Nasal ciliary ultrastructure and function in patients with primary ciliary dyskinesia compared with that in normal subjects and in subjects with various respiratory diseases

In an attempt to establish the relevance of ciliary ultrastructure to the pathophysiologic aspects of respiratory tract disease, we compared quantitatively the ultrastructure and function of cilia from healthy subjects (atopic and nonatopic nonsmokers, asymptomatic smokers) and patients with a variety of respiratory diseases (cystic fibrosis, chronic rhinitis, bronchiectasis associated with hypogammaglobulinemia, chronic bronchitis) with cilia from patients with primary ciliary dyskinesia (PCD). In healthy subjects and patients with non-PCD respiratory disease, approximately 5% of the cilia evaluated had ultrastructural abnormalities. Ciliary beat frequency was significantly higher in the chronic rhinitis group (15.3 +/- 1.2 Hz) than in the other non-PCD groups, which were within the normal range (12.5 +/- 1.7 Hz), and in all non-PCD cases ciliary wave form was normal. In each of these groups, normal mucociliary transport had been previously demonstrated. By contrast, in patients with PCD, the proportion of cilia with ultrastructural abnormalities was significantly greater than in the normal subjects and those with non-PCD respiratory disease (p less than 0.0001). In addition, beat frequency was significantly reduced, ciliary wave form was grossly abnormal, and pulmonary and nasal mucociliary transport were virtually absent. These findings demonstrate the relevance of ciliary ultrastructural abnormalities to altered ciliary function and lend support to the primary role of the demonstrated abnormalities in the respiratory tract disease of PCD.     

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.     

Multifaceted analysis of Japanese cases of primary ciliary dyskinesia: Value of immunofluorescence for ciliary protein detection in patients with DNAH5 and DNAH11 mutations

Multifaceted analysis is recommended for the diagnosis of primary ciliary dyskinesia (PCD). A 31-year-old woman had situs inversus, bronchiectasis, family history of PCD, and compound heterozygous mutations in DNAH5. Her cilia were immotile. Defects in the outer dynein arms were revealed by transmission electron microscopy and loss of DNAH5 proteins in the entire length of axonemes using immunofluorescence (IF). A 17-year-old boy had bronchiectasis and heterozygous mutations in DNAH11. His cilia were motile with normal ultrastructure. The loss of DNAH11 proteins at the proximal region of cilia was revealed by IF. IF could be useful to support PCD diagnosis.     

原发性纤毛运动障碍1例及文献复习

目的 提高对原发性纤毛运动障碍(PCD)临床与病理特点的认识.方法 分析我院收治的1例PCD患者的临床资料及诊治经过,并复习相关文献.结果 患者临床表现为慢性咳嗽、咯痰、喘息;胸部CT表现为双肺弥漫的小结节改变并伴局部支气管扩张;经纤维支气管镜肺活检电镜病理表现为纤毛结构异常,动力臂缺失.其表现符合PCD.结论 PCD是由纤毛功能和(或)结构缺陷导致的一种常染色体隐性遗传病,容易误诊,其诊断依赖于纤毛超微结构检测.目前尚无标准治疗方案,以对症治疗为主.     

原发性纤毛运动障碍1例及文献复习

目的 提高对原发性纤毛运动障碍(PCD)临床与病理特点的认识.方法 分析我院收治的1例PCD患者的临床资料及诊治经过,并复习相关文献.结果 患者临床表现为慢性咳嗽、咯痰、喘息;胸部CT表现为双肺弥漫的小结节改变并伴局部支气管扩张;经纤维支气管镜肺活检电镜病理表现为纤毛结构异常,动力臂缺失.其表现符合PCD.结论 PCD是由纤毛功能和(或)结构缺陷导致的一种常染色体隐性遗传病,容易误诊,其诊断依赖于纤毛超微结构检测.目前尚无标准治疗方案,以对症治疗为主.

Abstract：

Objective To improve the understanding of clinical and pathological characteristics of primary ciliary dyskinesia (PCD). Methods A case diagnosed with PCD was reported,and the related literatures were reviewed. Results The patient had cough,expectoration,and dyspnea. Chest CT scan showed diffuse nodules and local bronchieclasis. Transbronchial lung biopsy was done and transmission electron microscopy showed ciliary abnormalities and absence of dynein arms. Those findings were consistent with PCD. Conclusions PCD is an inherited disease characterised by functional and/or structural congenital abnormalities of cilia,and is often misdiagnosed. The diagnosis of PCD relies on the analysis of cilium ultrastructure. There is no specific therapy for PCD,and symptomatic treatment is recommended.     

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.     

Diagnostic yield of nasal scrape biopsies in primary ciliary dyskinesia: A multicenter experience

Examination of ciliary ultrastructure remains the cornerstone diagnostic test for primary ciliary dyskinesia (PCD), a disease of abnormal ciliary structure and/or function. Obtaining a biopsy with sufficient interpretable cilia and producing quality transmission electron micrographs (TEM) is challenging. Methods for processing tissues for optimal preservation of axonemal structures are not standardized. This study describes our experience using a standard operating procedure (SOP) for collecting nasal scrape biopsies and processing TEMs in a centralized laboratory. We enrolled patients with suspected PCD at research sites of the Genetic Disorders of Mucociliary Clearance Consortium. Biopsies were performed according to a SOP whereby curettes were used to scrape the inferior surface of the inferior turbinate, with samples placed in fixative. Specimens were shipped to a central laboratory where TEMs were prepared and blindly reviewed. Four hundred forty‐eight specimens were obtained from 107 young children (0–5 years), 189 older children (5–18 years), and 152 adults (> 18 years), and 88% were adequate for formal interpretation. The proportion of adequate specimens was higher in adults than in children. Fifty percent of the adequate TEMs showed normal ciliary ultrastructure, 39% showed hallmark ultrastructural changes of PCD, and 11% had indeterminate findings. Among specimens without clearly normal ultrastructure, 72% had defects of the outer and/or inner dynein arms (IDA), while 7% had central apparatus defects with or without IDA defects. In summary, nasal scrape biopsies can be performed in the outpatient setting and yield interpretable samples, when performed by individuals with adequate training and experience according to an SOP. Pediatr. Pulmonol. 2011; 46:483–488. © 2011 Wiley‐Liss, Inc.     

Freeze-fracture analysis of the respiratory cilia from the bronchial mucosa of a patient with primary ciliary dyskinesia

Respiratory cilia of the bronchial mucosa from a 5-year-old boy with clinical evidence of classical Kartagener's syndrome (situs inversus, bronchiectasis and sinusitis) were first examined by means of transmission electron microscopy for identification of the axonemal defects described as typical for primary ciliary dyskinesia (PCD). Additional oscillography was performed on the cilia in vitro, which showed absence of a coordinated ciliary beat frequency. After clear classification of the case as PCD, a freeze-fracture examination of the respiratory cilia was performed, which revealed a higher density of intramembrane particles on the outer fracture face (E-face) than on the inner fracture face (P-face). The results were discussed with regard to probable pathogenetic aspects on PCD.     

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.     

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.     

Nasal nitric oxide in atypical primary ciliary dyskinesia

BACKGROUND: Atypical cases of primary ciliary dyskinesia (PCD) may present with minimal transmission electron microscopy (TEM) defects. The diagnostic role of nasal nitric oxide (nNO) levels was evaluated in those patients. METHODS: Sixty-four children with recurrent pneumonia were studied with ciliary motion analysis, TEM, and nNO. RESULTS: Investigations indicated PCD in 12 patients, secondary ciliary dyskinesia (SCD) in 50 patients, and normal results in 2 patients. In 4 of 50 children with SCD, atypical PCD was considered possible. The mean (+/- SD) nNO was 130 +/- 46.95 parts per billion in children affected by PCD, 127.79 +/- 68.58 parts per billion in atypical patients, and 760 +/- 221 parts per billion in children with SCD. Three to 5 months later, the nNO level was 132.75 +/- 55.76 parts per billion in children with atypical disease and 778.00 +/- 197 parts per billion in children with SCD. CONCLUSION: Low levels of nNO may help to identify patients with atypical PCD.     

Nasal nitric oxide measurements for the screening of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) presents to general practitioners with symptoms pertinent to a variety of specialists because of the involvement of ciliated epithelium in the upper/lower respiratory tract, ears, eyes and genital tract. There is no easy, reliable screening test for PCD, and thus, the majority of patients remain undiagnosed. Nitric oxide (NO) is measurable in nasal air of normal subjects and found to be low in cystic fibrosis (CF) and very low in PCD. Recently, it was suggested to play an important role in regulating ciliary motility. The aim of this study was to evaluate whether measurements of nasal NO could be used to screen for PCD. Nasal NO was measured from the nasal cavity by a chemiluminescence analyser in subjects with PCD, healthy controls, CF, idiopathic bronchiectasis, Young's syndrome and lone sinusitis. Nasal NO was significantly lower in PCD (64.0+/-36.6) compared with normal controls (759+/-145.8), idiopathic bronchiectasis (734+/-163.7), CF (447.5+/-162.6), lone sinusitis (1487+/-734) and Young's syndrome (644+/-129.9). Nasal NO was also significantly lower in PCD than CF patients. Measurement of nasal nitric oxide may therefore be used clinically in various specialities to screen suspected patients for primary ciliary dyskinesia.     

Analysis of the diagnosis of Japanese patients with primary ciliary dyskinesia using a conditional reprogramming culture

BackgroundPrimary ciliary dyskinesia (PCD) is diagnosed through multiple methods, including transmission electron microscopy (TEM), a high-speed video microscopy analysis (HSVA), immunofluorescence (IF), and genetic testing. A primary cell culture has been recommended to avoid the misdiagnosis of secondary ciliary dyskinesia derived from infection or inflammation and improve diagnostic accuracy. However, primary cells fail to differentiate into ciliated cells through repeated passages. The conditional reprogramming culture (CRC) method, a combination of a Rho-kinase inhibitor and fibroblast feeder cells, has been applied to cystic fibrosis. The goal of this study was to evaluate the value of CRC in diagnosing PCD in Japanese patients.MethodsEleven patients clinically suspected of having PCD were included. Airway epithelial cells were obtained from an endobronchial forceps biopsy and cultured at the air-liquid interface (ALI) combined with CRC. Ciliary movement, ultrastructure, and mutated ciliary protein evaluation were performed using HSVA, TEM, and IF, respectively. Genetic testing was performed on some patients.ResultsCRC yielded dense and well-differentiated ciliated cells with a high success rate (～90%). In patients with PCD, the ciliary ultrastructure phenotype (outer dynein arm defects or normal ultrastructure) and IF findings (absence of the mutated ciliary protein) were confirmed after CRC. In DNAH11-mutant cases with normal ultrastructure by TEM, the HSVA revealed stiff and hyperfrequent ciliary beating with low bending capacity in CRC-expanded cells, thereby supporting the diagnosis.ConclusionsCRC could be a potential tool for improving diagnostic accuracy and contributing to future clinical and basic research in PCD.     

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.     

Long-term use of nebulized human recombinant DNase1 in two siblings with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is characterized by ultra-structural defects of the cilia. In this report, we describe the long-term use of nebulized dornase alfa in two siblings with PCD. This is the first report of long-term use of dornase alfa in PCD.     

Diagnosis of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetic disorder of ciliary structure or function. It results in mucus accumulation and bacterial colonization of the respiratory tract which leads to chronic upper and lower airway infections, organ laterality defects, and fertility problems. We review the respiratory signs and symptoms of PCD, as well as the screening tests for and diagnostic investigation of the disease, together with details related to ciliary function, ciliary ultrastructure, and genetic studies. In addition, we describe the difficulties in diagnosing PCD by means of transmission electron microscopy, as well as describing patient follow-up procedures.     

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.     

Orientation of human respiratory cilia

Ciliary orientation was studied on the respiratory epithelium of the nasal cavity or the sphenoidal sinus of ten adult nonsmokers without respiratory disease. The ciliary orientation was evaluated from micrographs by measuring the angle between the plane defined by the central tubules and reference line (with a semiautomatic image analyser (IBAS I]. The standard deviation of the angles of cilia population was counted in every field. The standard deviation of the measurements described the ciliary alignment. It varied from 12.1-41.2 degrees. The mean standard deviation was 27.3 +/- 7.4 degrees. 58% of all measured cilia were within +/- 0-20 degrees of the mean and 85% of cilia were within +/- degrees. However, a few cilia or small groups of cilia were found in most fields which differed dramatically from the main orientation. The size of these groups was always less than ten cilia. On the normal respiratory epithelium the standard deviation of ciliary orientation varies between +/- 10-40 degrees (at about 97% probability). For diagnostic conclusions more than 60 cilia should be measured.     

Disorders of ciliary motility

Clearance of mucus and other debris from the airways is achieved by 3 main mechanisms: mucociliary activity, coughing, and alveolar clearance. Disorders of ciliary structure or function results in impaired clearance, and result in chronic sinopulmonary disease manifested as chronic sinusitis, otitis media, nasal polyposis, and ultimately bronchiectasis. In addition, situs inversus, dextrocardia, and infertility can be associated with dysfunctional ciliary activity. The term primary ciliary dyskinesia has been proposed for the spectrum of these diseases. The term Kartagener syndrome applies to this syndrome when accompanied by infertility and dextrocardia or situs inversus. The more common types of ciliary dysmotility syndromes are characterized by missing dynein arms, central microtubule pairs, inner sheath, radial spokes, or nexin links. In addition to structural defects within the cilia, disordered ciliary beating and disordered ciliary arrays on epithelial cell surfaces have been described in this syndrome. Treatment includes rigorous lung physiotherapy, prophylactic and organism-specific antibiotics, and immunization against common pulmonary pathogens. Late stages of the disease may require surgical intervention for bronchiectasis or lung transplant for end-stage lung disease.     

Nasal and lower airway level of nitric oxide in children with primary ciliary dyskinesia.

Exhaled nitric oxide can be detected in exhaled air and is readily measured by chemiluminescence. It is thought to be involved in both the regulation of ciliary motility and host defence. Recently, upper airway NO has been found to be reduced in a small number of children with primary ciliary dyskinesia (PCD) and its measurement has been recommended as a diagnostic test for this condition. The aim of this study was to compare the levels of NO in the upper and lower airways in a larger number of children with proven PCD with those found in healthy children. Exhaled NO was measured in the upper airway by direct nasal sampling during a breath-hold and in the lower airway as the end-tidal plateau level, using a chemiluminescence NO analyser. Upper airway NO levels were significantly lower in PCD (n = 21) than in the healthy children (n = 60) (mean +/-SD, 97+/-193, 664+/-298 parts per billion (ppb), respectively, p<0.0001). In PCD, the lower airway NO levels were also reduced (2.17+/-1.18, 5.94+/-3.49 ppb, respectively, p<0.0001). The levels were not associated with steroid use and did not correlate with lung function. Although there was some overlap between normal children and those with primary ciliary dyskinesia with regard to lower airway NO, nasal NO discriminated between the two groups in all but one child in each group. Measurement of nasal NO therefore may be a useful screening test for primary ciliary dyskinesia.