Objectives

1. Define primary ciliary dyskinesia (PCD).
2. List the locations of cilia and flagella in the body and relate those locations to the disease manifestations of PCD.
3. Recognize the complexity of the genetic abnormalities that can cause PCD and how studies in single-celled eukaryotic organisms have provided a model for this syndrome.
4. List the clinical findings for which the clinician should consider the diagnosis of PCD.
5. Determine the role that ciliary motility analysis, electron microscopy, functional clearance studies, and exhaled nitric oxide have in establishing the diagnosis of PCD.

Key words

axoneme; bronchiectasis; dynein; immotile cilia syndrome; Kartagener’s syndrome; primary ciliary dyskinesia; situs inversus

Abbreviations

ATPase = adenosinetriphosphatase; NO = nitric oxide; PCD = primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a syndrome characterized by productive cough with bronchiectasis and sinusitis since early life and reduced fertility. PCD is a rare syndrome with an estimated incidence of 1:12,500 in the Caucasian population.¹ The name of the syndrome is informative. Primary indicates that PCD is a genetic disorder with manifestations present from early life and distinguishes it from acquired mucociliary disorders. Ciliary refers to the fact that all individuals with this syndrome have a defect in ciliary anatomy and/or function. Dyskinesia describes the abnormal or absent ciliary/flagellar movement that is the biologic basis for this syndrome. In this review, we present the background, history, genetics, clinical findings, diagnosis, treatment, and recent developments in our understanding of PCD.

Background

With minor variations, the ultrastructural organization of eukaryotic cilia and flagella is uniform across the phylogenetic spectrum and the protein components comprising the axoneme exhibit wide conservation. Ciliary beds are present in the epithelium lining the human respiratory airways, the vestibular epithelium of the brain, and the fallopian tubes, and flagella are present as organelles of locomotion in human sperm (Fig 1).

Cilia, when viewed in cross-section by electron microscopy, are approximately 300 nm in diameter. The internal structure of the cilium is highly organized and optimal function of the organelle is clearly mandated by organization of these components in the correct spatial orientation. Cross-sections of cilia reveal a radially symmetric pattern of nine peripheral microtubular pairs surrounding a central microtubular pair (Fig 2). One of the microtubules of each peripheral microtubular pair exhibits an inner and an outer dynein arm. Several other accessory structures, such as radial spokes and interdoublet links, also are evident in the cross-sectioned cilium. When viewed in longitudinal section, microtubules can be seen extending continuously along almost the entire length of the ciliary shaft. A longitudinal view of the axoneme provides an even more salient view of the dynein arms, particularly in freeze-fracture preparations. Here, both dynein arms are positioned at discrete intervals along the microtubular pairs.^2,3 The ciliary membrane also exhibits a specialized feature, best viewed in freeze-fracture preparations: the ciliary necklace, which appears as an array of membrane-associated particles at the base of each cilium. Although clearly related to effecting ciliary function, this structure has not been rigorously dissected at the molecular level. Both ciliary and flagellar activities are directed by the same basic mechanism, a molecular motor driven by the sliding interaction of axonemal microtubules and dynein adenosinetriphosphatases (ATPases). In order to accomplish the effective and return ciliary/flagellar stroke, the microtubular components of the axoneme undergo a sliding motility relative to one another. This sliding action is effected by a cycle of attachment, sliding, and release of dynein ATPases located on adjacent peripheral microtubular pairs. Accessory axonemal structures are thought to provide shear resistance that converts this sliding action to the bending/beating action of the intact axoneme.

In isolated experimental preparations of axonemes devoid of the native accessory structures present in the whole cilium, the microtubular pairs can be seen to slide away from their neighbors in the presence of adenosine triphosphate.^4-6 It is thought that the presence of the various accessory axonemal structures, such as radial spokes and interdoublet links, provide structural shear resistance to convert microtubular sliding to bending in the intact axoneme, thus conferring ciliary beat. Although present elsewhere in the cell and almost universally in eukaryotic cells, microtubules contribute prominently to the cytoskeletal architecture of cilia and flagella. Microtubules are assembled into a tubular helix from homodimers of a- and b-tubulin protofilaments, each with a molecular weight of approximately 55,000 d.⁷ There is evidence of posttranslational modification of axonemal a-tubulin by acetylation in Chlamydomonas, suggesting a fundamental difference between cytoplasmic and axonemal a-tubulin.^8,9 Similarly, dynein ATPases exhibit cilia and ciliated-cell specificity. Dynein heavy chains can be segregated into cytoplasmic and axonemal forms. There are one to three heavy chains in a functional dynein as well as a variable number of intermediate and light chains.¹⁰ Dynein heavy chains occur as cytoplasmic, inner axonemal arm, and outer axonemal arm forms.^11-13 Illustrative of the conserved nature of dynein heavy chain genes is the greater similarity of the homologs of a given chain across species than to other dynein heavy chains within the same species.

PCD: A Historical Perspective

The disparate symptoms comprising Kartagener’s triad—bronchiectasis, chronic sinusitis, and situs inversus—have been known for almost a century.^14,15 However, it was not until the advent of routine biological electron microscopy that Afzelius¹⁶ and others¹⁷ provided the first descriptions of abnormal ciliary ultrastructure associated with the syndrome and brought the term “immotile cilia syndrome” into common use. Subsequently, other investigators identified additional ultrastructural anomalies of PCD cilia^18,19 and characterized the “heterogeneity” of ciliary morphology associated with the syndrome.²⁰ A growing awareness that ciliary dyskinesia, rather than complete ciliary immotility, represented the functional characteristic of cilia from many affected individuals, as well as the observation that situs inversus appeared only in a segment of the patient population, led to the proposal that the term primary ciliary dyskinesia be used to describe the syndrome deriving from congenital ciliary abnormalities of which Kartagener’s triad is a subset (Fig 3).²¹

More recently, reports of ciliary abnormalities relating to PCD represent variations on the theme of earlier reports, although Rutland and deIongh²² have proposed that ultrastructural analysis of ciliary beat orientation is sufficient to confer a diagnosis of PCD. In contrast to normal ciliated epithelium, in which the central microtubular pair of adjacent cilia are aligned within 30 degrees of their neighbors, the cilia in this type of PCD are anchored on the epithelium in a haphazard orientation. This causes adjacent cilia to beat in random directions so that, while the cilia are not immotile, metachronal waves are not produced and therefore mucus is not cleared. This means that both individual cilium anatomy and the relative orientation of multiple cilia on the epithelium must be assessed as part of the electron microscopy evaluation. Because acquired ciliary defects occur commonly, the electron microscopic evaluation must examine a large number of cilia and take into account the types of defects that may represent acquired anomalies, such as compound cilia, megacilia, or random microtubular defects. Importantly, because acquired anomalies can also be seen in samples from patients with PCD, ciliary defects must be uniform to implicate PCD.

Subsequent to these clinical observations documenting dynein and radial-spoke defects in PCD, basic studies of Chlamydomonas reinhardtii revealed flagellar mutants that expressed patterns of axonemal defects structurally similar to those seen in PCD.^23-25 These observations combined with the rapid development and application of molecular genetic techniques in the late 20th century have led to a number of studies aimed at achieving a better understanding of the molecular basis of PCD.

The Search for PCD Candidate Genes

Among the organelles of the eukaryotic cell, cilia and flagella arguably provide the most striking example of the relatedness of structure and function; by extension, the failure of ciliary motility associated with the syndrome PCD provides a contrasting view of functional failure associated with structural disorganization. Inasmuch as the major proteomic constituents of ciliary and flagellar axonemes are highly conserved across the phylogenetic spectrum, it was hypothesized early on that examination of motility mutants of eukaryotic protists could provide new insights into candidate genes related to PCD.²⁶ Indeed, several studies using the candidate gene approach have provided promising insights into the molecular genetics of PCD.

The efforts to identify PCD candidate genes have also provided insights that may exclude certain candidates. A human axonemal dynein heavy chain gene, DNAH9, with considerable homology to a sea urchin axonemal heavy chain dynein, has been characterized.³⁹ However, in a genotype analysis performed in 31 PCD families, only two families were identified as having concordant inheritance of DNAH9 alleles in affected individuals. A mutation search revealed only polymorphic variants, suggesting that DNAH9 does not represent a PCD candidate gene. This experience points out the difficulty in associating genetic variations with genetic cause and effect.

The appearance of abnormal organ situs in a subset of patients with PCD also has prompted the search for a molecular-level explanation for this phenomenon. The transcription factor FOXJ1 (previously HFH-4 or FKHL13) is temporally related to ciliogenesis,³⁹ as well as to correct positioning of the viscera.⁴⁰ However, mutations of FOXJ1 have not been identified among patients with PCD,⁴¹ suggesting that axonemal assembly during ciliogenesis is a multigeneic event. In a similar vein, mutations of DNAH5 have been shown to be associated with randomization of left-right asymmetry.³² In terms of candidate genes, this is a noteworthy observation inasmuch as this gene encodes a protein highly similar to the Chlamydomonas g-dynein heavy chain.

Clinical Findings in PCD

It is important to recognize that PCD is a syndrome. All patients with PCD have in common the clinical manifestations of impaired mucociliary clearance, so they present with lifelong productive cough, sinusitis, and an increased risk for hearing loss, reduced fertility, and headaches. These organ-specific manifestations (Fig 1) largely reflect the location of ciliated epithelial cells (lung, sinuses, inner ear, brain ependyma, oviduct) or flagella (sperm). Because each specific genetic defect alters cilia and flagellar function in different ways, the clinical presentation of a specific individual will vary depending on the nature of the genetic cilia defect and the phenotypic dyskinesia conferred by the defect. As indicated earlier in the review, we are only beginning to relate specific individuals to specific gene abnormalities and clinical manifestations.

The most common presenting symptoms of PCD are cough and sinusitis with an onset at an early age. Productive cough since birth is crucial to establish from the history, since cough is the only collateral mechanism by which mucus is cleared from the airways of PCD patients.^42-44 In contrast to PCD, acquired productive cough (seen in non-PCD patients) caused by viral infection, smoking, or asthma occurs later in life and, with the exception of bronchiectasis, usually remits spontaneously or with appropriate treatment. Sputum from PCD patients is usually purulent and high in volume depending on the degree of airway colonization that is present. The same impaired mucociliary clearance that causes chronic cough results in poorly drained and chronically colonized or infected sinuses. Chronic sinusitis, characterized by chronic sinus drainage and nasal stuffiness, is almost universal in PCD and, like the productive cough, is usually a lifelong symptom.

Impaired fertility is another frequent but nondiagnostic finding in PCD. Infertility in men with PCD is very common likely due to both impaired sperm motility and seminal ductal obstruction. Although there are reports of successful conceptions from men with PCD,⁴⁵ it is rare and often paternity was not documented. In vitro fertilization has resulted in successful pregnancies using sperm from men with PCD.^46,47 Women with PCD have significantly reduced fertility with only three out of 12 being able to conceive in one small series.⁴⁸ Importantly for the clinician, an infertility history may suggest PCD and should be routinely part of the evaluation of a patient with symptoms of lifelong cough and/or chronic sinusitis. In addition to PCD, other conditions must be considered in the differential diagnosis including Young’s syndrome, variant cystic fibrosis, and congenital bilateral absence of the vas deferens.

Recent Developments in PCD

The initial description of Kartagener’s triad included situs inversus along with bronchiectasis and sinusitis. Because only one half of PCD patients have situs inversus and only one quarter of individuals with situs inversus have PCD, it raises the issue of the diagnostic value of finding situs inversus in determining whether a particular individual has PCD. It is becoming clear that the populations with PCD, Kartagener’s syndrome, and situs inversus have some overlap (Fig 3).

At this point, a few working assumptions can be made as the clinician considers the patient with chronic cough and sinusitis: (1) Approximately 50% of PCD patients will have situs inversus to complete the classic Kartagener’s syndrome triad. This means that the other 50% of PCD patients may have been missed. (2) Situs inversus may be present in individuals with no evidence of sinopulmonary disease who do not have PCD. (3) There are rare individuals with Kartagener’s triad who have no evidence of ciliary dysfunction or ciliary ultrastructural defects as can be detected with current techniques.

Establishing that an individual has PCD is important for several reasons: (1) The genetic cause of the patient’s symptoms is established so that appropriate education and counseling can begin; (2) additional invasive and expensive diagnostic tests can be avoided; (3) disease-management measures appropriate for chronic sinusitis and bronchiectasis can be put in place; and (4) complications such as pneumonia and hearing loss can be anticipated and hopefully prevented.

Establishing the Presence of PCD

The evaluation for possible PCD should be considered in any patient with lifelong productive cough, sinusitis, and impaired fertility. A stepwise approach is suggested below.

1. Exclude other causes of chronic productive cough and bronchiectasis:
   A. Cystic fibrosis: sweat chloride, cystic fibrosis genotyping
   B. Immunodeficiencies: quantitative immunoglobulins
   C. Foreign body: chest radiograph, chest CT scan, and bronchoscopy
   D. Gastroesophageal reflux: esophagogram and/or GI motility studies
   E. Infections: sputum culture for antibiotic-resistant pathogens
2. Perform a test of mucociliary function:
   A. Nasal saccharine transit time test⁴⁹ can be done in clinic and requires minimal training. Normal clearance time is ≤ 30 min, and > 30 min is abnormal. Repeat twice if abnormal or equivocal. False-positive results excluded by repeating tests on second day.
   B. Radionuclide clearance scan ^50,51(available only in certain centers)
3. Perform semen/sperm motility analysis (applicable for postpubertal male patients). Sperm may be absent from semen in PCD patients as a result of duct occlusion. The presence of motile sperm does not exclude PCD.⁴⁵ If the results are all normal, PCD is very unlikely. If abnormal and/or equivocal, proceed to step 4.
4. Measure ciliary motility:
   A. In vitro microscopy using nasal or bronchial epithelial biopsy tissue. Nasal tissue usually sufficient. Absent or low ciliary beat frequency (CBF) suggests PCD.⁵² May need to be repeated if no ciliated cells are obtained. Available only in certain centers.
   B. Finding beating cilia does not exclude PCD.²² Random cilia orientation with motile cilia can also cause PCD.
5. Evaluate cilia axoneme anatomy with transmission electron microscopy of nasal or bronchial ciliated epithelial cells:
   A. Handling and preparation of specimens is critical for good electron microscopic images
   B. Sufficient numbers of axonemes must be analyzed to exclude sampling errors
   C. Defects must be consistently seen in a majority of axonemes

Exhaled Nitric Oxide: An Emerging Diagnostic Tool?

The mucociliary escalator is elegantly evolved to efficiently limit and clear irritant gases and particulates from the respiratory airways. This is accomplished by regulation of both the volume and composition of the surface liquids bathing the mucosal surface as well as control mechanisms for the regulation of ciliary beat. While the coordinated interaction of secretion and ciliary activity is obvious, recent studies have begun to suggest that other constitutive and signaling cell functions may come into play to optimize mucociliary activity in periods of stress. To this end, there is considerable evidence for the influence of nitric oxide (NO) as a signaling molecule in a variety of cell functions,⁵³ including the regulation of ciliary beat.⁵⁴ Studies of exhaled NO levels from upper and lower airways in patients with PCD have consistently demonstrated reduced levels relative to those in normal control subjects and patients with cystic fibrosis.⁵⁵ Intermediate levels of exhaled NO have been documented in healthy PCD heterozygotes, suggesting a possible link between PCD and NO generation.⁵⁶ NO is produced through the activity of three isoforms of nitric oxide synthase on the substrate L-arginine. Two of these synthases, inducible (type II or iNOS) and endothelial (type III or eNOS), are present in normal airway epithelium.^57,58 In an effort to evaluate the potential for augmentation of low NO levels among patients with PCD, exogenous L-arginine, the substrate for nitric oxide synthase, was instilled into the upper and lower airways of groups of patients with cystic fibrosis and PCD.⁵⁹ These studies found that while NO concentrations increased significantly in both groups when they were given supplemental L-arginine, pulmonary function remained unchanged, suggesting that achievement of normal NO concentrations and therefore long-term therapeutic efficacy was not proven. Unfortunately, in this study, the pulmonary function was measured by spirometry and not mucociliary clearance. At this juncture, the findings of a very low nasal NO level may be helpful in identifying patients with possible PCD,⁶⁰ although the role NO plays in the course of the disease or NO’s possible role as a therapeutic agent remain undefined.

Treatment of PCD

The treatment of PCD, while largely symptomatic, is often delayed because of delayed diagnosis. Diagnosis of PCD is often delayed because respiratory infections are common in infants and because clinicians may not be familiar with the rare syndrome of PCD. For example, in a pediatric PCD referral center, the average age at diagnosis for PCD was 4.4 years despite evidence of neonatal distress in the majority of those individuals.⁶¹ As is true with all types of bronchiectasis, early diagnosis and aggressive treatment likely improves outcome in PCD patients.

The treatment of PCD is similar to the treatment of other forms of bronchiectasis, focusing largely on antibiotics and methods to enhance cough and sputum expectoration. Antibiotics clearly have a role in controlling bacterial colonization in PCD patients, who are typically colonized with Haemophilus influenzae, Pseudomonas, and coliforms. Although cyclic antibiotics are commonly used as prophylaxis for clinical exacerbations, there are no trials demonstrating an advantage for any specific antibiotic regimens. As with other individuals with bronchiectasis, the failure of a PCD patient to respond to empiric oral antibiotics should prompt the clinician to culture the sputum to exclude a multidrug-resistant or unusual pathogen. Although not proven in clinical trials, macrolide antibiotics may also have a role in the treatment of PCD independent of their antibiotic effects. Macrolides can stimulate ciliary motility in vitro^62,63 and, in one case report, appeared to improve clearance of secretions when given in combination with an inhaled b-agonist.⁶⁴ Because PCD patients tend to have some degree of airway obstruction during exacerbations, inhaled b-agonists, steroids, and theophylline have all been used as supportive treatments. One study suggests that when PCD patients are treated aggressively with daily chest physiotherapy, monthly spirometric monitoring, and sputum cultures with specific antibiotic treatment, they have stable lung function and fewer complications.⁶⁵ Exercise has also been used as a treatment for PCD patients and was found to increase peak flow rates better than treatment with inhaled b-agonists.⁶⁶ One explanation for this result is that exercise probably promoted vigorous coughing, and cough is the main way that airway secretions are cleared in individuals with PCD.

Natural and Treated History of PCD

Like much of our understanding of PCD, the small number of patients followed by any one center means that few data on the natural history of PCD are available. It is clear, however, that the natural history of PCD varies greatly and likely reflects the genetic heterogeneity of the syndrome. The best evidence for this is a study by Tamalet and colleagues,⁶⁷ who categorized 43 children with PCD based on cilia ultrastructural defects determined by electron microscopy. Those PCD individuals with absence of the cilia central complex had the worst outcomes, with higher incidences of respiratory tract infections and extensive bronchiectasis, and they often required surgical management of their bronchiectatic disease. In contrast, individuals with peripheral microtubule or dynein arm defects appeared to have a good prognosis. Certainly, many individuals with PCD live relatively normal lives, and occasionally PCD causes only minor complaints and is not recognized until late adulthood. In contrast, there are a few PCD individuals with advanced bronchiectasis in childhood that progresses to end-stage lung disease rapidly. Lung transplantation has been used successfully in PCD. Because of the infectious nature of advanced bronchiectasis, bilateral transplantation is the preferred approach to avoid contralateral infection of the new lung, similar to the transplantation strategy used for cystic fibrosis patients. Although it is controversial, living related lung donation has been used successfully in an individual with PCD.^68,69

Summary

PCD is an unusual but important cause of lifelong chronic productive cough with sinusitis, bronchiectasis, hearing dysfunction, and impaired fertility. The clinician needs to consider PCD in all patients with such lifelong symptoms, especially when situs inversus is present. A diagnosis of PCD should be considered when abnormal findings are noted in the history, physical examination, mucociliary clearance functional testing, and ciliary motility. Until more specific diagnostic tests are available, the confirmation of the diagnosis of PCD still depends on ultrastructural detection of cilia defects by electron microscopy. Very low nasal nitric oxide levels are emerging as a new and interesting tool to detect PCD, although this test is still a research tool at this time. Gene-oriented approaches that extend research done in flagellar organisms are now allowing identification of the specific genetic defects that cause PCD in humans. Understanding how these defects alter ciliary motility may provide new insights into diagnostic and therapeutic approaches that will allow early detection and improved symptom management in patients with PCD. Treatment of PCD is largely symptomatic, centering on control of airway infections and measures that enhance pulmonary toilet. These measures are likely to affect long-term outcome. While a great deal of variation occurs related to the genetic heterogeneity of PCD, when PCD is diagnosed early and treated aggressively, most patients will have preserved lung functions and can lead relatively normal lives.

Readers are referred to two Web sites for more PCD information (including videos of normal and PCD cilia):

http://pediatrics.med.unc.edu/div/infectdi/pcd/
http://www.pcdfoundation.org/

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