Two Distinct Clinical Presentations of Primary Ciliary Dyskinesia (PCD): Diagnostic Utility of Whole-Exome Sequencing in a Genetically Heterogeneous Disorder

Nasal Nitric Oxide: Clue to a Diagnosis of Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a rare hereditary disorder characterized by defective motile cilia and impaired mucociliary clearance in the respiratory tract. In recent years, significant advances have been made in both clinical and basic research on PCD, especially regarding the identification of newly discovered pathogenic genes. However, early diagnosis and treatment of PCD in China still face considerable challenges. In response, a new expert consensus on the diagnosis and treatment of PCD in China has been developed. This consensus was initiated by the Genetic and Rare Lung Disease Group (Preparatory) of the Chinese Thoracic Society of the Chinese Medical Association and the China Bronchiectasis Clinical Diagnosis and Research Alliance. This consensus is based on extensive opinion collection, literature review, online and offline discussions, and previous expert consensus on PCD in China. The consensus addresses 9 core issues related to PCD, including its clinical presentation, diagnosis, treatment, patient management, and follow-up. Nine recommendations have been formulated to improve the diagnostic and therapeutic approaches to PCD in China.Issue 1: Which high-risk populations should undergo PCD screening?Recommendation 1: It is recommended to screen for PCD in patients with a history of bronchiectasis plus any one of the following: situs inversus; chronic sinusitis or nasal polyps; recurrent or chronic otitis media; infertility/subfertility; consanguinity, or a sibling or first-degree relative with a confirmed PCD diagnosis (1C).Issue 2: What is the value of imaging examinations in the diagnosis and management of PCD?Recommendation 2: For the high-risk PCD populations mentioned above, we recommend performing active imaging investigations-including chest high-resolution computed tomography (HRCT), sinus CT, and cardiac ultrasonography-to aid in the early detection of clinical features indicative of PCD, such as bronchiectasis, sinusitis, and situs inversus. In addition, for patients diagnosed with PCD, we recommend regular follow-up chest HRCT to monitor the progression of pulmonary lesions (1C).Issue 3: What is the value of transmission electron microscopy (TEM) in diagnosing PCD?Recommendation 3: For patients with a high clinical suspicion of PCD, we recommend transmission electron microscopy (TEM) for the assessment of ciliary ultrastructure. TEM serves as a direct diagnostic tool for suspected PCD. A definitive diagnosis of PCD can be made if a class 1 defect is identified (1B). If a class 2 defect is observed, the results should be interpreted in conjunction with other diagnostic findings.Issue 4: What is the value of genetic testing in PCD diagnosis, and which genetic testing strategy should be used?Recommendation 4: For patients with a high clinical suspicion of PCD, we recommend performing genetic testing using whole-exome sequencing (WES) at an early stage. WES serves as a direct diagnostic tool for suspected PCD. A definitive diagnosis can be made when biallelic pathogenic or likely pathogenic variants in known PCD-related genes are identified. During WES data analysis, it is essential to include the assessment of copy number variations (CNVs) (1C).Issue 5: What is the value of nasal nitric oxide (nNO) measurement in diagnosing PCD?Recommendation 5: nNO measurement is recommended as an adjunctive diagnostic test for suspected PCD. Using a threshold of 77 nl/min, patients with nNO values below this threshold should undergo further confirmatory PCD testing (2B).Issue 6: What is the value of high-speed video microscopy analysis (HSVA) in diagnosing PCD?Recommendation 6: In clinical settings with access to HSVA, HSVA is recommended as an adjunctive diagnostic test for suspected PCD. A positive HSVA result warrants further confirmatory testing (2C).Issue 7: What is the value of immunofluorescence (IF) in diagnosing PCD?Recommendation 7: In clinical settings where IF is available, it can be recommended as an adjunctive diagnostic test for suspected PCD. A positive IF result warrants further confirmatory testing (2D).Issue 8: How should PCD patients be followed up?Recommendation 8: Regular annual follow-up is recommended for adult PCD patients. Key follow-up components include assessment of pulmonary, sinus, and otologic symptoms and function. Additional assessments should include evaluation of disease severity, immune status, quality of life, and psychological status. Furthermore, infertility/subfertility assessment and follow-up are recommended for PCD patients of reproductive age (1C).Issue 9: How can genetic counseling assist PCD patient families in reproductive planning and decision-making?Recommendation 9: Providing appropriate genetic counselling to families of PCD patients can help identify carriers and assess the risk of offspring having PCD for individuals of reproductive age, thereby assisting them in making informed reproductive decisions (1D).

The significance of nitric oxide (NO) in man was first investigated in the late 1980s, and NO has subsequently received great attention from biologists. Initially, this highly reactive gaseous molecule was seen as a mere noxious air pollutant. Closer investigation of its function in physiological processes, however, revealed that it took part in many different biologic processes. This multifunctionality led to its declaration as the molecule of the year in 1992. We now know NO to be a smooth-muscle relaxant in blood vessels, an inhibitor of platelet aggregation, a neurotransmitter, and a mediator in local defense (2, 3). In the airways, NO is an important molecule with different functions such as stimulation of ciliary motility, mediation in inflammation, bacteriostatic and virostatic activity, and regulation of bronchial airway tone and even pulmonary vascular tone (4–7). Further studies on other systems will probably reveal more processes in which NO plays a key role. Studies in healthy adults indicate that NO in nasal air is mainly produced in the epithelial cells of the nasal cavity, particularly in the paranasal sinuses (8). Many factors, such as smoking, drugs, physio-logical factors, and nasal and paranasal disorder, influence the level of NO measured in nasal air (6, 9, 10). The measurement technique is also of great importance (10, 11). NO measurement has begun to be used in experimental clinical settings, in order to clarify the clinical value of NO in diagnostic problems and therapeutic strategies for disorders such as primary ciliary dyskinesia (PCD) and various forms of sinusitis and allergy. The use of NO as a noninvasive diagnostic and therapeutic tool is the ultimate goal. Many cells within the (upper and lower) respiratory tract can produce NO, including endothelial cells, epithelial cells, neutrophils, and (alveolar) macrophages (12). First, l-arginine is taken up by the cells via cationic transporters (CAT) (Fig. 1). CAT1 is constitutively expressed (housekeeping), while CAT2 is induced by cytokines. Second, l-arginine is N-hydroxylated into NG-hydroxy-l-arginine (NOHA). Subsequently, a three-electron oxidation takes place, resulting in NO and l-citrulline. While NO diffuses to the lumen, l-citrulline can be reconverted to l-arginine via arginosuccinate inside the cell (13). NO metabolic pathway (13) (reproduced with permission). This pathway of generation of NO is regulated by a family of enzymes called nitric oxide synthases (NOS). Three isoforms of NOS have now been identified in man and are differentially distributed in organs and tissues (14). Constitutively expressed nitric oxide synthase (cNOS) consists of two isoforms, nNOS (NOS type 1) and eNOS (NOS type 3), respectively expressed in neurons and vascular endothelium. The activity of nNOS and eNOS is regulated by intracellular calcium/calmodulin concentrations. These isoforms have been localized in human alveolar type II cells and in transformed and primary cultures of human bronchial epithelial cells (15). Inducible NOS (iNOS or NOS type 2) is probably present in every (epithelial) cell, and is activated by proinflammatory cytokines and/or bacterial products (2). The inducible form of NOS is calcium independent. LPS alone increases the production of NO in human epithelial cells, but IFN-γ acts synergistically to enhance this response (15). Immunohistochemical and mRNA in situ hybridization show that NO synthase is expressed apically in the paranasal sinus epithelium, in contrast to the epithelium of the nasal cavity, where only weak NO synthase activity was found (16). The NOS of the paranasal sinuses most closely resembles the inducible isoform but has different characteristics from iNOS expressed elsewhere. These isoforms seem to be constantly expressed and active, and to be resistant to steroids. These properties are associated with constitutive, rather than with inducible, isoforms of NOS (16). A new nonenzymatic pathway has been discovered in man that produces NO by reduction of inorganic nitrite under specific conditions (17). These nonenzymatic reactions take place in the stomach, on the surface of the skin, in the ischemic heart, and in infected nitrite-containing urine. NO generated by this mechanism is likely to play a role in similar biologic events, as when produced from l-arginine by NO synthases. The exact origin of NO measured in nasal air and the relative contribution from other sources are not fully known. Not only is there the production within the nasal cavity and the paranasal sinuses, but there is also a contribution from other sources such as the ambient air and, more important, the lower respiratory tract (6–8, 10, 18, 19). Most studies indicate that the main production of nasal NO is in the paranasal sinuses (16, 20, 21). The first indication is the observation that there is a transient decrease in nasal NO measured from one nostril when air is continuously removed from one maxillary sinus, while air injected into the same sinus results in a transient elevation of nasal NO. This suggests a continuous flow of NO from the maxillary sinus to the nasal cavity (20). Another indication is the reduction of NO release from the paranasal sinuses by instillation of NO synthase inhibitor (L-NAME) into the maxillary sinus. Administration of L-NAME in the nasal cavity results in only a slight reduction of nasal NO levels (20). In patients who have impaired ostial patency, significantly lower nasal NO levels are measured. Impairment of ostial patency and thus lower nasal NO levels are seen in disorders such as Kartagener's syndrome and cystic fibrosis. In these cases, there is probably a lower contribution of NO flowing from the paranasal sinuses into the nose, in addition to a possibly decreased production of NO (8, 22). Moreover, nasal NO levels are high in man and other primates with paranasal sinuses, while, in contrast, the baboon, a primate which lacks paranasal sinuses, has very low nasal NO levels (21). The strong constitutive expression of iNOS in the sinus epithelium and the lack of expression in the nasal epithelium are another indication (16). There are indications that nasal NO levels in children rise until the age of 10 years, when they reach the normal value as in adults. This may be a sign of increasing pneumatization of the developing paranasal sinuses in growing children (16, 23). The role of bacteria in the production of nasal NO has also been suggested; however, most studies showed nasal NO release to be independent of the presence of bacteria, since systemic antibiotics had no effect on the nasal NO values of healthy adults, and the sterile nasal cavities of neonates delivered by cesarean section had measurable nasal NO levels (7, 24, 25). As, in recent years, a wide variety of physiological processes in which NO is involved have been thoroughly investigated, it became clear that NO is important within the system where it is produced. Although initially considered a noxious air pollutant, many scientists now agree on the important roles of NO in different organ systems, such as those of a neurotransmitter in the nervous system, a smooth-muscle relaxant, and an inhibitor of platelet aggregation in the cardiovascular system (6, 16, 26). In the airways, NO seems to be of great importance in local host defense and is a major mediator in many physiological and pathophysiological events, although the exact role of this pluripotent gas is far from fully known. It participates in host defense and inflammation, and as an airborne messenger in bronchial tonus and pulmonary vascular resistance. The role of NO in inflammation is contradictory. Some studies indicate a harmful role of NO in inflammation, whereas others indicate a positive influence (18). There is evidence that NO production is enhanced at sites of inflammation, leading to local increased NO levels, as in asthma, cystitis, and inflammatory bowel disease (18, 27). The harmfulness of NO may be due to extensive production of NO by iNOS in some inflammatory circumstances such as pertussis and asthma, leading to autotoxicity in the affected area (18). However, basal NO production in the upper respiratory tract by a continuous expressed iNOS, leading to fairly high NO levels, has no destructive effect on local airway epithelium, and is even physiological (16). On the contrary, NO production in the upper respiratory tract seems to serve as an important protection against local attack, not as a mere inflammatory mediator, but as a regulator of various protective activities in host defense. A remarkable illustration of the positive role of NO in inflammation was given by McCafferty et al., who found worse inflammation in iNOS knockout mice than in wild-type mice in an animal model of colon inflammation (28). The enhanced production of NO during local aggression against the airway epithelium suggests a role of NO in host defense. NO concentration in normal paranasal sinuses and even in the nasal cavity exceeds greatly NO concentrations that are bacteriostatic (i.e., 100 ppb) (6, 16, 29). Children who have low NO production, as in primary ciliary dyskinesia (PCD) and cystic fibrosis, also have recurrent airway infections, a fact which may be an indication of the (host) protective effect of NO. NO may also have virostatic activities, as indicated in a mouse model (30). There are also indications that NO is active against fungi and parasites, and it may also protect against tumor cells (31). NO is also a regulator of ciliary beat frequency in the upper airway epithelium (4, 5, 32). The lack of NO in nasal air in diseases caused by profound ciliary dysfunction, such as PCD, strongly suggests a relation between NO and ciliary motility with clinical implications. For example, in infection, increased NO production can lead to enhanced ciliary activity, resulting in an effective clearance of aggressive organisms and potentially noxious metabolic products. This can have beneficial results in host defense. Other findings suggest that NO enhances blood flow in the human nasal mucosa (33). Although its possible protective effect is not clear yet, further studies on this subject may elucidate the meaning of this finding. NO produced in the upper respiratory tract follows the airstream to the lower airways and lungs with inhalation. This supports the hypothesis that NO derived from the upper airways has physiological effects in the lung and acts as an aerocrine messenger. There is some evidence that inhaled (exogenous) NO, at concentrations as low as 100 ppb, significantly decreases pulmonary vascular resistance and improves arterial oxygenation in subjects with severe pulmonary disease (33). Other studies suggest that NO helps to decrease the bronchial tonus, although this might be a central rather than a peripheral airway effect (7). NO in gas phase at low concentrations, as in the human airways, is fairly stable and therefore can be detected and quantified. The most widely used technique for measurement of NO in exhaled air is the chemiluminescence method. This highly sensitive technique is based on the emission of electromagnetic radiation from excited NO2*. NO reacts with an excess of ozone (O3), resulting in NO2 with an electron in an excited state (NO2*), which returns to its basic energy by emitting a photon. The quantity of light emitted is proportional to the NO concentration and can be displayed online on-screen. The lower limit of measurement is 1 ppb. Nasal NO measurement is based on the same method as exhaled NO, but sampling can be done directly or indirectly from the nose (6, 10). Other methods that have been used to measure NO in human exhaled air are mass spectrometry and gas chromatography–mass spectrometry (6). The measurement technique that is used in a particular experiment is very important for the eventual value of the nasal NO level (10, 34). Even in the same population the NO level is dependent on the measurement technique (11). The most important factors are ambient NO; the method of measuring (i.e., sampling while breathholding or tidal breathing, soft palate closure, etc.); and the characteristics of the chemiluminescence analyzer, the sampling flow, and the intranasal flow (10, 11). For comparison of different values, it is important to have a notion of these factors. In 1997, the European Respiratory Society Task Force tried to determine a standard method in order to obtain more comparable and reliable values (10). However, scientists continue to use different experimental settings, and one should be aware of this in order to interpret and compare NO values from different studies. The values of oral and nasal NO in the exhaled air of controls measured by the chemiluminescence method vary among laboratories: oral NO ranges from 4 to 160 ppb, while nasal NO varies from 200 to 2000 ppb (12, 22, 23, 35–38). Another remarkable feature is that NO levels are always higher in the upper respiratory tract than in the lower airways in normal subjects (6, 8, 10, 12, 22, 24, 36, 38). The variety of NO values in different studies is due to different factors such as measurement techniques, physiological variations, and pathologic changes (9–11, 16, 23, 34, 39–41). A summary of the influences on nasal NO is given in Table 1. Nasal NO levels rise from birth until the age of 10 years, when they reach the normal adult level. This finding supports the paranasal origin of nasal NO, as in children development of paranasal sinuses results in higher nasal NO levels until the age of 10 years, when they reach their final constitution (16, 23, 43). Interestingly, Schedin et al. found nasal NO already present at birth, including those neonates delivered by cesarean section (25). When nasal NO levels were correlated with body surface, the concentration in children around 10 years of age was approximately twice as high as the nasal NO concentration in adults. The following two possible explanations have been proposed: 1)the surface of paranasal sinuses in children develops faster than the body surface 2)children excrete a larger proportion of NO in the nasal mucosa (16). Another study found that nasal NO levels in adults between 20 and 90 years of age were similar (23). Artlich et al. related levels of nasal NO to the body surface in preterm children and found that the NO excretion is similar to that of adults (about 3 nl/kg/min−1). They concluded that the lower NO levels in preterm children are due to the smaller volume of ventilated sinuses and smaller epithelial surface at that age (43). Mammals without sinuses have no age-related increase in nasal NO (44). Recently, Qian et al. contradicted Lundberg et al.'s conclusions. They showed that intranasal flow had a great influence on the result of NO measurement (16, 34). As there are many differences in ventilation and measurement techniques between children and adults, intranasal flow will not always be comparable. More work needs to be done to make measurements in children and adults more comparable, in order to draw conclusions about age-dependent NO differences (34). There is no evidence that nasal NO levels are sex-related (10, 34, 39). Variation in nasal NO levels in relation to the menstrual cycle has not yet been studied. Several studies show that nasal NO decreases during physical exercise (6, 10, 45). Lundberg et al. (6) showed that nasal NO decreased by 47% after 1 min of physical exercise. A maximal reduction of 76% was found at the end of the exercise period; thereafter, NO levels slowly increased. They reached normal basal levels in about 15–20 min. There are several possible reasons for this decrease in nasal NO. Firstly, changes in nasal cavity volume could result in lower NO levels by dilution of nasal air (46). This possibility has been rejected by a recent finding that nasal NO is independent of nasal cavity volume (47). Secondly, NO could be destroyed by reactive agents produced in the nasal mucosa during physical exercise. Thirdly, changes in NO could be caused by a reduction of blood flow in the nasal mucosa with a concomitant decrease in substrate supply to the highly producing NOS type 2 in the paranasal sinuses (6, 46). Smoking control subjects have somewhat lower exhaled NO and nasal NO values than age- and sex-matched nonsmokers. The reason for this could be related to the toxic effect of inhaled smoke on the downregulation in NOS and/or the disruption of NO-producing cells (6, 10, 23). When evaluating the effect of drugs on nasal NO, one should be aware of interactions among drugs, patients, and diseases. It is not always easy to determine whether the changes in nasal NO are caused by the drug or by the disease itself. Topical and systemic glucocorticoids showed no effect on the nasal NO levels in healthy people (6, 8, 48, 49). Antibiotics in healthy persons do not alter nasal NO levels (6, 8). Topical nasal decongestants, such as oxymetazoline, result in a decrease of nasal NO levels (6, 10, 40, 47, 50). The reason for this may be a reduction, caused by vasoconstriction, in substrate supply to the high-output NOS type 2 in the sinuses. Histamine seems to have no influence on nasal NO levels (51). Nasal NO levels in people suffering from an upper respiratory tract infection (URTI) do not differ from nasal NO levels in healthy people. Specifically, Ferguson & Eccles (50) and Lindberg et al. (23) found no significant differences in nasal NO levels during and after an episode of URTI. Lindberg et al. (23) found similar nasal NO levels in patients with URTI and healthy controls (23). Baraldi et al. reached the same conclusion when comparing children with and without URTI (41). The effect of allergic rhinitis on nasal NO is not consistent. Some researchers report higher nasal NO levels in patients with allergic rhinitis (9, 40, 42). This may be due to an upregulation of iNOS by local infection, resulting in higher NO production (9). Kharitonov et al. found that nasal NO levels in patients suffering from allergic rhinitis and treated with topical nasal glucocorticoids are even lower than nasal NO levels in controls (9). This led to the hypothesis that iNOS in nasal epithelial cells gives rise to increased nasal NO levels in allergic rhinitis and contributes to the normal NO production in basal circumstances, since topical nasal glucocorticoids normally do not reach the sinus cavity and decrease nasal NO values in allergic rhinitis to levels lower than nasal NO levels in controls. According to this hypothesis, iNOS in the nasal cavity, as its activity is altered by glucocorticoids, must be different from iNOS found in the paranasal sinuses, which is not influenced by glucocorticoids (9, 52). Lundberg et al. (36) and Henriksen et al. (53) found no alterations in nasal NO levels in patients with allergic rhinitis. The cause of these discrepancies is not very clear. One could speculate that the upregulation of iNOS in the nose leads to higher nasal NO levels in rhinitis, as is the case in local infections in the lower airways, such as asthma (9, 52, 54). In contrast, swelling of the nasal mucosa in rhinitis can lead to occluded sinus ostia, which results in a reduced passage of NO from the paranasal sinuses to the nasal cavity, where it is measured (40). An interesting finding supporting this view was made by Arnal et al. (40), who found increased nasal NO levels in patients with allergic rhinitis. But patients without symptoms at the moment of the measurement had even higher nasal NO levels than patients with symptoms. One could postulate that nasal NO levels in patients with symptoms are lower because of a reduced contribution of the NO produced in the paranasal sinuses, as a result of obstructed sinus ostia. In patients without symptoms, ostial patency is mostly better leading to a higher of NO from the paranasal sinuses into the nasal cavity (40). in the nasal NO level measure may be the result of in the may even This must be taken into when a given nasal NO value is Nasal NO levels seem not to be influenced by asthma (18, 22, 36, One can that asthma the upper respiratory tract to a than the lower airways, where increased NO levels are of glucocorticoids can NO levels by reduction of iNOS NO levels are considered to be a of airway measurement of NO levels in the lower airways could indicate the of (6, 22). Nasal NO levels seem to be decreased in patients suffering from but not studies are consistent. Lindberg et al. patients with sinusitis and found that nasal NO production was reduced by more than in comparison with healthy subjects (23). In contrast, Arnal et al. found no significant differences in their study of patients with sinusitis Lindberg et al. found similar nasal NO levels in patients after sinusitis and healthy subjects (23). nasal NO levels were measured by Baraldi et al. in children with These decreased nasal NO levels increased after with systemic nasal NO levels were to the levels of healthy children (41). It has not yet been whether low nasal NO levels in sinusitis result from reduced passage of NO via the sinus ostia, or whether the NO production is reduced in those patients (41). A low production of NO as a cause of low nasal NO levels in sinusitis is by the study of Lindberg et al., who found nasal NO levels to be low sinus or by sinus as by (23). In contrast to Lindberg et al.'s Baraldi et al. found only a reduced NO level in children with a of the sinus in the air derived from the nostril (41). The effect of nasal has not been The of nasal NO and nasal has been in only one Arnal et al. increased nasal NO levels in patients with nasal and to whereas patients with nasal without had significantly lower nasal NO The nasal NO concentration in patients with allergic was significantly higher than in patients with For a similar of sinus nasal NO was higher in allergic than in This that is an important in relation to the level of nasal NO in In the nasal NO concentration was correlated with the of alterations of the paranasal sinuses. This that the of the paranasal sinuses by the decreases the nasal NO with a similar of of the nasal NO levels was that sites of production other than the sinuses also to the nasal NO. It has been that also may to the NO production, as they also iNOS in their epithelial cells (2). can that the of paranasal sinus and the allergic strongly influence the nasal NO level in nasal studies report very low nasal NO levels in patients suffering from cystic (12, 22, This may be the result of reduced NO production by destroyed epithelial cells or reduced NOS An increased NO into the sinus and a reduced NO passage from the sinuses to the nasal cavity may be another possible (12, 22, Kartagener's syndrome is a and They are part of In patients with PCD, nasal NO levels are (8, 12, 52). explanations are reduced NO production by a reduced from the nasal and paranasal and reduced passage of NO via the sinus (8, 12, 22, 52). In studies on PCD, significantly lower nasal NO levels in than in disease controls. nasal NO values, however, do not We found that the in have no significant influence on the nasal NO level et al., of NO can an interesting and diagnostic and therapeutic However, to be done in order to make it a in This noninvasive measurement can be even in It could be used as an easy for the of In the therapeutic may It is to that drugs will be used to or decrease NO production in such a that it can have a positive influence on However, there is to be in the various physiological and pathologic factors, such as that nasal NO, particularly the should on and on measurements more reliable and comparable. NO is a gaseous the significance of which in man to be investigated in the late the it has the attention of many who have revealed its significance in various physiological and pathologic processes. It has functions in the cardiovascular system, the nervous system, and the upper and lower In the airways, NO levels in the upper respiratory tract are higher ppb) than those in the lower respiratory tract The chemiluminescence which is based on a of NO with resulting in the emission of is the most widely used measurement technique for NO. NO has a major influence on airway by mediation in ciliary activity, inflammation, host bronchial and pulmonary vascular resistance. It is also considered to be an aerocrine messenger between the upper and lower such as physical smoking, and some drugs influence physiological nasal NO concentrations. conditions such as allergic rhinitis, nasal cystic fibrosis, and lead to altered nasal NO concentrations. of nasal NO can be at and can be used to for disease or to the effects of However, the clinical of the measurement of nasal NO in different physiological and pathologic conditions to be it can be used as a diagnostic on the function of NO in and is its in diagnostic and therapeutic of some

Sir, Primary ciliary dyskinesia (PCD) is a rare autosomal recessive condition affecting the structure and function of motile cilia, thereby resulting in impairment of mucociliary clearance.[1] The common manifestations in children include neonatal respiratory distress, early-onset chronic wet cough, and recurrent respiratory infections, leading to bronchiectasis, recurrent rhinosinusitis, and middle ear infections.[1] Around one-half of PCD patients have dextrocardia and/or situs inversus totalis.[2] In the reproductive age group, PCD can be associated with male infertility and subfertility in females.[1] Diagnosis of PCD involves identification of the clinical phenotype and a combination of tests that require expertise to conduct and interpret the results.[3] The upfront screening tests are nasal nitric oxide measurement and high-speed video microscopy of nasal brushings for ciliary beat pattern and frequency [Figure 1].[3] Hallmark ciliary ultrastructure abnormalities and/or bi-allelic disease-causing mutations in known PCD genes would be diagnostic.[3] PCD can occur in the presence of normal ciliary ultrastructure, and at present, genetic testing can identify mutations in more than 80% of PCD patients.[4] Due to lack of awareness among clinicians and nonavailability of diagnostic tests, PCD is underdiagnosed and the exact frequency of PCD in India is still not known. We report a 14-year-old boy with PCD due to DRC1/CCDC164 mutation for the first time from the Indian subcontinent.Figure 1: High-speed video microscopyA 14-years-old boy, born to nonconsanguineous parents, presented to us with purulent nasal discharge, persistent productive cough, and recurrent respiratory infections with wheeze since infancy. He was born at term and did not have respiratory distress in the neonatal period. His weight and height were on the third centile. He had purulent nasal discharge and auscultation of his chest revealed bilateral crackles and wheeze. High-resolution chest tomography revealed right upper lobe collapse and centrilobular opacities. Sweat chloride estimation and serum immunoglobulin profile were within normal limits. Primary ciliary dyskinesia rule,[5] a predictive score with seven simple questions to predict the likelihood of having PCD, was 4, i.e., the likelihood of PCD was not high. Nasal nitric oxide analysis using portable electrochemical device, NIOX VERO®(Circassia, Sweden), was very low at 11.6 ppb (3.5 nl/min). High-speed video microscopy analysis (HSVA) of nasal brushings revealed motile cilia with stiff motility (reducing bending capacity) and a ciliary beat frequency of 14 Hz. The HSVA videos were reviewed by PCD team, Southampton University Hospital, UK. EDTA peripheral blood sample was sent for targeted next-generation targeted sequencing of the genes known to be associated with PCD. A homozygous nonsense variation in exon 10 of the DRC1 gene (chr2:26667625G>A; depth: 70x) that results in a stop codon and premature truncation of the protein at codon 402 (p. Trp402Ter; ENST00000288710.2) was detected. He is being managed by a multidisciplinary team and is under follow-up. PCD can be caused by bi-allelic mutations in the DRC1/CCDC164 gene.[6] This disorder characteristically starts in infancy with chronic sinopulmonary infections due to abnormal ciliary function.[6] The available literature shows that individuals with DRC1/CCDC164 mutations do not have any abnormal left–right body symmetry.[6] Our patient did not have any left–right body asymmetry. HSVA from our patient showed uniformly stiff cilia. These findings are similar to previous reports in PCD with documented DRC1/CCDC164 mutation.[6] We did not perform transmission electron microscopy due to lack of this facility at our center. PCD with normal body symmetry makes diagnosis even more difficult and late. When there is a strong suspicion based on other clinical features, we should proceed with diagnostic testing for PCD. Early diagnosis of PCD and prompt intervention have the potential to stall the progression of disease. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Dr. Antony Terance Benjamin received NIOX VERO® equipment as a research grant from Circassia, Sweden. Conflicts of interest There are no conflicts of interest. Acknowledgments We would like to acknowledge Dr. Louis Balan, Molecular Pathologist, Coimbatore, India, Dr. Janice Coles, PCD Diagnostic Service, University Hospital Southampton, UK, and Medgenome Labs, Bangalore, India.

Primary ciliary dyskinesia (PCD) is a rare genetic heterogeneous disorder mainly characterized by impaired mucociliar clearance and chronic respiratory symptoms. Although DNAH5 is commonly implicated in PCD, several DNAH5 variants remain unclassified. The proband was studied by high-speed videomicroscopy, transmission electron microscopy, whole exome sequencing and immunofluorescence. Protein structural analysis was performed through structural models obtained by X-ray crystallography and cryo-electron microscopy. The family was studied by Sanger sequencing of the variants, high-speed videomicroscopy and immunofluorescence. The patient carry, in heterozygosity, the DNAH5 c.5290T > C p.(Ser1764Pro) missense variant of uncertain significance and the pathogenic truncated variant DNAH5 c.4237C > T p.(Gln1413*). He was clinically diagnosed in adulthood with PCD, confirmed following nasal nitric oxide measurement, high-speed videomicroscopy and transmission electron microscopy, all of which revealed hallmark PCD defects, with decreased nasal nitric oxide levels (30nl/min) and ciliary beating frequency (0.66Hz), a dyskinetic ciliary beating pattern (62.5% total immotility) and a class-1 ultrastructure. Immunofluorescence analysis demonstrated reduced DNAH5 expression, and protein structural models predicted that the variant of uncertain significance causes an unstable protein. Family analyses confirmed a trans-inheritance and uncovered a brother with a similar PCD phenotype, the same two variants and similar reduced DNAH5 expression. Results support a pathogenic role for the c.5290T > C p.(Ser1764Pro) variant and elucidate the effects of the other variant. These results underscore the importance of integrating clinical, ultrastructural, molecular and protein expression analyses to clarify and contribute to PCD diagnosis, besides now serving as potential markers for diagnostics and targeted therapies.

BackgroundDiagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic and clinical spectrum of the Palestinian PCD population.MethodsIndividuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) and/or PCD genetic panel or whole-exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including forced expiratory volume in 1 s (FEV1) Global Lung Index z-scores and body mass index z-scores.Results68 individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. 45 individuals from 40 families had 17 clinically actionable variants and four had variants of unknown significance in 14 PCD genes. CCDC39, DNAH11 and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had a median age of 10.0 years at diagnosis, were highly consanguineous (93%) and 100% were of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%) and situs inversus (43%). Lung function at diagnosis was already impaired (FEV1 z-score median −1.90 (−5.0–1.32)) and growth was mostly within the normal range (z-score mean −0.36 (−3.03–2.57). 19% individuals had finger clubbing.ConclusionsDespite limited local resources in Palestine, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity.

Accuracy of Clinical Phenotype for Diagnosing Adults With Primary Ciliary Dyskinesia.

Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain significance in approximately 30% of tested individuals. We aimed to assess genotype-phenotype correlations in adults with bronchiectasis, clinical suspicion of PCD, and inconclusive whole-exome sequencing results using transmission electron microscopy (TEM) and ciliary image averaging by the PCD Detect software. We recruited 16 patients with VUS in CCDC39, CCDC40, CCDC103, DNAH5, DNAH5/CCDC40, DNAH8/HYDIN, DNAH11, and DNAI1 as well as variants in the PCD candidate genes DNAH1, DNAH7, NEK10, and NME5. We found normal ciliary ultrastructure in eight patients with VUS in CCDC39, DNAH1, DNAH7, DNAH8/HYDIN, DNAH11, and DNAI1. In six patients with VUS in CCDC40, CCDC103, DNAH5, and DNAI1, we identified a corresponding ultrastructural hallmark defect. In one patient with homozygous variant in NME5, we detected a central complex defect supporting clinical relevance. Using TEM as a targeted approach, we established important genotype-phenotype correlations and definite PCD in a considerable proportion of patients. Overall, the PCD Detect software proved feasible in support of TEM.

RATIONALE: Primary Ciliary Dyskinesia (PCD), a genetic disorder of motile cilia, is associated with a range of clinical features including neonatal respiratory distress, chronic sino-oto-pulmonary disease, and laterality defects, including situs inversus totalis (approximately 50%) and heterotaxy (at least 6%). Heterotaxy is a disorder of ambiguous organ laterality frequently associated with congenital heart disease. Clinical symptoms in PCD can overlap with symptoms from congenital heart disease with heterotaxy. Nasal nitric oxide (nNO) levels are very low in PCD, but have not been studied systematically in heterotaxy. PCD-specific symptom criteria and nNO cut-off levels would help guide appropriate PCD screening within heterotaxy populations. METHODS: Two heterotaxy populations (age ≥5 years) were recruited. First, our multi-center consortium assessed individuals referred for suspected PCD due to sino-oto-pulmonary symptoms, including participants with heterotaxy. Second, one site (UNC) evaluated heterotaxy individuals followed in a cardiology clinic and never referred for suspected PCD. Both populations were investigated with symptom questionnaires and nNO measurements during palate closure. Those referred for suspected PCD also had electron microscopy (EM) of nasal cilia and genetic testing for PCD mutations. “Classic PCD” was diagnosed in participants with classic EM cilia defects and/or 2 PCD causing genetic mutations. Otherwise, participants were classified as “not classic PCD” and stratified by nNO level – those with nNO 100nL/min. RESULTS: Of 336 individuals referred for PCD symptoms, 30 (8.9%) have heterotaxy, including 15 with congenital heart disease. 15/30 have “classic PCD” and all of these have nNO 100nL/min (median 280; 137-338nL/min). The 12 cardiology clinic patients all have nNO >100nL/min (median 199; 127-287nL/min). Clinical features more prevalent in heterotaxy with “classic PCD” versus heterotaxy with nNO >100nl/min are: year-round wet cough (100% versus 26%, p<0.0001), year-round nasal congestion (93% versus 42%, p=0.001), bronchitis/pneumonia (93% versus 47%, p=0.003), recurrent otitis media (87% versus 53%, p=0.02), and chronic sinusitis (73% vs 37%, p=0.03). No significant differences were noted for clinical features in heterotaxy with “classic PCD” versus with low nNO levels. CONCLUSION: In heterotaxy, PCD-specific clinical features include year-round wet cough, year-round nasal congestion, bronchitis/pneumonia, otitis media, and sinusitis. nNO seems a sensitive screening test for PCD within heterotaxy. Presence of PCD specific clinical features and/or low nNO should prompt further PCD evaluation.

Primary ciliary dyskinesia (PCD) is a hereditary disorder with structural and/or functional impairment of the cilia of the whole body. PCD is characterized by chronic sinusitis, bronchiectasis, male infertility and ectopic pregnancy due to impaired ciliary motility. The prevalence of PCD is estimated at 1 in 20,000 live births. Cases with situs inversus are termed “Kartagener’s syndrome” and usually these diagnoses are not difficult to make. However, in cases without situs inversus, the diagnosis can be very difficult. As such, it is most likely that PCD without situs inversus is underdiagnosed at the present time. The diagnosis of PCD is made when the patients have characteristic clinical features and (1) structural abnormalities of the cilia observed with electron microscopy and/or (2) presence of mutations in one of the genes associated with PCD. Electron microscopic diagnosis is not easy because ultrastructural ciliary defects can also be found in a few cilia among the healthy populations. Measuring levels of nasal nitric oxide (NO) concentration is a very useful tool in screening for PCD, because low NO values are a feature of PCD. Hand-held devices using tidal breathing are as useful as stationary devices. The sensitivity and specificity of nasal NO levels of <250 ppb for diagnosing PCD were 97% and 90%, respectively. Some other features can help to diagnose PCD. Frontal sinuses and sphenoid sinuses are hypoplastic. Having a combination of rhinosinusitis, middle ear and lung diseases increases the probability of PCD. The effectiveness of endonasal sinus surgery to treat rhinosinusitis associated with PCD is controversial.

BackgroundKartagener syndrome is a subtype of primary ciliary dyskinesia that may exhibit various symptoms including neonatal respiratory distress and frequent infections of the lung, sinus and middle ear because of the impaired function of motile cilia. In addition to typical symptoms of primary ciliary dyskinesia, patients with Kartagener syndrome also show situs inversus. It is an autosomal recessive disorder which is mostly caused by mutations in DNAH5. Kartagener syndrome is often underdiagnosed due to challenges in the diagnosis process. As next-generation sequencing becomes widely used in clinical laboratories, genetic testing provides an accurate approach to the diagnosis of Kartagener syndrome.Case presentationA 7-year-old female patient presented with runny nose of 6 years duration and recurrent cough with phlegm of 2 years duration. Kartagener syndrome was diagnosed through diagnostic tests such as nasal nitric oxide (NO) concentration and transmission electron microscopy, and after performing other exams that corroborated the diagnosis, such as computed tomography, bronchoscopy and hearing test. Whole-exome sequencing was performed for the patient and both parents. The pediatric patient was diagnosed as Kartagener syndrome with the typical symptoms of ciliary dyskinesia including bronchiectasis, sinusitis, conductive hearing loss and situs inversus along with a reduced nasal NO concentration and ciliary abnormalities. The patient carried two novel compound heterozygous mutations in DNAH5, NM_001369:c.12813G > A (p. Trp4271Term) and NM_001369:c.9365delT (p. Leu3122Term). Both mutations lead to premature stop codons and thus are pathogenic. The p. Trp4271Term and p. Leu3122Term mutations were inherited from the father and the mother of the patient individually. A literature review was also conducted to summarize DNAH5 mutations in pediatric patients with Kartagener syndrome across different ethnic groups.ConclusionsOur study provides a good example of the diagnosis of Kartagener syndrome in pediatric patients using a series of diagnostic tests combined with genetic testing. Two novel loss-of-function mutations in DNAH5 were identified and validated in a pediatric patient with Kartagener syndrome.

Primary ciliary dyskinesia (PCD) is a rare inherited disease affecting motile cilia lining the respiratory tract. Despite neonatal respiratory distress as an early feature, diagnosis is typically delayed until late childhood. Our objective was to identify characteristics that differentiate PCD from common causes of term neonatal respiratory distress. This was a case-control study. Patients with PCD born after 1994 attending a regional PCD clinic who had a history of neonatal respiratory distress (n = 46) were included. Controls (n = 46), term neonates with respiratory distress requiring a chest radiograph, were randomly selected from hospital birth records and matched on gender, birth month/year, and mode of delivery. Multiple logistic regression was used to determine the association between neonatal characteristics and PCD diagnosis. The diagnostic performance of the best predictive variables was estimated by calculating sensitivity and specificity. PCD cases required more oxygen therapy (39 cases, 29 controls, P = .01), longer duration of oxygen therapy (PCD mean = 15.2 days, control mean = 0.80 days, P < .01), had later onset of neonatal respiratory distress (PCD median = 12 hours, control median = 1 hour, P < .001), and higher frequency of lobar collapse and situs inversus (PCD = 70% and 48% respectively, control = 0% for both, P < .001). Situs inversus, lobar collapse, or oxygen need for >2 days had 87% (95% confidence interval: 74-94) sensitivity and 96% (95% confidence interval: 85-99) specificity for PCD. When encountering term neonates with unexplained respiratory distress, clinicians should consider PCD in those with lobar collapse, situs inversus, and/or prolonged oxygen therapy (>2 days).

Nasal Nitric Oxide Measurements To Screen Children for Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is typically an autosomal recessive disease characterized by recurrent infections of the lower respiratory tract, frequent and severe otitis media, chronic rhinosinusitis, neonatal respiratory distress, and organ laterality defects. While severe lower respiratory tract infections and bronchiectasis are common in Inuit, PCD has not been recognized in this population. We report a case series of seven Inuit patients with PCD identified by genetic testing in three Canadian PCD centers. Patients ranged from 4 to 59 years of age (at time of last evaluation) and originated in the Qikiqtaaluk region (Baffin Island, n = 5), Nunavut, or Nunavik (northern Quebec, n = 2), Canada. They had typical features of PCD, including neonatal respiratory distress (five patients), situs inversus totalis (four patients), bronchiectasis (four patients), chronic atelectasis (six patients), and chronic otitis media (six patients). Most had chronic rhinitis. Genetic evaluation demonstrated that all had homozygous pathogenic variants in DNAH11 at NM_001277115.1:c.4095+2C>A. The discovery of this homozygous DNAH11 variant in widely disparate parts of the Nunangat (Inuit homelands) suggests this is a founder mutation that may be widespread in Inuit. Thus, PCD may be an important cause of chronic lung, sinus, and middle ear disease in this population. Inuit with chronic lung disease, including bronchiectasis or laterality defects, should undergo genetic testing for PCD. Consideration of including PCD genetic analysis in routine newborn screening should be considered in Inuit regions.

Rationale: Primary ciliary dyskinesia (PCD) is a heterogenous disease that is difficult to diagnose. American Thoracic Society (ATS) guidelines recommend PCD testing with an appropriate phenotype of ≥2 of 4 key clinical symptoms (year-round wet cough or nasal congestion appearing before 6 months old, neonatal respiratory distress at term birth, an organ laterality defect), using nasal nitric oxide (nNO) measurement and genetic testing, with ciliary electron microscopy (EM) as needed. Accuracy of this approach has not been robustly explored in adults. Methods: This retrospective analysis explores patients ≥18 years old, referred for possible PCD to the McGill University Health Center between 2013 and 2024. All performed nNO measurement per PCD Foundation protocol. Key clinical symptoms were systematically collected but modified for year-round wet cough or nasal congestion “since early childhood”. Additional PCD-related symptoms relevant in adults were also systematically collected. Those with ≥2 key clinical symptoms or low nNO (&amp;lt;77 nL/min) underwent PCD genetic and EM testing. Participants were classified per final diagnosis, including: 1. Definitive PCD with disease-causing genetic variants or classic EM defect, 2. Probable PCD with low nNO but inconclusive genetics/EM and no alternative diagnosis, 3. Unlikely PCD with normal nNO, an alternative diagnosis, and negative genetics/EM, when performed. Results: Of 158 referred adults, 57 (36%) displayed ≥2 key clinical symptoms while 45 (28%) had low nNO values. However, 29 (18%) participants could not recall their neonatal histories. Genetic or EM testing was performed in 82 (52%) and 48 (30%) patients, respectively. Overall, 41 (26%) had definitive PCD (88% through genetics, 12% through EM), 6 (3%) had probable PCD, and 111 (70%) had unlikely PCD. Low nNO was seen in 36 (88%) of definite PCD versus 3 (3%) with unlikely PCD. Prevalence of key clinical symptoms and additional PCD-related symptoms relevant to adults differed significantly between diagnostic groups. [Table 1] Conclusion: The ATS guidelines have a high diagnostic yield in adults when modifying key clinical criteria to include wet cough or nasal congestion since early childhood. However, many adult patients cannot recall their neonatal histories, possibly decreasing accuracy. Several additional PCD-related symptoms seem helpful in adult diagnosis.