Diagnostik der primären ziliären Dyskinesie

Primary ciliary dyskinesia (PCD) is a phenotypically and genetically heterogeneous genetic disorder. The respiratory disease phenotype which is characterized by upper and lower airway infections results from inborn defects of respiratory cilia responsible for defective mucociliary clearance. Randomization of left/right body asymmetry is responsible for situs inversus (Kartagener’s syndrome) in half of affected individuals. As a screening test nasal nitric oxide measurement can be used. Establishment of the diagnosis currently relies on electron microscopy, high-resolution immunofluorescence analysis, and/or direct evaluation of ciliary beat by light microscopy. Recently mutations in the four genes DNAI1, DNAH5, TXNDC3, and DNAH11 that all encode for outer dynein arm proteins have been linked to recessive PCD. For diagnostic testing especially DNAH5 and DNAI1 mutation screening is useful, because they are responsible for more than 50% of PCD cases with outer dynein arm defects. Rarely mutations in RPGR (PCD + retinitis pigmentosa) and OFD1 (PCD + complex mental retardation syndrome) have been identified in X-linked recessive PCD variants.     

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 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.     

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 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.     

Diagnostik der primären ziliären Dyskinesie

Characteristics

Primary ciliary dyskinesia (PCD) is a rare congenital disease of the cilia which is mostly manifested in the respiratory system.

Diagnostics

When there is a clinical suspicion of the presence of PCD and/or a positive screening result with reduced nasal nitrogen oxide (NO) values, further diagnostic measures should be initiated as soon as possible. As first diagnostic measure for confirmation or exclusion of PCD, high-speed videomicroscopy analysis (HVMA) should be carried out. If the findings are suggestive of PCD transmission electron microscopy (TEM) of the ciliary structure and high-resolution immunofluorescence (IF) microscopic analysis of the cilia should follow. Mandatory for the diagnosis are at least two congruent pathological findings from HVMA, TEM or IF. When a PCD variant with no evidence of ultrastructural defects is present, an identical pathological beating of cilia must be demonstrated with HVMA on three independent occasions. Following that, a targeted genetic clarification should be attempted based on the findings for HVMA, TEM and IF. A clear genetic result can also confirm the diagnosis.

Approach

When PCD is suspected contact with a diagnostic center should be initiated. A reference center for PCD diagnostics will evaluate uncertain findings.     

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.     

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.     

Nitric oxide levels and ciliary beat frequency in indigenous New Zealand children

New Zealand children's morbidity from respiratory disease is high. This study examines whether subclinical ciliary abnormalities underlie the increased prevalence of respiratory disease in indigenous New Zealand children. A prospective study enrolled a group of healthy children who were screened for respiratory disease by questionnaire and lung function. Skin-prick tests were performed to control for atopy. Exhaled and nasal NO was measured online by a single-breath technique using chemiluminescence. Ciliary specimens were obtained by nasal brushings for assessment of structure and function. The ciliary beat frequency (CBF) (median CBF, 12.5 Hz; range, 10.4-16.8 Hz) and NO values (median exhaled NO, 5.6 ppb; range, 2.3-87.7 ppb; median nasal NO, 403 ppb; range, 34-1,120 ppb) for healthy New Zealand European (n=58), Pacific Island (n=61), and Maori (n=16) children were comparable with levels reported internationally. No ethnic differences in NO, atopy, or CBF were demonstrated. Despite an apparently normal ciliary beat, the percentage of ciliary structural defects was 3 times higher than reported controls (9%; range, 3.6-31.3%), with no difference across ethnic groups. In conclusion, it is unlikely that subclinical ciliary abnormalities underlie the increased prevalence of respiratory disease in indigenous New Zealand children. The high percentage of secondary ciliary defects suggests ongoing environmental or infective damage.     

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.     

Primäre ziliäre Dyskinesie

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder with defective structure or function of normally motile cilia, leading to chronic upper and lower respiratory tract infections, fertility problems and organ site abnormalities. The PCD is a genetically heterogeneous condition entailing a broad range of different disease variants. Diagnosing these different PCD phenotypes requires a combined approach using complementary methods for detection of defects of ciliary function, ultrastructure and composition as well as low nasal nitric oxide values and biallelic genetic mutations. To date, mutations in 31 different genes have been linked to PCD permitting a genetic diagnosis in approximately 60?% of cases. Due to the lack of adequate trials evidence-based knowledge on the epidemiology, disease course and management of PCD is currently lacking. An international PCD registry has been developed to overcome these limitations (www.pcdregistry.eu) and is currently recruiting patients. Current treatment regimens have to rely on expert opinions and on experience gained from other respiratory diseases. The management of PCD includes surveillance of pulmonary function, culturing upper and lower airway secretions and diagnostic imaging. Daily airway clearance techniques as well as prompt antibiotic treatment of infections are the cornerstones of PCD treatment regimens.     

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.     

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 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.     

Primary Ciliary Dyskinesia Due to Microtubular Defects is Associated with Worse Lung Clearance Index

Purpose

Primary ciliary dyskinesia (PCD) is characterised by repeated upper and lower respiratory tract infections, neutrophilic airway inflammation and obstructive airway disease. Different ultrastructural ciliary defects may affect lung function decline to different degrees. Lung clearance index (LCI) is a marker of ventilation inhomogeneity that is raised in some but not all patients with PCD. We hypothesised that PCD patients with microtubular defects would have worse (higher) LCI than other PCD patients.

Methods

Spirometry and LCI were measured in 69 stable patients with PCD. Age at testing, age at diagnosis, ethnicity, ciliary ultrastructure, genetic screening result and any growth of Pseudomonas aeruginosa was recorded.

Results

Lung clearance index was more abnormal in PCD patients with microtubular defects (median 10.24) than those with dynein arm defects (median 8.3, p?=?0.004) or normal ultrastructure (median 7.63, p?=?0.0004). Age is correlated with LCI, with older patients having worse LCI values (p?=?0.03, r?=?0.3).

Conclusion

This study shows that cilia microtubular defects are associated with worse LCI in PCD than dynein arm defects or normal ultrastructure. The patient’s age at testing is also associated with a higher LCI. Patients at greater risk of obstructive lung disease should be considered for more aggressive management. Differences between patient groups may potentially open avenues for novel treatments.

     

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.     

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.     

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.     

RPGR mutations might cause reduced orientation of respiratory cilia

RPGR gene encodes retinitis pigmentosa guanosine triphosphatase regulator protein, mutations of which cause 70% of the X‐linked retinitis pigmentosa (XLRP) cases. Rarely, RPGR mutations can also cause primary ciliary dyskinesia (PCD), a multisystem disorder characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis, and male subfertility. Two patients with PCD_RP and their relatives were analyzed using DNA sequencing, transmission electron microscopy (TEM), immunofluorescence (IF), photometry, and high‐speed videomicroscopy. The Polish patient carried a previously known c.154G>A substitution (p.Gly52Arg) in exon 2 (known to affect splicing); the mutation was co‐segregating with the XLRP symptoms in his family. The c.824 G>T mutation (p. Gly275Val) in the Australian patient was a de novo mutation. In both patients, TEM and IF did not reveal any changes in the respiratory cilia structure. However, following ciliogenesis in vitro, in contrast to the ciliary beat frequency, the ciliary beat coordination in the spheroids from the Polish proband and his relatives carrying the c.154G>A mutation was reduced. Analysis of the ciliary alignment indicated severely disturbed orientation of cilia. Therefore, we confirm that defects in the RPGR protein may contribute to syndromic PCD. Lack of ultrastructural defects in respiratory cilia of the probands, the reduced ciliary orientation and the decreased coordination of the ciliary bundles observed in the Polish patient suggested that the RPGR protein may play a role in the establishment of the proper respiratory cilia orientation. Pediatr Pulmonol. 2013; 48:352–363. © 2012 Wiley Periodicals, Inc.     

Die prim?re zili?re Dyskinesie

Primary ciliary dyskinesia (pcD) is a rare inherited autosomal-recessive condition characterized by chronic bronchitis, recurrent pneumonia, bronchiectasis, recurrent otitis and sinusitis. Half of the pcD patients additionally develop a situs inversus viscerum, which is also referred to as Kartagener’s syndrome. High frequency video analysis of ciliary motility, transmission electron microscopy, immunofluorescence imaging and genetic analysis are important diagnostic methods. Exhaled nasal nitric oxide (NO) and examination of the ciliary beat pattern by phase contrast microscopy are screening procedures. Basic principles of therapy are physiotherapy and antibiotic treatment of exacerbations. Topical steroids, β-agonists, acetylcysteine, rhDNAse and phosphodiesterase-4 inhibitors are also sometimes used. In rare conditions surgical interventions are necessary.