Clinical phenotype and current diagnostic criteria for primary ciliary dyskinesia

Eleonora Dehlink1, Claire Hogg2, Siobhan B. Carr1, and Andrew Bush 1,3

Department of Paediatrics, Royal Brompton Hospital, London, UK

Department of Paediatric Respirology, Royal Brompton Hospital, London, UK

Department of Paediatric Respirology, Imperial School of Medicine at National Heart and Lung Institute, London, UK

Abstract:

Introduction: Primary ciliary dyskinesia (PCD) is a rare, mostly autosomal-recessive disorder of motile

cilia, characterized by chronic lung disease, rhinosinusitis, hearing impairment, and subfertility. PCD is

still often missed or diagnosed late since symptoms overlap with common respiratory complaints, but

should be considered if one or more of the cardinal clues are present.

Areas covered: We provide an overview on clinical presentations of PCD and clues for when to consider

PCD, these include unexplained neonatal respiratory distress, persistent rhinitis from the first days of

life, situs anomalies, or otorrhoea following tympanostomy tube insertion. Diagnosis is on the basis of

clinical suspicion, and an algorithm of nasal nitric oxide, ciliary beat pattern and frequency , transmission

electron microscopy , immunofluorescence of ciliary proteins and genetic studies. However, there is no

one gold-standard test as yet. We reviewed the current literature based on PubMed and Ovid databases

literature search.

Expert commentary: There is a need for increased awareness about PCD beyond specialist respiratory

clinicians and a need for standardization of PCD diagnostics internationally. Early diagnosis means that

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inappropriate treatment based on misdiagnosed conditions can be avoided, and the onset of

bronchiectasis may be delayed.

Key words: heterotaxy, rhinorrhea, neonatal respiratory distress, infertility, congenital heart disease,

chronic cough

1 Introduction:

In 1976, Bjorn Afzelius described “A human syndrome caused by immotile cilia”, describing six men and

a woman with frequent bronchitis and sinusitis, four of them had situs inversus and the men had

immotile spermatozoa, [1]. Both sperm tails and respiratory cilia were lacking dynein arms and had

impaired motility. Afzelius hypothesized that these symptoms were linked by inborn defects of motile

cilia, thus demonstrating the defect underlying the triad of signs and symptoms described by Kartagener

almost half a century earlier: the classical triad of sinusitis, bronchiectasis, and situs inversus [2]. The

clinical picture of primary ciliary dyskinesia (PCD) has since evolved from his observations. It has since

been discovered that a number of other conditions and syndromes are caused by pathology in other

types of cilia, referred to as ciliopathies. Here we will focus on the clinical features and diagnostic clues

for primary ciliary dyskinesia (PCD), the congenital condition caused by defects of motile cilia, which is

almost exclusively inherited as an autosomal-recessive trait. Although an inborn defect, PCD patients

may be diagnosed late due to the non-specific symptoms of PCD and the overlap with other, more

common respiratory ailments. In contrast to PCD, external factors like infection or tobacco smoke

exposure result in transient impairment of ciliary motility and -ultrastructure [secondary ciliary

dyskinesia]. We will elaborate on the current diagnostic criteria of PCD and recent advances in

diagnostic techniques. We will also briefly discuss the approach to patients with other ciliopathies where

respiratory complications co-exist.

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1.1 Ciliary ultrastructure and function

Ciliary biology is complex and a detailed description is beyond the scope of this review; we give a

summary here, in order to allow a better understanding of the pathology of PCD and the characteristic

findings in ciliary diagnostics.

Cilia are hair like organelles projecting from cells. They are formed by a cytoskeletal scaffold, the

axoneme, built of doublets of microtubules, covered by a membrane contiguous with the plasmalemma.

The basal body anchors and orientates the cilium in the cell membrane. Microtubules are linked by a

number of proteins crucial for ciliary function.

Cilia can be divided into three classes: primary, which are non-motile mechano- and chemosensory

organelles widely distributed throughout the body; nodal, which are found in the embryonic node

where they determine organ situs; and motile, which propel fluids along epithelial surfaces or single

organisms through a liquid medium. This type of cilia is dysfunctional in PCD. Motile cilia extend from

the epithelial surface of many mucus membranes numbering hundreds per cell (Figure 1 A and B) and

create a unidirectional, coordinated beat. They are made up of nine outer doublets and a central pair of

microtubules, often referred to as the classic 9+2 structure (Figure 1 C and D). Each outer microtubule

pair is connected to the central pair by radial spokes. Inner (IDA) and outer dynein arms (ODA) protrude

from the outer doublets. Dynein composition varies along the axoneme. For example, the ODA heavy

chain, DNAH9, localizes only to the distal end of the cilium [3]. The Dynein arms contain ATPases which

drive motility by sliding microtubule doublets relative to each other, creating a cycle of movement

consisting of a forward beating stroke and a whip-lash backward recovery beat. Dynein motor activity,

i.e. ciliary movement, is regulated by the Nexin-dynein regulatory complex (DRC), connecting outer

microtubule doublets. During the forward beating stroke, the tips of the cilia engage with the respiratory

mucus and whip back in the periciliary fluid layer during the recovery phase [4]. Respiratory cilia

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typically beat at a frequency of 8 to 20 Hz, which is dependent on temperature, and chemical and

mechanical stimulation. Lower temperatures will reduce the beat frequency, hence temperature-

controlled microscope stage chambers are usually used for high-speed video microscopy analysis [5].

Structural and functional defects of motile cilia within the lower respiratory tract, Eustachian tube and

nasal sinuses, and the oviducts lead to reduced mucociliary clearance or reduced fluid movement (there

are motile cilia in the cerebral ependyma, but these do not move mucus). The result is chronic

infections, such as otitis media with effusion associated with conductive hearing loss, sinusitis, and

chronic suppurative lung disease characterized by recurrent bacterial infection, and bronchiectasis. The

role of motile cilia in the cerebral ventricles is not completely understood. It is presumed that they effect

normal flow of cerebro-spinal fluid, hence the association with ventriculomegaly and hydrocephalus.

However, most PCD patients do not have hydrocephalus.

Recent research suggests that ciliary beating is not a perpetual, monotonous pattern, but is modulated

by external stimuli sensed via specific receptors. Motile cilia on airway epithelia express bitter taste

receptors of the T2R family. Activation of these receptors by bitter compounds denatonium, thujone,

salicin, quinine, and nicotine, triggers calcium-ion influx and increases ciliary beat frequency by 25% in

vitro [6]. In cystic fibrosis (CF), the Pseudomonas aeruginosa-derived quorum sensing molecule N-(3-

oxododecanoyl)-l-homoserine lactone has been shown to activate the bitter taste receptor T2R38 [7].

Hence in vivo, increased ciliary beat frequency, upon sensing bacterial products, via these receptors may

be the first line of defense by improving clearance of bacteria from the airway.

Sperm flagella have a similar structure to respiratory cilia, therefore ciliary defects often impair sperm

motility leading to infertility. However, not all PCD is associated with immotile spermatozoa, suggesting

that they are either under different genetic control or there is genetic redundancy [8, 9].

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Nodal cilia in the embryonic node lack the central pair and radial spokes, which results in a circular

movement in contrast to the uni-directional whip-lash movement of motile cilia. These cilia determine

organ laterality during embryonic development. Since they lack the central pair and radial spokes, PCD

causing gene mutations affecting these two structures are not associated with laterality defects [10].

Primary cilia generally have nine outer microtubule pairs, no central pair [9+0], and (unlike nodal and

motile cilia) are usually immotile [11]. There are some types of primary cilia that have a central pair of

microtubules, e.g. the inner ear sensory cilia [12]. Primary cilia have numerous functions as mechano-,

chemo-, osmo-, and photo-sensors [13, 14]. They are also critically involved in a number of signaling

pathways during development, e.g. the sonic hedgehog (SHH)- Wnt-pathways [15], the signaling

cascades of platelet-derived growth factor receptor α (PDGFRα), fibroblast growth factor (FGF), or mTOR

[16]. Defects in primary cilia can cause a wide range of diseases, termed ciliopathies. Ciliopathies may

manifest in defects of neural tube formation, bone structure, sensory organs such as the retina, inner

ear, the olfactory system, and development of polycystic kidneys, liver and pancreas. The underlying

defects are often associated with disruption of the complex intraflagellar transport. This mechanism is

crucial for the building, maintenance, and signaling function of cilia, since they lack the machinery to

manufacture proteins and all proteins must be transported from the cytoplasm to the tip of the cilium

(anterograde), and from the tip back to the cytoplasm again (retrograde) [17, 18]: In retinitis

pigmentosa, defects in the Retinitis Pigmentosa GTPase Regulator (RPGR), a protein crucially involved in

trafficking in rod and cone cilia, lead to death of photoreceptors and consequent blindness. In Bardet-

Biedl syndrome, disruptions of the Bardet-Biedl syndrome protein complex, which shuttles membrane

proteins into the primary cilium, gives rise to a variety of organ manifestations (Table 2).

In clinical practice, the dichotomous classification of cilia is often not as clear. In Usher’s syndrome,

forexample, although it is classified as a nonmotile ciliopathy, nasal ciliary function is also impaired [19],

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suggesting single mutations can affect both, motile and primary cilia. Recently, motile cilia dysfunction

and low nasal NO have also been described in patients with Sensenbrenner syndrome and Jeune

syndrome, two ciliopathies that may have respiratory problems due to restrictive lung diseases caused

by thoracic deformities [20]. These patients have compound heterozygous mutations in WDR35, a gene

that had only been known to be required for primary cilia function. However, WDR35 siRNA knockdown

in human respiratory epithelial cultures disrupted motile ciliogenesis, indicating that mutations in

WDR35 can perturb both motile and primary cilia function.

1.2 Epidemiology

The prevalence of PCD is difficult to determine, with an estimated incidence of 1 in 15,000 to 1 in 30,000

live births according to Orphanet [21]; others suggest between 1:10,000 [22] and 1:40,000 [23]. These

figures are likely to underreport the true prevalence, since milder cases and particularly those without

situs inversus may not get diagnosed. A survey across European centers showed that reported cases in

children between the age of 5 and 14 years were highest in Cyprus, Switzerland and Denmark, with 111,

47, and 46 per million, respectively. Overall, 48 % of these patients had situs inversus [24]. Currently,

international databases are being established to gather more information about the prevalence of this

condition [25].

2. Clinical phenotypes of PCD

2.1 When to suspect PCD?

PCD can present at any time from antenatal to adult life. Hence all clinicians, but especially

obstetricians, paediatricians, ENT specialists, pulmonologists, fertility specialists, cardiologists,

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neurologists and neurosurgeons should be aware of the diagnostic clues of PCD and refer patients for

testing. It is not uncommon that problems outside a particular specialty are missed or recognized late,

resulting in missed or late diagnosis of PCD, e.g. that the snuffly child with complex congenital heart

disease has underlying PCD may be missed.

Because many clinical symptoms of PCD are rather non-specific and there is significant overlap with

common respiratory ailments, PCD is often considered quite late and referral may be delayed

significantly. The other important implication of the non-specific presentation of PCD is that clinical skills

are increasingly important in determining who to refer for the ever more complex array of diagnostic

tests; it is unfeasible for every child with a cough or runny nose to have transmission electron

microscopy performed, for example. A recent study by the North American Genetic Disorders of

Mucociliary Clearance Consortium has identified laterality defect, unexplained neonatal respiratory

distress, early onset, year round nasal congestion, and early onset, year-round wet cough as clinical

features statistically predictive of PCD in children and young people referred due with a high suspicion of

PCD [26]. These and other diagnostic clues for PCD at different ages are summarized in Table 1.

Although there is no study proving that early diagnosis improves outcome, it is likely that diagnosis and

treatment before the development of irreversible bronchiectasis would be beneficial; and it could also

prevent the deployment of inappropriate treatment, for example inhaled corticosteroids for a mistaken

diagnosis of asthma. There is growing evidence that lung function is significantly worse in patients who

are diagnosed late or in adulthood [27, 28]. Alanin et al. found that patients who were diagnosed after

preschool age (>6 years) were more frequently colonized with Pseudomonas aeruginosa [29]. It should

be noted though that the detrimental effect of Pseudomonas aeruginosa on longitudinal lung function in

PCD is less well established than in CF [30].

2.2 Antenatal features

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As described above, cilia in the embryonic node determine organ situs. Hence mirror image

arrangement of organs, heterotaxy, polysplenia (left isomerism) or asplenia (right isomerism) on

antenatal ultrasound scanning can be a diagnostic clue to PCD, especially in association with complex

congenital heart disease [31]. These babies should be referred for PCD diagnostics after birth. It is

important to counsel parents though that not all individuals with mirror image arrangement actually

have PCD [32]. In our diagnostic clinic, only 25% of individuals with situs inversus referred for testing

actually have PCD, and this is widely reflected in other centers. This is because heterotaxy has a more

complex, largely non-monogenic etiology [33, 34]. Mirror image arrangement is found in 40-50% of PCD

patients [31, 35], and around 12.1% of PCD patients show heterotaxy of some degree [36]. Ciliary

dysfunction can affect the development of the cerebral ventricles, hence babies with cerebral

ventriculomegaly should be considered for referral as well, although hydrocephalus due to ciliary

impairment, which is well described in animal models [37, 38], is rare in humans [39-42]. A positive

family history of PCD or other ciliopathies should always prompt a referral for testing of the newborn,

even in the absence of antenatally detected situs anomalies. Chorionic villus sampling for genetic testing

can be considered if the relative’s genotype is known.

2.3 Neonatal presentation

All babies with situs anomalies, whenever the diagnosis is made, should be investigated for PCD. PCD

with heterotaxy is also strongly associated with complex congenital heart disease (CHD). Early

assessment of ciliary function in CHD patients with heterotaxy is important for their pre- and

postoperative management, as well as early institution of treatment to try to prevent bronchiectasis.

CHD patients with heterotaxy have a higher rate of postsurgical respiratory complications, and morbidity

and mortality than those with normal situs [43, 44]. This may be because they have defects of motile

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cilia underlying their cardiovascular diagnosis. However, it must be said that there is no study showing

beneficial effects of an early diagnosis of ciliary dysfunction on postsurgical outcomes.

In the absence of situs anomalies, diagnostic clues for PCD can be less obvious. An important indicator of

PCD is unexplained neonatal respiratory distress in otherwise healthy term infants; a recent case-control

study showed that up to 84% of PCD patients have a history of unexplained oxygen requirement after

birth [45]. Children who were diagnosed with PCD later in life had a longer duration of oxygen

requirement than controls (mean 15.2 days versus 0.8 days), a higher frequency of lobar collapse (48%

versus 0%), and a later onset of respiratory distress (12 hours versus 1 hour). Chronic rhinorrhea from

the first day of life is almost a hallmark of PCD, and most parents will remember if a baby had a runny

nose from the first day of life. This must be distinguished from the baby who gets a first viral cold and

rhinitis in the first few weeks of life.

2.4 Presentation in Childhood

Many PCD patients will present in childhood. A review of medical records in a British paediatric PCD

referral center showed that the median age at referral was 4 years [46]. A survey of 223 PCD centers

across 26 European countries revealed that the median age at diagnosis in Europe was 5.3 years. The

age at diagnosis seems to be largely affected by two factors: whether or not the child presents with

obvious signs like situs inversus and where in Europe the child presents. In children with mirror image

arrangement the median age at diagnosis was 3.5 years (still a very long delay!), whereas children with

situs solitus were diagnosed significantly later, at median 5.8 years of age. With regards to regional

differences, the survey showed that children in Western Europe, including Britain, were diagnosed at a

younger age (5.0 and 4.8 years) than children in regions with lower health expenditures, e.g. Eastern and

Southern Europe (6.8 and 6.5 years) [24].

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In the absence of situs anomalies, the presentation of PCD in childhood is often non-specific, and many

features of PCD are so commonly seen in preschool children that PCD is often considered late and

delayed referral is common. It needs to be emphasized that situs anomalies are not a prerequisite of

PCD, and taking a focused history is essential in the diagnostic workup. Clues from the patient’s history

are neonatal dyspnea/distress, pneumonia or rhinorrhea starting in the neonatal period (above),

recurrent ear infections and middle ear effusions resulting in hearing loss and speech delay. Persistent

malodorous otorrhoea following insertion of ventilation tubes with minimal impact on hearing loss [47,

48], a history of atypical asthma or wheeze not responsive to conventional treatment, as well as a family

history of PCD or atypical asthma should all warrant further investigation. Agenesis of paranasal sinuses

may also be found [49]. As a mostly autosomal recessive trait, PCD is more common in consanguineous

families.

Typical features on presentation include chronic daily wet cough (which should be distinguished from

recurrent acute cough), persistent rhinitis or nasal congestion, recurrent otitis media with effusion

otorrhoea and hearing impairment. Most cases of conductive hearing loss resolve by the age of 12 years.

Up to 70% of PCD patients have some degree of bronchiectasis [50], and upper and lower respiratory

tract problems require intensive treatment, consisting of daily chest physiotherapy, nasal douching, and

a low threshold for antibiotic treatment. Although generally rare in children with PCD [51], we and others

are increasingly seeing patients with nasal polyposis. Polyps often contribute significantly to general

morbidity and some patients may require polypectomy.

2.5 Presentation in Adolescence and Adult Life

Patients who are diagnosed as adults will have similar features to paediatric patients although middle

ear effusions and hearing loss may have resolved. There may be a history of hearing impairment in

childhood and complications after insertion of ventilation tubes, including persistent otorrhoea and lack

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of improvement in hearing loss. Taking a detailed history is crucial. Most adults with PCD will have

established bronchiectasis and a productive cough, chronic rhinosinusitis, and some will have nasal

polyposis [52]. PCD in adult life is less well characterized than childhood PCD, and longitudinal data are

scarce. Longitudinal data from the adult PCD service at the Royal Brompton Hospital indicates that the

older the age at diagnosis the lower the forced expiratory volume in 1 second (FEV1%) predicted and the

higher prevalence of infection with Pseudomonas aeruginosa [28] . Also lung function decline over time

seems to be determined by the severity of bronchiectasis and the type of ciliary defect, i.e. patients with

microtublar defects tend to have the greatest decline in lung function [28, 53].

Some adult patients will be diagnosed in the context of investigations for infertility, which affects

approximately 50% of men with PCD [8]. In women, impaired ciliary function in the fallopian tubes can

cause sub-fertility or ectopic pregnancy due to impaired transport of the ovum [54-56].The exact

prevalence of fertility problems in women with PCD has not yet been established.

3 Ciliopathies

With our growing understanding of ciliary biology and function beyond the role of motile cilia, the

spectrum of conditions known to be associated with defects in other types of cilia has been expanding

exponentially. Ciliopathies can be non-syndromic or syndromic, and can affect all organ systems

comprising primary cilia, such as the cardiovascular system, kidneys, liver, retina, skeleton, and cerebral

ventricles. Reviewing the full spectrum of ciliopathies is beyond the remit of this article, but it is

important for clinicians looking after these patients to bear in mind that if there are respiratory

symptoms, such as persistent rhinitis and wet cough, they should be referred for evaluation of ciliary

motility. Similarly, chest physicians should be vigilant to signs and symptoms of ciliopathies beyond the

respiratory tract and refer PCD patients to appropriate specialists.

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Respiratory symptoms in CHD patients can easily be attributed to the underlying cardiac disease, hence

it is important to be aware of this association. Polycystic kidney disease (PKD), nephronophtisis, and

renal dysplasia have been found to be associated with an increased prevalence of bronchiectasis [57,

58]. Ciliopathies are caused by an abundance of genetic defects, and depending on the resulting cilia

defect can manifest in more than one organ system [59-61]. Likewise, the same family can have more

than one ciliopathy syndrome in the kindred.

An overview of ciliopathies is presented in Table 2 but have been reviewed in detail elsewhere [62].

4. Diagnostic testing for PCD

PCD is rare, and as such is usually considered as one of a number of differential diagnoses after more

common respiratory conditions have been excluded, unless there are hallmark features of PCD present,

such as situs inversus. There is no “gold standard” test for diagnosing PCD, and no currently available

single test or combinations of tests is accurate enough for making a diagnosis under all circumstances.

Many cases can be diagnosed very easily, but there are borderline cases which are difficult to resolve, as

is the situation with CF, for example. Hence diagnosis is usually made by a combination of tests together

with a suggestive clinical history (Figure 2). All PCD tests require specialized equipment and highly skilled

staff, and should only be performed in experienced centers. The diagnostic challenge starts earlier

though, namely identifying patients who merit referral for diagnostics because most symptoms of PCD

are non-specific. A tool designed to help in making a decision for referral has recently been validated in

adults: PICADAR (PrImary CiliARy DyskinesiA Rule) is a seven-variable-predictor model that uses clinical

information (“full-term gestation”, “neonatal chest symptoms”, “admission to neonatal intensive care”,

“chronic rhinitis”, “ear symptoms”, “situs inversus” and “congenital cardiac defect”) to calculate a

numeric score to predict a patient’s likelihood of testing positive for PCD. The higher the score, the more

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likely a patient is to have PCD. In this first study on patients consecutively referred for PCD testing,

PICADAR had a sensitivity and specificity of 90% and 75% respectively, for a cut-off score of 5 points, and

the area under the curve for the internally and externally validated tool was 0.91 and 0.87 [63]. It may

prove a valuable tool in helping respiratory specialists decide whom to refer to PCD centers. Validation

in paediatric patients is awaited.

4.1 Nasal NO (nNO)

Expression of nitric oxide synthase-2 is reduced in PCD, resulting in very low nNO [64]. A low nNO

measure by itself, with a 30 nL/minute cut-off, has good sensitivity and specificity (91% and 96%,

respectively) [65]. In most North American PCD centers however, a cut-off of 77 nl/minute is used,

which showed excellent sensitivity and specificity (98% and >99.9%, respectively) in multi-center studies

using a standardized protocol [66]. Some authors advocate nNO as a screening test for PCD since it is

easy to measure in clinical routine, and normal nNO together with a low risk history almost excludes

PCD. However, there are patients with an unequivocal diagnosis of PCD who have a normal nNO. Nasal

NO is usually measured during a velum closure maneuver such as a breath hold, which limits its use to

patients of school age. Some studies have explored nNO measured during tidal breathing, and a recent

meta-analysis concluded that either technique can be used with high diagnostic accuracy [67]. Although

this paper suggests that nNO could be used in younger children as soon as they manage to perform

steady tidal breathing, results of this meta-analysis should be interpreted with caution. For the analysis

of the diagnostic performance of nNO testing during breath hold, twelve studies comprising a total of

325 PCD patients and 711 non-PCD subjects were included, but only seven studies comprising 210 PCD

patients and 471 non-PCD subjects were eligible for analysis of the tidal breathing technique. Summary

sensitivity and specificity were similar for both techniques, with a sensitivity of 0.95 (95% confidence

interval (CI) 0.91-0.97) and specificity of 0.94 (95% CIs 0.88-0.97) for the breath hold technique, and a

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sensitivity of 0.93 (95% CIs 0.89-0.97) and specificity of 0.95 (95% CIs 0.82-0.99) for the tidal breathing

technique. However, the lower 95% CI limit for specificity of the tidal breathing technique (82% versus

89% for the velum-closure technique) suggests that considerably more patients who do not have PCD

will have low nNO readings and will need further diagnostic testing. Therefore decisions to proceed to

other diagnostic testing should not be influenced by tidal breathing nNO measurement alone, and

diagnostics must be undertaken where clinical suspicion dictates. It is an advantage that nNO

measurements during tidal breathing require only passive cooperation from the patients, and can hence

be performed on infants, and normative values have recently been proposed [68]. A number of

conditions can also result in low nNO, ranging from any form of nasal obstruction to chronically

debilitating conditions like CF [69, 70] and diffuse panbronchiolitis [71]. A recent analysis of 282

consecutive referrals to a tertiary diagnostic center for PCD concluded that nNO had good sensitivity and

specificity in the referral population, but would over diagnose PCD if extended to a general population

[72].

However, nNO can be normal in some ultrastructural defects, and with a sensitivity of 91%, a normal

nNO in isolation would miss about 10% of PCD cases [65]. Hence most experts advise interpreting nNO

levels in combination with other diagnostic tests, as described below.

4.2 Ciliary motility assessed via high-speed video microscopy analysis

Analysis of the ciliary beat pattern and frequency in ciliated epithelium obtained by nasal or

bronchoscopic brush biopsies, via high-speed video microscopy analysis (HSVMA) and digital recording,

has excellent sensitivity and specificity for PCD [65]. Particular beat patterns have even been linked to

specific ultrastructural defects [4, 65, 73](Table 2). However, some mutations may cause very subtle

changes [74]or even result in a range of beat pattern anomalies, consistent with the variable severity of

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the ultrastructural defect [75]. Brush biopsies from patients with nasal congestion and infection, during

nasal oxygen therapy or after a nasogastric tube has been in place are often of poor quality and barely

ciliated, increasing the risk of misinterpretation. Repeat brushings or primary cell culture [76] should

always be performed when results are inconclusive. Following cell culture, secondary ciliary dyskinesia is

reduced compared to the initial sample, while primary ciliary dyskinesia is still evident.

4.3 Techniques to assess ciliary structure - transmission electron microscopy, electron microscopic

tomography, and immunofluorescence microscopy

4.3.1 Transmission electron microscopy

In up to 80% of patients, PCD is caused by defects of the ciliary ultrastructure which can be detected by

electron microscopy, but electron microscopy in isolation will not detect all PCD patients, since in some

cohorts up to 33% may have normal ciliary ultrastructure [77]. Any ciliary component can be defective

or missing, with lack of outer dynein arms (up to 43%) and a lack of both outer and inner dynein arms

(up to 24%) being the most common, followed by central pair defects with transposition, inner dynein

arm defects with microtubular disarrangement and isolated inner dynein arm defects [78] (Figure 3,

Table 3). However, isolated defects of inner dynein arms should always be treated with caution, since

these may be secondary defects and patients may have normal ultrastructure on repeat testing,

questioning the diagnosis of PCD [79]. Rarely there is a reduced generation of motile cilia resulting in

only one or two cilia per cell (Table 3). Diagnosis may need to be confirmed using primary cell culture to

exclude secondary loss of cilia. Of note, secondary ultrastructural changes are also common in

secondary ciliary dyskinesia [78]. In asthma, for instance, the number of secondary defects correlates

with disease severity [80]. Secondary changes include compound cilia (where several axonemes are

found within one membrane), absence of one or both of the central pair of microtubules, microtubular

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doublet disarrangement, single microtubules, extra microtubules and disorientation. Culture of ciliated

epithelium can be useful in eliminating secondary ultrastructural defects [81].

4.3.2 Electron microscopic tomography

Recently, electron tomography has allowed for 3D reconstruction of cilia, which allows for a more

detailed analysis in cases with normal transmission electron microscopy and a high clinical suspicion of

PCD, for example, in patients with a central pair and transposition defect. Electron tomography has

shown that the central pair rotates at the base of the axoneme and this can appear as an apparent

intermittent loss of the central pair in longitudinal section[82]. Other defects with normal electron

microscopy which are detected by electron microscopic tomography include DNAH11 defects, in which

only the heavy chain of the outer dynein arm is missing [74], or the absence of Hydin, in which this

technique revealed that projections associated with the central pair apparatus are absent [10], and

Nexin link defects. Currently, electron tomography is highly specialized and time-consuming and as such

is reserved for difficult to diagnose cases only.

4.3.3 Immunofluorescence microscopy

Some ciliary proteins can be directly stained for by immunofluorescence microscopy, and their absence

[94] or mislocalization [3] can confirm the diagnosis of PCD. An example from our diagnostic laboratory

is presented in Figure 4. Immunofluorescent microscopy is not yet widely available or used in routine

diagnostics, but shows excellent accuracy in research settings [3]. It has a number of advantages, such as

a much shorter turnaround time than transmission electron microscopy in most centers, allowing faster

and more cost-effective diagnosis, but to date there is no published evidence for its accuracy in a

diagnostic setting. Defects of one specific ciliary element could be the result of mutations in multiple

genes. Since immunofluorescence microscopy detects the protein downstream from genes, it may have

a higher chance of detecting abnormalities than looking for a single or very few gene mutations.

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However, only a small number of ciliary-protein specific antibodies are currently available, so some

cases of PCD will be missed by immunofluorescence .

4.4 Genetics

PCD is genetically heterogeneous. Cilia are complex, composed of many different proteins, and

therefore many genes can potentially cause PCD. Thirty-five genes have been associated with PCD to

date (Table 3). It is estimated that in about 65% of patients with a clinical PCD phenotype a known PCD

mutation can be found [83]. However, since diagnosis is difficult, and especially in mild cases, it is

impossible to be exact about the percentage of PCD cases which can be accounted for by the current

known genes.

Genetic testing panels for PCD are already used clinically and are both sensitive and specific in

identifying a large proportion of PCD patients. In our PCD cohort at the Royal Brompton Hospital, the

commonest disease-causing mutations are DNAH5 (18.5%), DNAH11 (9.3%), and CCDC40 (6.6%) in 151

genotyped patients. DNAH5 mutations are found in half of our cases with outer dynein arm defects

(51%). Half of our patients with normal electron microscopy findings have DNAH 11 mutations (48%),

and around one third of patients with inner dynein arm mutations with microtubular disarrangement

have CCDC 40 mutations (35%). An overview of PCD-mutations, the affected protein, and the resulting

ultrastructural defect are given in Table 3. Gene discovery programs for rare diseases, including PCD, are

enjoying an unprecedented focus and drive, e.g. the Genomics England 100,000 genome project. This

large national sequencing program aims to recruit thousands of patients, and their families, with rare

diseases to identify new disease-causing genes. These data may offer patients in whom no known

mutation had previously been found a genetic diagnosis of PCD. Improved genetics will also open up the

possibility of antenatal diagnosis. It should be noted though, that novel genetic mutations may not

17

necessarily cause functional defects, hence assessment of cilia motility and structure will be needed to

establish gene-function links. It is also likely that many new mutations may only account for a small

number of patients each, resulting in a large number of eventual disease causing genes.

4.5 “Atypical PCD” - Difficult diagnoses in patients with unclear test results

No diagnostic test available today, or combinations thereof, is capable of diagnosing all PCD. There will

be difficult cases with discordant or even negative test results despite a typical clinical phenotype. These

cases certainly merit follow up and repeat testing at regular intervals as well as testing for differential

diagnoses. We believe it is in the best interest of the symptomatic patient to assume it is PCD, treat

disease manifestations as they occur, and ensure general pulmonary health measures are adopted.

Airway clearance through regular exercise and physiotherapy, avoidance of pollutants and tobacco

smoke, immunizations including the annual influenza vaccine, and having a low threshold for antibiotic

treatment for increased respiratory symptoms should all be employed for any patient with a clinical

phenotype consistent with PCD.

5. Overall summary and conclusions

PCD is a rare disease, and although awareness of the condition is increasing, it is likely still

underdiagnosed. Patients with a classical manifestation of situs inversus are more likely to be diagnosed

early than those with more non-specific symptoms, such as chronic wet cough and rhinorrhea.

Awareness of PCD across medical specialties is crucial, since PCD patients can present at literally any age

with symptoms ranging from unexplained neonatal respiratory distress, chronic wet cough and

rhinorrhea during childhood, to unexplained bronchiectasis, chronic sinusitis or male infertility in adult

life. There should be a low threshold for referral in patients with a high risk history or hallmark signs like

situs inversus. A number of complex syndromes and congenital heart diseases are also associated with

18

ciliary dysfunction, and these patients are at higher risk of respiratory complications. PCD diagnosis is

complex and requires expertise, and should therefore only be performed at specialist centers. A

combination of diagnostic tests together with the typical history and symptoms is needed to make a

diagnosis of PCD. If diagnostic tests are negative, but there still is a high suspicion of PCD, re-testing

and/or expanding the panel of diagnostic tests is indicated. Although beyond the remit of this review, it

is important to stress that treatment of PCD requires multidisciplinary teams, and every patient should

have access to a dedicated PCD service (as with CF).

6. Expert commentary

Currently there is no consensus on a diagnostic algorithm of PCD, and late referral and diagnosis is

common. The European Respiratory Task Force on Primary Ciliary Dyskinesia in Children has published a

consensus statement on diagnostics and treatments in children in 2009 [84], and an updated version is

in preparation. The Genetic disorders and mucociliary clearance consortium in North America has

recently published consensus recommendations for testing, monitoring, and treating PCD patients [85].

These recommendations have been adopted by the governing board of the PCD Foundation. With

networks of scientists, clinicians, and patients growing on a national and international level

(http://bestcilia.eu, http://syscilia.org), we anticipate that multi-center and international PCD registries

will thrive over the coming years.

7. 5-year view

We anticipate that the formation of international networks and multi-center studies will result in the

discovery of more PCD-causing mutations and increase awareness and harmonize the standard of PCD

diagnostics across Europe and North America, resulting in the implementation of standardized

diagnostic algorithms. New screening tools such as diagnostic predictive models could help clinicians in

19

primary care to identify patients who should be referred for PCD diagnostics, and new diagnostic

techniques such as immunofluorescence microscopy could be a simpler and more accessible test.

Diagnostic delay should decrease, and patients should have access to specialized diagnostic centers and

clinical services.

8. Key issues – 8-10 bullet points

PCD is a rare disease and remains underdiagnosed

Although an inborn condition, PCD patients can present at any age, from antenatal diagnosis of

situs inversus to diagnosis of bronchiectasis and male infertility in adults

Diagnostic clues that should prompt further investigations include situs inversus totalis or

heterotaxy, unexplained neonatal respiratory distress in term babies, children with chronic

productive cough, bronchiectasis of unknown cause or severe upper airway disease, and male

infertility with immotile spermatozoa.

Early diagnosis is crucial to try to minimize the development of bronchiectasis and irreversible

lung damage.

Diagnosing PCD is complex, usually made by a combination of diagnostic tests, each requiring

specialized equipment and skilled staff with experience in assessing ciliary function and

structure, and should therefore be performed at specialized PCD diagnostic centers.

A negative test does not exclude PCD. If there is a high suspicion of PCD from a clinical point of

view, re-testing and/or expanding the panel of diagnostic tests is indicated. Such patients should

be treated as if they have PCD, with airway clearance, general lung health measures and

antibiotics for respiratory infection

20

Defects in primary cilia can cause a variety of organ manifestations and syndromes, termed

ciliopathies. Some ciliopathies have been associated with impaired function of motile cilia,

putting patients at risk of respiratory complications.

All patients with a confirmed or probable diagnosis of PCD should have access to dedicated PCD

clinical services.

Acknowledgements

We thank A. Shoemark, A. Dewar, T. Cox, M. Dixon and T. Burgoyne for providing microscopy images

and illustrations for this article, and Hanah Mitchison and Sunayna Best for genetics data of the

Brompton PCD cohort.

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31

Table 1. Clinical features associated with PCD and diagnostic clues suggestive of PCD

Clinical signs and symptoms associated

with PCDDiagnostic clue for PCD

History Consanguinity

Family history of PCD, atypical

asthma, unexplained

bronchiectasis/respiratory

problems, other ciliopathies

Antenatal Situs inversus totalis or heterotaxy

Cebrebral ventriculomegaly

NeonatalUnexplained respiratory distress in term

infants, rhinitis from the first days of life

Prolonged oxygen requirement,

lobar collapse, unexplained

neonatal pneumonia

Situs anomalies, splenic anomalies Heterotaxy, asplenia, polysplenia

Cardiac defects/complex congenital heart

diseaseassociated with heterotaxy

Hydrocephalus

Childhood Chronic rhinitis From day 1 of life

Chronic cough Can be wet or wheezy, children

often do not expectorate

Intractable wheezeBeing treated for atypical

asthma

Recurrent otitis media with effusion Resistant to treatment

Chronic otitis media with exsudate Post tympanostomy otorrhoea

32

Conductive hearing lossLack of improvement after

tympanostomy

Bronchiectasis No other cause identified

Adolescence/Adult life Chronic sinusitis

Bronchiectasis

Male infertility Immotile spermatozoea

Sub-fertility in females and/or ectopic

pregnancies

33

Table 2. Overview of Ciliopathy syndromes

Cilioapthy syndrome Features

Alstrom’s syndrome

Cone-rod dystrophy, obesity, sensineuronal hearing loss, dilated

cardiomyopathy, insulin resistance syndrome, developmental delay,

male hypogonadism.

Bardet-Biedl syndrome

Cone-rod dystrophy, polydactyly, obesity, hypogonadism, renal disease,

developmental delay, brachydactyly or syndactyly, hypertonia, anosmia,

diabetes, fibrocystic liver disease, Hirschsprungs disease, cardiovascular

anomalies, and sometimes craniofacial abnormalities

Joubert syndromeHypotonia, ataxia, psychomotor developmental delay, irregular breathing

pattern, oculomor apraxia with cerebellar and brainstem abnormalities.

Meckel-Gruber

syndrome and

Overlap with Joubert syndrome plus posterior fossa defects, cystic

dysplastic kidneys, polydactyly, and hepatic duct proliferation

Jeune asphyxiating

thoracic dystrophy

Narrow thorax with short ribs, hypoplastic ileal wings, trident acetabular

roofs and rhizoelic limb shortening. Polydactyly, brachydactyly,

hydrocephalus, retinins pigmentosa and renal- and liver disease can

occur

Ellis van Creveld

syndrome

Overlap with Jeune asphyxiating thoracic dystrophy, but nail and teeth

dysplasia, characteristic appearance of the upper lip and often cardiac

defects

Sensenbrenner Similar in appearance to Ellis van Creveld, but has additional features of

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syndrome renal cysts with dolicocephaly, sparse and slow-growing hair, epicanthal

folds, disorders of dentition, brachycephaly and a narrow thorax

Leber’s congenital

amaurosis

Early presenting poor visual function, nystagmus, photophobia,

keratoconus, hperopia

Senior-Loken syndrome Retinitis pigmentosa, renal disease

Orofacial digital

syndrome

Facial and digital malformations, cystic kidney disease, CNS

abnormalities, organ situs disorders.

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Table 3: Common PCD-causing mutations, resulting defect and microscopy findings

Gene Ref Ciliary component affected Ultrastructural

defect

Ciliary beat

pattern

DNAH5 [3] ODA: Heavy chain dynein Absent or

shortened ODA

Mostly immotile

with some

residual

movement

DNAH11 [74] ODA: Heavy chain dynein in

proximal region of the axoneme

Normal

Absent heavy

chain in proximal

portion in ET

Hyperkinetic,

reduced

amplitude with

stasis in the

proximal portion

and/or

completely

immotile.

DNAI1

DNAI2

[86]

[87]

ODA: Intermediate chain dynein Absent ODA Immotile

DNAL1 [88] ODA: Light chain dynein Absent ODA Impaired motility

RSPH1

RSPH4A

RSPH9

[89] Radial spoke head proteins intermittent

central-pair loss

and transposition

Circling on top

view

RSPH3 [90] Radial spoke component

(unknown)

Reduction in

number of radial

Some immotile

cilia

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spokes,

intermittent

central MT pair

abnormalities

Motile cilia with

reduction in

beat amplitude

CCDC39

CCDC40

[91]

[92]

Nexin-Dynein Regulatory

Complex assembly

IDA and

microtubule

disorganization

Stiff cilia with

reduced

amplitude

CCDC65

CCDC164

[93]

[73]

Nexin-Dynein Regulatory

Complex

Subtle

microtubule

disorganization

Hyperkinetic,

subtle reduction

in beat

amplitude

GAS8 [94] Nexin-Dynein Regulatory

Complex

Normal/

Subtle

microtubule

disorganization

Subtle reduction

in beat

amplitude

HYDIN [10] Central microtubular pair

projection protein

Majority of cilia

normal, some

central pair

abnormalities.

Absent C2b

projection in ET

discoordinated

CCDC103 [75] outer dynein arm assembly

factor

Variable:

Absent IDA +

ODA

missense

mutation

Partial absence

IDA + ODA

Variable:

Immotile cilia

Missense

mutation

reduced

amplitude, static

patches and

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Normal

ultrastructure

normal beat

pattern

CCDC114

CCDC151

ARMC4

[9]

[95]

[96]

ODA docking complex Absent ODA Immotile

ZMYND10

LRRC6

DNAAF5

(HEATR2)

C21ORF59

DNAAF1

(LRRC50)

DNAAF2 (KTU)

DNAAF3

DYX1C1

(DNAAF4)

SPAG1

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

Proteins involved in cytoplasmic

assembly of dynein arm

complexes (“preassembly”

proteins)

Absence or

partial absence of

IDA + ODA

Immotile

TXND3

(NME8)

[106] Component associated with

ODA

Heterogenous

ultrastructure:,

normal, absent or

shortened ODA

Immotile

CCNO

MCIDAS

[107]

[108]

Factors involved in human

multiciliated cell differentiation

Ciliary depletion

(1 or 2 per cell)

CCNO:

Mislocalized

basal bodies

Reduced cilia

numbers. May

be

misinterpreted

as secondary

ciliary absence

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MCIDAS:

Mislocalized

basal bodies

additional lack of

axonemal

components

OFD1

RPGR

[109]

[110]

PCD co-segregation with other

syndromes: Retinitis

pigmentosa GTPase regulator

(RPGR) – associated with X-

linked retinitis pigmentosa:

OFD1: centrosome and basal

body protein - associated with

x-linked orofacialdigital

syndrome.

Normal

ultrastructure,

RPGR: disturbed

cilia orientation,

OFD1:

disorganized

ciliary beat

ODA: outer dynein arms, IDA: inner dynein arms, MT: microtubules, ET: electron tomography.

39

Figure 1. Normal ciliary ultrastructure. A. Light microscopy DIC image of single ciliated cell. Unstained. B.

Transmission Electron Microscopy image of normal columnar epithelium from a nasal brushing. C.

Transmission Electron Microscopy of normal ciliary ultrastructure. D. Diagram of ciliary ultrastructure.

(all courtesy of A. Shoemark). Nexin-DRC: Nexin-dynein regulatory complex.

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Figure 2. Diagnostic pathway in the United Kingdom National PCD Service. On clinical suspicion of PCD,

all patients should have other causes of chronic suppurative lung disease excluded. Differential

diagnoses include cystic fibroisis, asthma, immunodeficiencies, chronic pulmonary aspiration, and

persistent bacterial bronchitis. First line testing confirms a diagnosis of PCD if 2 or more tests are

positive (pathway on the right). If first line tests cannot confirm PCD, i.e. findings are inconclusive, but

there is a high clinical suspicion, second line tests are performed (bottom pathway). If first line tests are

negative, but there remains a clinical suspicion of PCD, first line test are repeated (left upper pathway).

If first line testing excludes PCD, differential diagnosis should be reconsidered (left bottom pathway).

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Videomicroscopy

Figure 3. Electronmicroscopy images of PCD defects. A. Inner and outer dynein arm defect, B. Outer

dynein arm defect, C. Inner dynein arm and microtubular disorganisation, D. central complex

defects. (A and B courtesy of A. Shoemark, C courtesy of A. Dewar & T. Cox and D, courtesy of M.

Dixon and T. Burgoyne)

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Figure 4. A. Localization of ciliary proteins studied by immunofluorescence stainings. B.

Immunofluorescence microscopy image from a patient with PCD with a dynein assembly defect (lower

panel) and a healthy control (upper panel). DAPI (blue) depicts the cell nucleus, acetylated α tubulin

(green) stains cilia microtubules, anti-DNALI1 antibody (red) stains light intermediate chain dynein of

IDA. The merged image illustrates absent DNALI1 staining from the axoneme. (courtesy of A. Shoemark)

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