigated the ciliary beat frequency (CBF) of 36 Chinese patients who had undergone allogeneic BMT. The CBF was significantly lower in the BMT group compared to controls (P < 0.001). The reduction in CBF was more severe in patients with cGVHD and BO compared with their counterparts (P = 0.048 and P = 0.077, respectively). There was a correlation between CBF with forced expiratory flow rate FEF (P = 0.024) and forced expiratory volume FEV (P = 0.044). We conclude that abnormal ciliary clearance is a common feature after allogeneic BMT, particularly among patients with BO and cGVHD. Further studies are indicated to evaluate this important phenomenon, which could be an important cause of the susceptibility for BMT recipients to respiratory infections.     

Pulmonary radioaerosol mucociliary clearance in diagnosis of primary ciliary dyskinesia

BACKGROUND: Methods relying on nasal ciliary motility for the diagnosis of primary ciliary dyskinesia (PCD) are often hampered by secondary ciliary dyskinesia. A functional test for pulmonary mucociliary clearance, which is not influenced by secondary nasal ciliary defects, would be a valuable tool in a PCD workup. METHODS: The diagnostic validity and repeatability of a pulmonary radioaerosol mucociliary clearance (PRMC) test for the diagnosis of PCD was assessed in the following three sequentially performed substudies: (1) a preliminary cross-sectional study of PRMC in patients with known PCD; (2) a prospective blinded trial of patients referred for suspicion of PCD; and (3) an implementation study of PRMC as a routine method used in a PCD workup. PRMC was studied after (99m)Tc-albumin colloid aerosol inhalation, and the results were compared to (1) the results of nasal ciliary motility studies, (2) ciliary ultrastructure, and (3) the final clinical diagnosis. The repeatability of PRMC was assessed in 14 patients. RESULTS: A total of 95 patients, 5 to 74 years of age, were included in the study (57 patients in whom PCD was diagnosed, 26 non-PCD patients, and 12 patients referred for PCD workup without a conclusive workup result). In substudy 1, 14 of 15 patients with known PCD showed impaired PRMC; the results were inconclusive in 1 patient. In substudy 2, among 59 patients referred for PCD workup PRMC test results, compared to nasal ciliary motility, showed a sensitivity of 88% and a specificity of 100%. In substudy 3, among 21 patients referred for PCD investigation who were included in a routine PCD workup after PRMC implementation, 71% of PRMC test results were in alignment with nasal ciliary motility. Repeatability of interpretation was seen in 13 of 14 cases. A conclusive PRMC after only one test was found in 81 of 95 patients (85%). CONCLUSION: PRMC is a noninvasive functional test for total tracheobronchial mucociliary clearance with a high sensitivity and specificity for PCD, a high rate of conclusive results after only one test and a further ability to separate PCD from focal pulmonary mucociliary defects.     

Central microtubular agenesis causing primary ciliary dyskinesia

Primary ciliary dyskinesia is an autosomal recessive disorder characterized by chronic upper and lower respiratory tract symptoms. We report the diagnosis of primary ciliary dyskinesia associated with a circular ciliary beat pattern in three siblings. This beat pattern is consistent with a ciliary transposition defect, where a peripheral microtubule doublet is transposed to the center of the ciliary axoneme to replace the absent central microtubule pair. However, in these siblings, ultrastructural analysis of the cilia revealed an absence of the central microtubule pair only. This variant of transposition with a circular ciliary beat pattern has not been described previously. In addition, this defect, together with the transposition defect, may help explain the mechanism of the circular beat pattern and also the absence of situs inversus in these patients.     

Ciliary assessment in bronchiectasis

Bronchiectasis is a common condition among the Oriental population and affected patients suffer from chronic sputum production punctuated by recurrent infective exacerbations. Cilia are minute structures present on the surface of respiratory and other epithelial cells that beat continuously to maintain a sterile mucosal surface in the respiratory tract. Patients with primary ciliary dyskinesia could potentially develop recurrent sinotrachrobronchitis, bronchiectasis, serous otitis media, hydrocephalus, and male infertility. The assessment of cilia has, however, received little attention until recently and generally involves elaborate methods that require complex and expensive technology. This brief article discusses application of the saccharine test, light microscopy assessment of ciliary beat, and transmission electron microscopy assessment of the ultrastructure of cilia. The rationale and indications for ciliary assessment are also listed along with illustrations showing ciliary structure, equipment required for sampling and assessment of cilia, and transmission electron micrographs of ciliary ultrastructural abnormalities.     

Cystic fibrosis serum does not inhibit human ciliary beat frequency

The effect of cystic fibrosis (CF) serum on human respiratory tract ciliary beat frequency (CBF) was studied to investigate the possible relevance of CF-serum-induced disruption of rabbit tracheal ciliary motility to human disease. Nasal ciliated epithelium from patients with CF and from normal subjects was incubated at 37 degrees C in CF serum, in normal human serum (NHS), or in nutrient medium. CBF was measured photometrically at time intervals up to 3 h. The effect of CF serum on human trachea and of CF serum, NHS, and rabbit serum on rabbit tracheal CBF was also studied. Mean CBF for all the normal and CF nasal cilia incubated in CF serum, NHS, or nutrient medium was not significantly different and did not fall during 3 h. Human tracheal CBF did not fall during 3 h. The CBF for rabbit trachea in rabbit serum did not slow and rabbit trachea in NHS slowed after 150 min. Rabbit trachea in CF serum beat more slowly than all the other groups (p less than 0.001) progressing to ciliostasis between 45 and 150 min. We conclude that human respiratory tract ciliary motility, as measured by CBF in vitro, is unaffected by factors in CF serum that impair rabbit tracheal ciliary activity.     

Update of respiratory tract disease in children with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by abnormal ciliary structure and function leading to impaired mucociliary clearance and chronic progressive sinopulmonary disease. Upper and lower respiratory tract manifestations are cardinal features of PCD. This review summarizes the current state of knowledge of respiratory tract disease in individuals with PCD and highlights the challenges in identifying and quantifying lung disease in very young children with PCD. No specific therapies are available to correct ciliary dysfunction in PCD. Treatment is not evidence based, and recommendations are largely extrapolated from cystic fibrosis and other conditions with impaired mucociliary clearance. There is a pressing need to develop and validate outcome measures, including patient-reported outcomes, that could be used to evaluate potential therapies in PCD. This review concludes with recommendations for clinical endpoints and outcome measures and a prioritized list of treatments to study in PCD clinical trials.     

Primary ciliary dyskinesia with CCDC39 variants displaying specific ciliary ultrastructure and movement concordant with the genotype: A case report

Primary ciliary dyskinesia (PCD) is a genetic disease with chronic airway infection and inflammation caused by ciliary ultrastructural defects and impairment in ciliary function.We present an adult case of PCD with compound heterozygous nonsense variants in CCDC39. The ciliary ultrastructure findings using electron microscopy and ciliary movement using high-speed video analysis matched the genotype. This is the first case report of PCD with CCDC39 variants in Japan demonstrating specific ciliary ultrastructure and movement related to the genotype.     

Cystic fibrosis compared with the immotile-cilia syndrome. A study of mucociliary clearance, ciliary ultrastructure, clinical picture and ventilatory function.

Patients with cystic fibrosis (CF) were investigated for mucociliary clearance (with and without stimulation by terbutaline), clinical picture, ventilatory function and ultrastructure of cilia. The results were compared with those of patients with congenitally immotile cilia (immotile-cilia syndrome). Mucociliary clearance could be demonstrated in all the seven CF patients who succeeded in inhaling the test aerosol. Ciliary ultrastructure from a deceased CF patient was normal. Patients with the immotile cilia syndrome had no substantial clearance and defective cilia. The CF patients coughed more during the clearance measurements than any other group studied earlier, and their coughing was effective. One patient succeeded in avoiding coughing in both measurements and had faster clearance when he got terbutaline than when he got the vehicle. Although younger, the CF patients tended to be more obstructed in their lungs and more handicapped than the patients suffering from the immotile-cilia syndrome. The latter patients had more discomfort from rhinitis, sinusitis and otitis than had the CF patients. An impairment of the mucociliary transport rate is hence unlikely to be a primary pathogenic factor for the respiratory tract disease in CF patients.     

Mislocalization of DNAH5 and DNAH9 in respiratory cells from patients with primary ciliary dyskinesia

RATIONALE: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by recurrent infections of the airways and situs inversus in half of the affected offspring. The most frequent genetic defects comprise recessive mutations of DNAH5 and DNAI1, which encode outer dynein arm (ODA) components. Diagnosis of PCD usually relies on electron microscopy, which is technically demanding and sometimes difficult to interpret. METHODS: Using specific antibodies, we determined the subcellular localization of the ODA heavy chains DNAH5 and DNAH9 in human respiratory epithelial and sperm cells of patients with PCD and control subjects by high-resolution immunofluorescence imaging. We also assessed cilia and sperm tail function by high-speed video microscopy. RESULTS: In normal ciliated airway epithelium, DNAH5 and DNAH9 show a specific regional distribution along the ciliary axoneme, indicating the existence of at least two distinct ODA types. DNAH5 was completely or only distally absent from the respiratory ciliary axoneme in patients with PCD with DNAH5- (n = 3) or DNAI1- (n = 1) mutations, respectively, and instead accumulated at the microtubule-organizing centers. In contrast to respiratory cilia, sperm tails from a patient with DNAH5 mutations had normal ODA heavy chain distribution, suggesting different modes of ODA generation in these cell types. Blinded investigation of a large cohort of patients with PCD and control subjects identified DNAH5 mislocalization in all patients diagnosed with ODA defects by electron microscopy (n = 16). Cilia with complete axonemal DNAH5 deficiency were immotile, whereas cilia with distal DNAH5 deficiency showed residual motility. CONCLUSIONS: Immunofluorescence staining can detect ODA defects, which will possibly aid PCD diagnosis.     

Dissimilar expression of axonemal anomalies in respiratory cilia and sperm flagella in infertile men

Infertility by sperm immotility may be a consequence of axonemal defects, and it is sometimes associated with respiratory disease as in the primary ciliary dyskinesia. The purpose of this study was to investigate the respiratory epithelium of 13 infertile patients with flagellar ultrastructural anomalies whether they suffered or not from respiratory disease. Only one patient had severe respiratory disease. The beat frequency of nasal cilia (9 to 13 Hz) was considered normal in 11 of the 13 patients. All the cilia were found abnormal in one patient. In 12 of 13 infertile men (92.3%), significant ultrastructural cilia anomalies were found (greater than 7%) even though the men did not suffer from any respiratory disease. Axonemal anomalies were found at both levels either of the same type (four cases) or only partially similar (two cases) or totally different (six cases). The most frequently abnormal substructure of cilia was the central complex (10 of 12) even in cases exhibiting a different abnormal pattern at the flagellar level. Findings are discussed according to those found in animal mutants. Our results suggest that ciliary and flagellar axonemes should be controlled both by common and by different groups of genes.     

The effect of aging on nasal mucociliary clearance, beat frequency, and ultrastructure of respiratory cilia

The increased susceptibility of the elderly to lower respiratory tract infection cannot be fully explained. Although mucociliary clearance, which is affected by ciliary beating and ultrastructure, plays a crucial role in the defense of the airways against inhaled microbes, little is known of the effects of aging on these parameters. We studied the nasal mucociliary clearance (NMCC) time, ciliary beat frequency, and ultrastructure of respiratory cilia in a cohort of healthy volunteers (age range 11 to 90 yr). Ciliary beat frequency of ciliated nasal epithelial cells was obtained via an established photometric method, and NMCC time was measured with the saccharine test. There was a correlation of ciliary beat frequency (r = -0.48, p = 0.0001) and NMCC time r = 0.64, p < 0.001) with increasing age. Transmission electron microscopy revealed an increase in the percent of subjects exhibiting microtubular disarrangement and single central microtubules with aging (p = 0.002 and p = 0.005, respectively). Subjects older than 40 yr of age had significantly slower ciliary beat frequency, higher percent of ciliary cross-sections displaying single tubules, and longer NMCC time than their younger counterparts (p < 0.05). These findings may help explain the frequent occurrence of respiratory infection in the elderly.     

The emerging genetics of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive, rare, genetically heterogeneous condition characterized by oto-sino-pulmonary disease together with situs abnormalities (Kartagener syndrome) owing to abnormal ciliary structure and function. Most patients are currently diagnosed with PCD based on the presence of defective ciliary ultrastructure. However, diagnosis often remains challenging due to variability in the clinical phenotype and ciliary ultrastructural changes. Some patients with PCD have normal ciliary ultrastructure, which further confounds the diagnosis. A genetic test for PCD exists but is of limited value because it investigates only a limited number of mutations in only two genes. The genetics of PCD is complicated owing to the complexity of axonemal structure that is highly conserved through evolution, which is comprised of multiple proteins. Identifying a PCD-causing gene is challenging due to locus and allelic heterogeneity. Despite genetic heterogeneity, multiple tools have been used, and there are 11 known PCD-causing genes. All of these genes combined explain approximately 50% of PCD cases; hence, more genes need to be identified. This review briefly describes the current knowledge regarding the genetics of PCD and focuses on the methodologies used to identify novel PCD-causing genes, including a candidate gene approach using model organisms, next-generation massively parallel sequencing techniques, and the use of genetically isolated populations. In conclusion, we demonstrate the multipronged approach that is necessary to circumvent challenges due to genetic heterogeneity to uncover genetic causes of PCD.