Primary Ciliary Dyskinesia Diagnostic and Phenotypic Features

Transcription

1 Primary Ciliary Dyskinesia Diagnostic and Phenotypic Features Peadar G. Noone, Margaret W. Leigh, Aruna Sannuti, Susan L. Minnix, Johnny L. Carson, Milan Hazucha, Maimoona A. Zariwala, and Michael R. Knowles Department of Medicine and Pediatrics and the Center for Environmental Medicine, the University of North Carolina School of Medicine, Chapel Hill, North Carolina; and Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary structure/function. We hypothesized that the major clinical and biologic phenotypic markers of the disease could be evaluated by studying a cohort of subjects suspected of having PCD. Of 110 subjects evaluated, PCD was diagnosed in 78 subjects using a combination of compatible clinical features coupled with tests of ciliary ultrastructure and function. Chronic rhinitis/ sinusitis (n 78; 100%), recurrent otitis media (n 74; 95%), neonatal respiratory symptoms (n 57; 73%), and situs inversus (n 43; 55%) are strong phenotypic markers of the disease. Mucoid Pseudomonas aeruginosa (n 12; 15%) and nontuberculous mycobacteria (n 8; 10%) were present in older ( 30 years) patients with PCD. All subjects had defects in ciliary structure, 66% in the outer dynein arm. Nasal nitric oxide production was very low in PCD (nl/minute; vs in normal control subjects). Rigorous clinical and ciliary phenotyping and measures of nasal nitric oxide are useful for the diagnosis of PCD. An increased awareness of the clinical presentation and diagnostic criteria for PCD will help lead to better diagnosis and care for this orphan disease. Keywords: primary ciliary dyskinesia; ciliary ultrastructure; nitric oxide; situs inversus; Pseudomonas aeruginosa Primary ciliary dyskinesia (PCD) is a genetic disease associated with defective ciliary structure and function and chronic otosino-pulmonary disease (1, 2). Situs inversus occurs randomly in approximately 50% of subjects with PCD (3, 4). The prevalence is estimated at approximately 12,000 to 17,000, as extrapolated from radiographic studies in Norway and Japan involving situs inversus in association with bronchiectasis, though precise figures for the United States are lacking (5, 6). Diagnosis relies on a combination of clinical evaluation and electron microscopic analysis of ciliary ultrastructure and may be difficult to establish in some subjects (7 9). Diagnostic delay leading to inadequate therapy may result in poorer outcomes for patients with PCD (10). Despite first descriptions of the ciliary defect in PCD in (Received in original form March 12, 2003; accepted in final form November 24, 2003) Supported by grants HL04225, HL34322, RR00046, the American Lung Association of North Carolina, and the American College of Chest Physicians. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through cooperative agreement CR with the Center for Environmental Medicine, Asthma, and Lung Biology at the University of North Carolina at Chapel Hill, it has not been subjected to the Agency s required peer and policy review, and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Correspondence and requests for reprints should be addressed to Peadar G. Noone, M.D., Level 8, Respiratory Department, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK. peadar_noone@med.unc.edu This article has an online supplement, which is accessible from this issues s table of contents online at Am J Respir Crit Care Med Vol 169. pp , 2004 Originally Published in Press as DOI: /rccm OC on December 4, 2003 Internet address: the mid-1970s, and some published literature from Europe and Australia, the disease has been little studied in North America to date (1, 11, 12). The clinical phenotype in PCD is broad and overlaps with other chronic airways diseases. We hypothesized that assembly of a large cohort of subjects with PCD from North America was possible. We further hypothesized that a rigorous evaluation, targeted toward the major clinical and biologic markers of the disease would help better characterize and categorize patients for subsequent genetic and other physiologic testing. We tested for the ciliary structural and functional defects occurring in PCD, specifically defects of the outer and inner dynein arms and central apparatus (7, 9). As an adjunctive test, we measured nasal nitric oxide (NO) levels to evaluate its use as a diagnostic marker in a cohort of subjects with PCD across a wide age spectrum (8, 13 15). We hypothesized that the data from such a large cohort of subjects would lead to a better understanding of the patterns of clinical presentation in PCD, hopefully leading to better efforts to recognize the disease earlier and also standardize clinical care for the primary problems associated with the disease. Insights into the genetics of PCD are currently evolving, and this new knowledge will likely facilitate the diagnostic work up in patients, as well as lead to a better understanding of the pathophysiology of defective ciliary structure and function (16 18). Emerging definition of ciliary ultrastructure offers an excellent opportunity to test candidate genes in this genetically heterogeneous disease (19). One current strategy to facilitate genetic studies is to stratify groups of subjects for genetic testing according to ultrastructural ciliary defect rather than solely by clinical phenotype. The data set generated from this type of study allows categorization of subjects by ciliary structural defect and thus facilitates parallel studies addressing the molecular basis of PCD (17). Less commonly, other ultrastructural abnormalities may include microtubular transposition, or random ciliary orientation, or no ultrastructural abnormality may be seen even with a compatible phenotype, thus supporting the concept of atypical disease (20 23). Some of the results of these studies have been previously reported in the form of abstracts (24 26). METHODS Study Population Subjects with suspected PCD were recruited for the study from around North America. All studies were performed at the General Clinical Research Center at the University of North Carolina at Chapel Hill under the auspices of the Committee on the Protection of the Rights of Human Subjects. Clinical Evaluation/Evaluation of Ciliary Structure and Function Clinical evaluations included details of oto-sino-pulmonary signs and symptoms (8). Spirometry was performed, sputum was obtained if possible for microbiologic analysis, and available radiographs were reviewed. Ciliated epithelial cells were obtained from the inferior surface of the inferior nasal turbinates using a nasal curettage technique, and ciliary structure and function were assessed using previously described techniques (9, 27 29). Three reviewers examined the electron microscopic photomicrographs in a blinded fashion for the presence or absence or

2 460 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL shortening of dynein arms and for gross abnormalities in the central apparatus (central pairs/nexin links/radial spokes). Subjects were classified as having absent or shortened outer dynein arms, absent or shortened inner dynein arms alone, central apparatus abnormalities, or normal ciliary structure. Video microscopy recordings of ciliary beat from the samples were examined in a blinded fashion, and ciliary activity (ciliary beat frequency, cilia bending, and ciliary beat coordination) was scored in a semiquantitative manner, from 0 (absent ciliary activity) to 9 (normal ciliary activity), according to a previously described technique, and compared with ciliary activity of healthy control subjects (30). Nasal NO Studies Nasal production of NO (31) was measured directly by inserting a NO analyzer sampling line (at a constant rate of 0.5 liters per minute) into one nostril (model 270B; Siever, Boulder, CO). The subject was asked to inhale deeply and then to close the soft palate to prevent crosscontamination of the nasal sample by air from the pharynx. The measurement was terminated once the NO concentration reaches a plateau lasting for at least 10 seconds, approximately after 20 to 30 seconds of acquisition time. Six measurements per subject were obtained, with the final mean data for each subject reported as steady state production (nl/minute) (17). Levels were also obtained from nonsmoking healthy control subjects, from subjects with cystic fibrosis (CF) (32), and from nonsmoking healthy parents of subjects with PCD. After the evaluation, subjects were deemed to have PCD (patients who may have atypical PCD were excluded) depending on the completeness of the clinical phenotype and the results of the tests of ciliary structure/function. Nasal NO levels were compared between the groups with PCD versus control subjects with and without disease. Subjects in whom other diseases were diagnosed were excluded from the study: n 15, subjects with atypical asthma (n 3), CF (n 1), allergic bronchopulmonary aspergillosis (n 1), Young s Syndrome (n 1), and idiopathic bronchiectasis (n 9). Four non-white subjects were evaluated (three African American, one Asian Pakistani). The three African Americans had idiopathic bronchiectasis, whereas the Asian Pakistani had PCD (see below). Statistical tests were performed using the statistical program Stata (Stata Corporation, College Station, TX); specific tests are noted for each data analysis, with the level of significance set at RESULTS General Excluding the patients with alternate diseases (see above), 94 subjects (n 53 females) from 68 families were seen at the University of North Carolina from 1994 to Subjects of all ages (years) were evaluated, ranging from the neonatal period up to late adulthood: mean age, 26.8 (median age 29, range 0 73). After the complete evaluation, 78 subjects (n 42 females; n 47 adults age 18 years) were classed as having PCD, associated with well-defined ultrastructural defects on ciliary analysis. Of the 78 subjects with PCD, 43 (55%) had situs inversus totalis (one female had dextrocardia and polysplenia) (33). One additional male (27 years) with situs inversus was evaluated and found to be otherwise completely healthy (asymptomatic, normal chest radiograph, FEV 1 102% predicted, and normal dynein arm structure/activity) and thus was excluded from further analysis because he likely has non PCD-associated situs inversus (5). Classic PCD (n 78) Clinical disease spectrum. All subjects had a clinical history of lower airway disease, manifested by cough productive of sputum, wheeze, or shortness of breath (Table 1). All subjects had evidence of chronic upper airway symptoms/chronic rhinitis by history (nasal discharge, episodic facial pain, and anosmia), physical examination and/or by sinus radiographs. Forty-two subjects had the necessary studies to fully establish the diagnosis of sinusitis, which was based on previous sinus surgery having been performed (n 19 adults and 10 children) or sinus radiographs showing evidence of chronic sinusitis (n 3 adults and 10 children) (34). Seventy-four subjects (95%) had a history of recurrent otitis media requiring multiple interventions, including repeated courses of antibiotics. A proportion of these had undergone placement of myringotomy tubes, although documentation justifying the procedure was unavailable (58; 74%). Fifty-seven subjects (74%) had a specific history of a neonatal respiratory syndrome, defined as excess pulmonary congestion, excess mucus, or a requirement for intravenous antibiotics, supplemental oxygen, or an intensive care admission for neonatal pneumonia (35, 36). These data probably underestimate this neonatal symptom complex because older subjects had poorer recollection of this aspect of their histories. Bronchiectasis was primarily diagnosed clinically on the basis of a history of chronic excess mucopurulent sputum production associated with finger clubbing (n 20 with clubbing, all adults), and, where available, computed tomographic scans of the thorax (n 32, 27 adults) or with clear abnormalities on chest radiographs (n 13) were also used to support the diagnosis. Using these clinical and radiologic criteria, bronchiectasis was present in 65 subjects (83%). There was an age correlation for the diagnosis of bronchiectasis, consistent with a time factor in its development (young subjects tending not to manifest symptoms or signs of bronchiectasis); the mean age (years SEM) for those with clinical bronchiectasis was significantly greater that for those without ( vs , p , regression analysis). Lobectomies had been performed on eight subjects (seven adults), three of whom had very severe disease at the time of the evaluation at the University of North Carolina. Spirometry was performed in 70 subjects with PCD. The mean FEV 1 (% predicted SD) was 69 25, range 24 to 123, and median 73. As might be expected, there was a significant relationship between age and lung function, FEV 1 declining with age (p ; unadjusted regression) (37). The slope of a regression line of FEV 1 plotted versus age for this cohort of patients of all ages with PCD was (i.e., a TABLE 1. CLASSIC PRIMARY CILIARY DYSKINESIA Cough Bronchiectasis* Sinusitis Otitis Media NRS Situs Inversus FEV 1 Nasal NO n(% ) n (% ) n (% ) n (% ) n (% ) n (% ) (% predicted) (nl/min) Adults ( 18 yr; n 47 [31 females]; median age, 36 yr; range, yr) 47 (100%) 46 (98) 22 (47) 43 (92) 30 (65) 22 (46) Pediatric subjects ( 18 yr; n 31 [11 females]; median age, 8 yr; range, 1 17 yr) 31 (100%) 19 (61) 20 (65) 31 (100) 27 (87) 21 (68) Definition of abbreviations: NO nitric oxide; NRS a syndrome of neonatal respiratory symptoms. n 78 (42 females); age (years): mean 27; median 29; range Continuous data SD are presented. * All subjects reported chronic cough. See text for definitions of bronchiectasis (excess purulent sputum and/or radiographic evidence) and NRS. All 78 subjects had a history of chronic upper airway symptoms/chronic rhinitis; see text for definition of sinusitis i.e., prior surgery, computed tomography/sinus radiographs showing evidence of chronic sinusitis. 100% of those who had computed tomography scans had evidence of chronic sinusitis.

3 Noone, Leigh, Sannuti, et al.: Clinical and Ciliary Phenotype and Nasal NO Levels in PCD 461 drop of 0.8% per year), as compared with a slope-decline of up to approximately 3.6% for patients with CF in North America (38 41). It must be borne in mind that this cohort of patients with PCD represents subjects of all ages and spans puberty, with possible implications for changes in lung function (42). Thirteen patients (all 30 years of age) were considered to have very severe disease (respiratory failure). All these subjects had an FEV 1 less than or equal to 40% predicted, plus were postlung transplantation subjects (n 2) or were listed for lung transplantation (n 5), or had chronic use of oxygen/oxyhemoglobin desaturation (to 85% or less) at rest or on exercise. Approximately 25% of the total cohort had severe disease using these criteria (i.e., 38% of adults 18 years of age). Of the 13 subjects with respiratory failure, 12 had digital clubbing. The two who underwent lung transplantation have had good outcomes at 2 and 4 years respectively, with two (twin siblings) and three other unrelated patients currently on the waiting list (4). Subjects with severe disease as defined previously had a range of dynein arm defects, evenly divided between inner and outer arm defects (see below). Sputum for microbiologic testing was obtained in 59 subjects (75%) with PCD and some had medical records of chronic sputum sampling ( 3) with available data on dominant organisms (Table 2). Subjects in whom sputum was obtained were slightly older (years SEM) on average than those in whom sputum was not obtained, although this did not reach statistical significance (29 2 [median 30] vs [median 9], p 0.06, regression analysis). Cough, or deep pharyngeal swabs, if performed, may have increased the recovery of organisms from the younger subjects. The breakdown (by absolute number because there was overlap in some subjects) for those yielding a sample by specific bacteria was as follows: Haemophilus influenzae.: n 22, Staphylococcus aureus: n 13, Pseudomonas aeruginosa (smooth): n 15, mucoid P. aeruginosa:n 12, and nontuberculous mycobacteria: n 8(Mycobacterium avium complex: n 3, M. abscessus: n 2, M. gordonae: n 2, mixed M. avium complex/m. gordonae: n 1). Some subjects cultured more than one organism in the same sample: for example, smooth P. aeruginosa (n 9) with another organism, most commonly nontuberculous mycobacteria (n 4). There was a relationship between sputum positivity for mucoid P. aeruginosa and age (older subjects 30 years). These data are illustrated in Figure 1, which depicts FEV 1 (% predicted) as a function of age, with labeling for sputum culture results where available, according to the bacteria isolated. It should be noted that eight subjects cultured oropharyngeal flora only in the sample obtained. This may relate to only one sample being obtained in the visit to the University of North Carolina, and repeated sampling may have had a higher yield of organisms. In the subjects for whom data were available on repeat testing of sputum, it was evident that Figure 1. FEV 1 (% predicted) as a function of age in subjects with classic primary ciliary dyskinesia (PCD) (n 70) plotted by dominant sputum microbiology (some subjects cultured more than one organism). Closed circles represent Haemophilus influenza (n 14), and open circles represent Staphylococcus aureus (n 3). Closed squares represent smooth Pseudomonas aeruginosa (n 10), and open squares represent mucoid P. aeruginosa (n 12, 35% of subjects, most over the age of 30 years). Closed half circles represent nontuberculous mycobacteria (n 8 [5 of whom also cultured smooth P. aeruginosa]). Open stars represent subjects in whom sputum culture was negative for bacterial pathogens (n 8), closed stars represent subjects in whom no sample was obtained (n 15). Thirteen subjects had respiratory failure and an FEV 1 less than or equal to 40% predicted. P. aeruginosa and mucoid P. aeruginosa were commonly present in the lower airway on a chronic basis (11/15 and 11/12, respectively, Table 2). Testing for CF (via sweat electrolytes and cystic fibrosis transmembrane regulator testing) was negative in all subjects culturing P. aeruginosa. On review of the patients histories of ever having specific treatment directed against H. influenza or P. aeruginosa or nontuberculous mycobacteria, less than half (38/78; 49%) of the subjects had had such directed treatment. Although fertility in males was not consistently tested, only one adult male reported being a biological father, having achieved a successful pregnancy with a female partner using artificial insemination of his own sperm, consistent with reports of fertility in some males (43). This male had an outer dynein arm defect and was a compound heterozygote for mutations in an intermediate chain dynein, DNAI1 (17). Adult females reported a range of fecundity, some achieving pregnancy with no difficulty, TABLE 2. SPUTUM MICROBIOLOGIC CHARACTERISTICS OF SUBJECTS WITH PRIMARY CILIARY DYSKINESIA (59 SUBJECTS WITH AT LEAST ONE SPUTUM SAMPLE) Haemophilus influenza Pseudomonas aeruginosa Mucoid P. aeruginosa Staphylococcus aureus Nontuberculous Mycobacteria n(%) n (%) n (%) n (%) n (%) Adults (n 44) One sample* 10 (22) 12 (27) 12 (27) 6 (14) 8 (18) Repeated samples 5 (11) 10 (23) 11 (25) 1 (2) 5 (11) Pediatric subjects (n 15) One sample* 12 (80) 3 (20) 1 (6) 7 (46) 0 (0) Repeated 2 (13) 1 (6) 0 (0) 2 (13) 0 (0) * Results of a single specimen of sputum. Results of several ( 3) sputum cultures on a subset of the same subjects, some data obtained from medical records, and regular clinical visits.

4 462 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL others reporting consistent infertility despite unprotected intercourse, also consistent with previous reports (8, 44, 45). Classic PCD: ciliary ultrastructure and ciliary activity. Seventysix subjects with PCD had abnormal ciliary ultrastructure, by definition (Table 3). Thirty-four subjects had a defect in outer dynein arm, 18 had defects in both outer and inner dynein arms, and 23 had a defect in the inner dynein arm alone, with normal outer dynein arm. One subject appeared to have a defect in the central complex (7, 22). Ciliary activity scores (mean SD) in PCD (n 33) were (range 0 8.6, median 1). By comparison, ciliary scores in healthy control subjects (n 52) were (range 2.9 9, median 8.6) (p , unpaired t test). The breakdown of ciliary activity by specific ciliary defect is shown in Figure 2. Analysis of variance testing showed a significant relationship between ciliary activity scores and ciliary structure category, p , with the worst ciliary activity seen in subjects with no dynein arms (n 10) (Figure 2). A subset of the PCD subjects had had more rigorous tests of mucociliary clearance done as a part of a separate study (12). In all subjects, there was no effective mucociliary clearance, although clearance was effective during controlled (reproducible) repeated cough efforts, consistent with previous reports (46). Two subjects declined a nasal scrape but were considered to have PCD because one was homozygous for mutations in the DNAI1 gene (see below for genetic tests completed to date), associated with defective outer dynein arms (17), and the other subject had a complete clinical phenotype, including otitis media, bilateral lower lobe bronchiectasis, expectoration of P. aeruginosa and M. abcessus, and situs inversus. Thus, the diagnosis of classic PCD seems secure in both subjects. However, to be rigorous in terms of definition, measures of nasal NO from this last individual were not included in the NO analyses (although both in fact had low nasal NO, 12 and 20 nl/minute). Nasal NO Levels Nasal NO was measured in 64 of the subjects with PCD and compared with normal healthy control subjects (n 27, mean age 37 2) and disease control subjects with CF (n 11, mean age 24 2). Levels were also measured in healthy parents of subjects with PCD (n 44, mean age 44 2). Levels of NO (nl/minute SD) were severely reduced in subjects with PCD as compared with normal subjects (Figure 3): versus (p ). Of note, NO levels did not relate to dynein arm defect within the PCD group, both subgroups having equally low levels (Figure 3), suggesting an all or no relationship between the cilia structural defect and the defect in NO production. Levels of NO in the subjects with CF were lower ( ) than in normal subjects, as reported previously (32). Despite subjects with CF having lower nasal NO than normal subjects, levels were significantly still higher than in subjects with PCD TABLE 3. FREQUENCY OF CILIARY STRUCTURAL DEFECTS IN CLASSIC PRIMARY CILIARY DYSKINESIA (n 76*) Structural Ciliary Defect Frequency Percent ODA alone IDA alone Both ODA and IDA Central apparatus 1 4 Total Definition of abbreviations: IDA inner dynein arm; ODA outer dynein arm; PCD primary ciliary dyskinesia. *n 2 others with complete classic PCD phenotype/mutations in a gene associated with PCD (DNAH1) declined nasal scrape samples for analysis. Defects of the central pair, radial spokes, or nexin links. Figure 2. Ciliary activity scores for subjects with PCD (by ciliary structural defect), as compared with healthy control subjects. Significant differences (analysis of variance, followed by unpaired t test) were seen between all subjects with PCD and normal subjects (p 0.001). ODA outer dynein arm defect; IDA inner dynein arm defect; ODA/IDA both dynein arms defective. (p ). Interestingly, for the other control group, the healthy parents of subjects with PCD (obligate heterozygotes, expected to be carriers for mutations associated with PCD) had lower than expected nasal NO levels, and the levels were intermediate between those of the subjects with PCD and normal subjects ( , range , median 279), suggesting that there is a relationship between carrier status and NO production intermediate between those subjects with overt cilia defects (PCD) and subjects without PCD who have apparently normal cilia structure and function. Analysis of variance testing showed a significant relationship between nasal NO levels and the different cohorts (p 0.001, corrected for multiple comparisons). There was a significant difference between levels in parents (carriers) as compared with those in subjects with PCD (p ). Levels in parents were also significantly lower than those in normal subjects (p 0.02). There was no relationship between age and nasal NO in any of the subject groupings (regression analysis) (47). There was no difference between PCD subjects with Kartagener s Syndrome (situs inversus) and those without situs inversus for age, FEV 1, sputum bacteriology, nasal NO levels, or dynein arm defects. DISCUSSION PCD is a clinical syndrome that overlaps with other disorders and may present to different medical subspecialties depending on the dominant clinical manifestations (8), (48). The diagnosis may be missed, or delayed, if the disease is not associated with all the classic features, particularly situs inversus. Delay in recognizing the disease may lead to adverse consequences for patients, in terms of inadequate programs of care or inappropriate treatment (10). Even when the diagnosis is successfully made, the disease may be poorly understood outside specialized centers, and thus adequate care and follow up may be lacking. This is at least partly due to the fact that the disease is

5 Noone, Leigh, Sannuti, et al.: Clinical and Ciliary Phenotype and Nasal NO Levels in PCD 463 Figure 3. Nasal nitric oxide (NO) levels in subjects with PCD (n 61 in total; n 16 with both DA defective, n 32 with ODA defective, and n 13 with IDA defective). The data are compared with healthy control subjects (n 27), subjects with CF (n 11), and parents (heterozygotes) of subjects with PCD (n 35). No differences were seen between any of the PCD subgroups, but significant differences (analysis of variance, followed by direct unpaired t test comparison) were seen between PCD and between parents and PCD/healthy control subjects (p ). Levels of nasal NO in subjects with CF were also less than those in normal subjects (p ) as previously reported but higher than in those with PCD (p ). perceived as very rare, although data for the precise incidence and prevalence are unavailable. Estimates from the published literature regarding situs inversus and its association with respiratory disease (specifically bronchiectasis) suggest that the numbers of subjects with some form of PCD may be quite substantial, that is, as many as 12,000 to 17,000 in the United States alone (5, 6). Another reason why PCD is not well understood may relate to the relative lack of detail on diagnosis and treatment in the standard general adult medical and pulmonary textbook, though treatment is relatively well covered in the pediatric textbooks (49 51). Finally, although there has been research performed in adults with PCD in Europe and elsewhere, with subsequent attention to clinical care, relatively little focus has been devoted to clinical care or research in North America to date (8). This study was thus undertaken to rigorously prospectively examine multiple components of the phenotype, from both a clinical and biologic point of view, in a cross-sectional study of a large cohort of subjects, and to gain initial insights into the status of PCD in North America. Initially, recruitment was slow, but as subjects with PCD in many parts of the North American continent became aware of the program at the University of North Carolina, and with the development of a patient advocacy group with a PCD website, and with easier internet access, enrollment steadily increased. Currently, the Chapel Hill PCD programs are backlogged with multiple patients and families anxious to participate, in many instances to gain clinical insights into their condition. Of note, only one non-white subject (an Asian Pakistani) emerged as having PCD, suggesting that gene mutation associated with PCD occurs predominantly in whites, or the data may reflect selection bias for good care and referral. Supporting the latter are anecdotal reports of a high prevalence of PCD amongst Asians living in Britain, possibly related to the tradition of consanguineous unions in that culture. The clinical phenotype of the subjects with what we defined as PCD (for the purposes of this study) and associated with ciliary structural defects (almost exclusively dynein arm defects) is similar to previous reports (1, 8, 48). The prevalence of situs inversus in this study at approximately 50% is in concordance with the randomness of laterality in PCD (4). Chronic upper and lower airway disease are part of the classic description of the disease and for this study were considered sine qua non features for diagnosis (52 54). Documented sinusitis was common in PCD, and patients had frequently undergone surgical procedures (37%). Otitis media in childhood, often requiring myringotomy tubes (though this procedure may be regarded as controversial in terms of its implementation in PCD) is another strong feature of PCD, a symptom complex that may regress somewhat in later life but that may result in some hearing loss (10). As has been recognized, these patients may present to oto-rhino-laryngology clinics, and a high index of suspicion is warranted when confronted with a phenoptype suggestive of PCD. Bronchiectasis may not be present in very young children and tends to be age dependent as with the decline in lung function. Routine performance of high-resolution computed tomographic scanning to more accurately delineate any potential bronchiectasis was beyond the scope of this study and would have likely increased the prevalence of the diagnosis of bronchiectasis. Many of the younger patients, or those with milder disease, had not had high-resolution computed tomographic scans and may in fact have bronchiectasis. Of note, no child less than 18 had finger clubbing. The incidence of prior lobectomies for this genetic, generalized disease of the lungs was also high at approximately 10% of those seen, especially in patients with severe disease. Although the slope-decline of lung function (FEV 1 ) with age in this cross-sectional small data set of subjects with PCD appears to be less than that of CF ( 25 50% that of CF), supporting the perception that this is a milder disease than CF, nonetheless it was particularly noteworthy that a significant proportion of patients had severe disease with respiratory failure, a smaller proportion either having or being considered for lung transplantation (38 40). Finally, a predominant symptom complex includes an early neonatal syndrome resembling wet lung, presumably reflecting the importance of functional cilia in clearing lung liquid in this early period after birth (35, 36). Although fertility was not systematically studied, most males from this cohort reported infertility (one achieved a pregnancy via simple artificial insemination of his own sperm), and some females appeared to be subfertile, although others were apparently fully fertile. This is consonant with anecdotal experience with PCD and the reports in the literature (43, 45). A systematic study of the microbiology of airway secretions in PCD has not been reported previously in a cohort of patients with PCD. In this study, although limited by the fact that samples were obtained in large part only once, sputum flora in PCD somewhat resembles that of CF, but with important age differences (55). H. influenza is the commonest organism in PCD from childhood into early adulthood, with or without S. aureus in a proportion of samples. Smooth P. aeruginosa is also common (25% in this cohort) in PCD, even at a young age ( 5 years of age), but the appearance of mucoid P. aeruginosa in 12 of the subjects with PCD is delayed compared with that of CF, first appearing after age 30 in the majority (11/12 of positive samples) of this cohort of subjects (one 15-year-old recently cultured mucoid P. aeruginosa for the first time). In CF, mucoid P. aeruginosa is typically seen at an earlier age, sometimes as young as during infancy (55). Its presence in PCD is interesting, as mucoid P. aeruginosa is usually regarded as a marker of CF and viewed as rare in non-cf lung disease. In this study, CF was excluded in all patients with PCD with mucoid P. aeruginosa by sweat

6 464 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL tests, nasal potential difference tests, and negative cystic fibrosis transmembrane regulator mutation analysis. Because the organism was isolated in a significant proportion of subjects with PCD in this study ( 20% of all PCD sputum samples and 35% of samples obtained from those 30 years), it appears that the change to mucoid-producing strains of P. aeruginosa may occur in a manner similar to CF but later in the course of the disease. This suggests that chronic failure of the mucociliary apparatus may predispose to colonization/infection with these organisms. Nontuberculous mycobacteria were also isolated in a significant proportion of cases ( 10%), again generally in older patients, and again affords interesting comparisons with CF, where up to 20% of patients with CF in some centers will grow these organisms from sputum cultures (56, 57). In three subjects (two M. abscessus and one M. avium complex), nontuberculous mycobacteria infection was considered pathogenic according to American Thoracic Society guidelines and required an aggressive multidrug regimen (58). Although some subjects had regular sputum sampling (most showing chronic colonization with organisms; Table 2), many of the subjects who attended for the study reported that their clinical care neither included regular surveillance of sputum for culture nor regular pulmonary function testing, and only 50% of subjects (39/78) had had at least one course of intravenous antibiotics. Given the spectrum of bacteria seen in this study, it seems reasonable to recommend that patients with PCD should be treated like patients with CF. Specifically, the program of treatment should include regular airway clearance, three to four times a year sputum samples monitoring, oral or systemic antibiotic coverage directed against the predominant organism, and pulmonary function surveillance (59). Directed antibiotic therapy is also appropriate, and with inexorable declines in lung function in some patients, despite aggressive treatment, lung transplantation is an option, as seen in some of the subjects in this study. We found a predominance of outer dynein arm defects in this cohort of subjects with PCD, as previously reported (7). In a study incorporating a rigorous analysis of ciliary structure and function in PCD originating from Australia, ciliary defects included dynein arm defects as well as central and peripheral tubule defects and abnormalities of ciliary orientation (22). We decided to categorize the current PCD cohort by the most obvious defects as seen in transmission electron microscopy primarily that of outer and inner dynein arms and central apparatus defects. Although abnormalities in the inner dynein arm may be difficult to ascribe to genetic abnormalities in some subjects with PCD, the consensus of the blinded review in this study was consistent, that is, the inner dynein arm was defective in all ciliary cross sections of subjects included in that category. In two subjects, the inner dynein arm was difficult to discern, and a previously described computerized technique was employed to help decide. In both cases the inner dynein arm was visible using this methodology, but the central pair was consistently abnormal (9, 29). A disease of interest also is the bronchiectasis found in Polynesian Island natives, characterized by a range of defects in cilia structure. Because situs inversus does not appear to occur in that syndrome, it appears to be due to different mechanisms than the genetic-based disease PCD, which is the subject of this report (60). Although it would be interesting to compare and contrast the two entities at several levels, there have been few clinical descriptors of the disease in Polynesians, no measures of nasal NO, and no genetic mutations sought or found in Polynesians to our knowledge. Measures of ciliary activity derived from video microscopy recordings of fresh biopsy samples from PCD and control subjects provide a semiquantitative insight into ciliary function in vivo (30). The observation that the subjects with loss of both dynein arms had the lowest ciliary activity scores is consonant with the notion of the outer arm particularly being associated with ciliary beat frequency, in contrast to the inner dynein arm, which appears to relate more to ciliary bending than frequency (7, 61, 62). Two of the eight subjects with inner dynein arm defects had near-normal ciliary activity (Figure 2). A recent study, using high-speed digital video imaging analyses of epithelial samples from patients with PCD, found a relationship between ciliary ultrastructural defect type and ciliary beat pattern (63). In that study, cilia with an outer dynein arm defect alone and those with both outer and inner arm defects had a higher incidence of ciliary immotility than those with inner arm defects alone, which tended to be associated with ciliary bending defects (reduced amplitude and stiffness). In vivo measures of mucociliary clearance in the absence of cough would be an alternative measure, to provide more information on whole lung clearance in these subjects because normal appearance on video microscopy in vitro may not translate into effective mucociliary clearance in vivo (12, 46). Some patients seen in the course of the study did not have classic features of PCD, yet had sufficient features suggestive of abnormal airway host defense to suggest a possible variant or atypical form of the disease (in the absence of other known causes of bronchiectasis) (64). Presumably the remaining subjects with clear-cut PCD and reasonably normal-looking ciliary activity (Figure 2) had functional abnormalities in cocoordinated and functional ciliary clearance but no recognizable abnormalities on electron microscopic analysis of cilia ultrastructure (7, 64). We elected to exclude the subjects without classic features of PCD from the data analysis, yet these subjects may be of interest in future studies. All, for example, had CF excluded by sweat tests, nasal potential difference tests, or cystic fibrosis transmembrane regulator mutation analysis. Given the genetic heterogeneity in PCD related to the complexity of ciliary structure and function, it seems plausible that there should exist milder forms of the disease with apparently incomplete phenotypes or with apparently normal ciliary structure and function as measured semiquantitatively by the techniques we employed for this study. This scenario has been well described in CF, with several genetic variants of the disease associated with milder phenotypes (65, 66). Because the genetic spectrum of PCD is still emerging, it remains to be seen whether mutations in genes other than those associated with dyneins/central apparatus structures are responsible for disease in these cases or alternately genetic mutations resulting in some residual dynein arm function. Examples of genes that plausibly could be associated with a milder phenotype include those with residual dynein arm function, radial spoke genes, or other ciliary genes (23, 67 69). Nasal NO has been reported to be low in PCD (13 15, 17, 31). The observation has been confirmed in this large cohort of subjects with PCD across a wide age and sex range in this study. All subjects with PCD had nasal NO measure below 100 nl/ minute, with clear separation from measures in healthy control subjects. Of particular note, there was no difference in NO levels between the subgroups of subjects with different ultrastructural abnormalities, that is those with outer versus inner versus both dynein arm defective, suggesting an all or none phenomenon irrespective of the dynein arm category associated with PCD, nasal NO levels are subsequently reduced. Although there is some overlap with CF, the difference on average between PCD and CF is still significant, and because CF is usually straightforward to diagnose, there should be little confusion between the two diseases. Therefore, use of NO as a relatively simple screening tool seems justified (15, 31). It can be performed from about the age of 5 years with appropriate coaching, and the results are very reproducible. The mechanism of the low nasal NO in PCD

7 Noone, Leigh, Sannuti, et al.: Clinical and Ciliary Phenotype and Nasal NO Levels in PCD 465 is still unknown and may relate to altered ciliary activity, altered expression of NO synthase isoforms, or chronic inflammation in the sinus antra (70 75). Patients with maxilliary sinusitis of nonspecific cause also have low levels of nasal NO and low immunohistochemical staining with NO synthase (75). Although we did measure nasal NO in such patients, the CF control group in the study do suffer from sinusitis almost invariably, and abnormalities in the NO/NO synthase system have also been reported in CF (72, 76). Despite some overlap in some of the phenotypic features of all these diseases, the extremely low levels of NO seen in this and other studies appears to be unique to PCD (31). Ciliary activity may play a role in NO production, perhaps induced by the shear stress of ciliary bending (77). Of note, healthy (including specifically no sinusitis) nonsmoking parents of subjects with classic PCD have intermediate levels of NO as compared with PCD and healthy control subjects. This suggests a genetic link to the reduction in nasal NO production and argues against an inflammatory or sinus obstruction origin to the reduction. The parents have no clinical symptoms or signs of PCD and have normal pulmonary function (FEV 1 [% predicted], ). Quantitative measures of mucociliary clearance would be of great interest in this group of subjects to see if nonclinically obvious alterations in mucociliary clearance are associated with this intermediate level of nasal NO production. There have been significant advances in the elucidation of the molecular basis of PCD recently, with disease-causing mutations identified in DNAH1 and DNAH5. DNAH1 is a human intermediate chain dynein orthologous to IC78, and DNAH5 is a heavy chain dynein orthologous to -heavy chain, both in Chlamydomonas reinhardtii. Chlamydomonas mutants of either the IC78 or the heavy chain are associated with defective outer dynein arms. DNAH1 is located on chromosome 9p13-p21, and composed of 20 exons, encoding 699 amino acids. To date, diseasecausing mutations have been detected in six (13%) of PCD families (n 47 studied) (16, 17, 78). A splice mutation, 219 3insT represents 6 of the 12 reported mutant alleles. DNAH5, on the other hand, is located on chromosome 5p15- p14 and is composed of 79 exons, and the first alternative exon encodes 4,624 amino acids. Thus far, disease-causing mutations have been detected in eight (32%) PCD families (n 25 studied) (18). Allelic heterogeneity has been observed with respect to the mutations discovered, and so far, DNAI1 and DNAH5 account for about 19% of mutations in PCD patients with outer dynein arm defects. Thus, PCD patients with outer dynein arm defects phenotypically have mutations in genes encoding for outer dynein arm proteins. In summary, PCD may be diagnosed using a combination of a careful clinical history together with an examination of ciliary structural analysis and measures of nasal NO. Ciliary ultrastructure and functional studies may appear normal in some instances, despite a strong phenotype otherwise. Conversely, PCD may be excluded if the phenotype is very weak (absence of lifelong otosino-pulomonary disease) with normal nasal NO levels. Patients with PCD appear to be an unmet clinical need in North America. These patients resemble patients with CF in many ways, yet have no structured resources similar to that of CF. Anecdotally, many of the participating subjects ( 70%) remarked that they had never or rarely had measures of pulmonary function, sputum culture tests, and never had systemic treatment with aerosolized or intravenous antibiotics, despite the existence of fairly severe disease in some cases. Physicians dealing with patients who have, or may have PCD, should adopt a specific diagnostic and therapeutic approach. Treatment of the severe form of the disease should include regular monitoring of lung function and sputum microbiology and aggressive airway clearance and antibiotics (including use of regular intravenous antibiotics if required). Milder, atypical forms of PCD may comprise a subset of patients with the disease, yet requiring a similar diagnostic and management strategy for the present. As has occurred in the field of CF over the past decade, molecular advances in the future may assist in the diagnosis of all suspected of having the disease and assist in genetic counseling. Conflict of Interest Statement : P.G.N. has no declared conflict of interest; M.W.L. has no declared conflict of interest; A.S. has no declared conflict of interest; S.L.M. has no declared conflict of interest; J.L.C. has no declared conflict of interest; M.H. has no declared conflict of interest; M.A.Z. has no declared conflict of interest; M.R.K. has no declared conflict of interest. Acknowledgment : The authors acknowledge the technical assistance of Rhonda Pace, B.S., and editorial assistance of Elizabeth Godwin and Lisa Brown. The authors thank all the subjects and referring physicians who participated in this study. References 1. Afzelius BA. A human syndrome caused by immotile cilia. Science 1976; 193: Mossberg B, Afzelius BA, Eliasson R, Camner P. On the pathogenesis of obstructive lung disease: a study on the immotile cilia syndrome. Scand J Respir Dis 1978;59: Afzelius BA. Inheritance of randomness. 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