Background CCDC39 and CCDC40 genes have recently been implicated in primary ciliary dyskinesia (PCD) with inner dynein arm (IDA) defects and axonemal disorganisation; their contribution to the disease is, however, unknown. Aiming to delineate the CCDC39/CCDC40 mutation spectrum and associated phenotypes, this study screened a large cohort of patients with IDA defects, in whom clinical and ciliary phenotypes were accurately described.
Methods All CCDC39 and CCDC40 exons and intronic boundaries were sequenced in 43 patients from 40 unrelated families. The study recorded and compared clinical features (sex, origin, consanguinity, laterality defects, ages at first symptoms and at phenotype evaluation, neonatal respiratory distress, airway infections, nasal polyposis, otitis media, bronchiectasis, infertility), ciliary beat frequency, and quantitative ultrastructural analyses of cilia and sperm flagella.
Results Biallelic CCDC39 or CCDC40 mutations were identified in 30/34 (88.2%) unrelated families with IDA defects associated with axonemal disorganisation (22 and eight families, respectively). Fourteen of the 28 identified mutations are novel. No mutation was found in the six families with isolated IDA defects. Patients with identified mutations shared a similar phenotype, in terms of both clinical features and ciliary structure and function. The sperm flagellar ultrastructure, analysed in 4/7 infertile males, showed evidence of abnormalities similar to the ciliary ones.
Conclusions CCDC39 and CCDC40 mutations represent the major cause of PCD with IDA defects and axonemal disorganisation. Patients carrying CCDC39 or CCDC40 mutations are phenotypically indistinguishable. CCDC39 and CCDC40 analyses in selected patients ensure mutations are found with high probability, even if clinical or ciliary phenotypes cannot prioritise one analysis over the other.
- Kartagener syndrome
- electron microscopy
- respiratory medicine
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Primary ciliary dyskinesia (PCD) (MIM 244400) is a rare inherited disorder characterised by abnormal ciliary motility,1 and affecting 1 in 15 000 to 30 000 individuals.2 Ciliary dysfunction, which in most cases results from structural defects, is responsible for impaired mucociliary transport leading to early upper and lower respiratory tract infections.3 4 In addition, given the key role of motile cilia in the establishment of left–right asymmetry during embryogenesis, ∼50% of patients display a situs inversus (thereby defining Kartagener syndrome (MIM 244400)).4 Infertility is also frequently observed in male patients, due to functional and structural abnormalities in sperm flagella, whose axonemal structure is very similar to that of motile cilia.5
The axoneme, which represents the core of motile cilia and flagella, consists of nine peripheral microtubules connected by nexin links and radial spokes, and a central complex composed of two single microtubules surrounded by the central sheath.6 Attached to the peripheral doublets, the inner and outer dynein arms (IDAs and ODAs, respectively) are multiprotein ATPase complexes that are essential for normal ciliary and flagellar movements.6 Another important regulator of motor activity is the dynein regulatory complex (DRC) that, as shown recently,7 corresponds to the nexin links. In theory, mutations in each of the genes encoding key components of the axoneme could lead to PCD. As expected, various axonemal ultrastructural defects have been reported in this disorder.8 A second level of heterogeneity is attested to by the fact that mutations in distinct genes may result in the same ultrastructural defect.9 This, for example, is the case for DNAI1, DNAI2, DNAH5, TXNDC3 and DNAL1 mutations in patients with a PCD phenotype characterised by an absence of ODAs,10–14 and RPGR, DNAAF1/LRRC50, and DNAAF2/KTU mutations in patients with an absence of ODAs and IDAs.15–18 Other genes have been involved in central complex defects (RSPH9 and RSPH4A)19 or in Kartagener syndrome with normal ciliary ultrastructure (DNAH11).20 As for the so-called IDA defects, they can be isolated or associated with axonemal disorganisation in several ciliary sections. In our experience, this latter phenotype accounts for about 16% of PCD cases8; it was found to represent 14% to up to 29% of PCD cases in other PCD cohorts.4 21 22 Noteworthy, no gene has been found to be implicated in the isolated absence of IDAs. However, just recently, mutations in two genes—CCDC39 and CCDC40—have been found in patients with a complex ultrastructural defect characterised by the absence of IDAs and defects of nexin links and radial spokes, leading to axonemal disorganisation.23 24
These latter data raise the larger question of the overall contribution of CCDC39 and CCDC40 to PCD related to an absence of IDAs associated or not with axonemal disorganisation. Another so far open question is to know whether patients with CCDC39 mutations are phenotypically distinguishable from those with CCDC40 mutations. Answering these two questions also has important consequences for the general strategy to follow in order to better guide the molecular analysis to be performed in patients with a suspicion of PCD. To address these issues, we screened CCDC39 and CCDC40 for mutations in a cohort of patients with a PCD phenotype characterised by an absence of IDAs associated or not with axonemal disorganisation. Genotype–phenotype correlations were subsequently performed in 43 patients, 15 of whom had previously been identified with mutations in CCDC39,23 after an extensive description of their clinical and ciliary features that were also assessed by quantitative studies and statistical analyses.
Patients and methods
The patients were recruited through the French National Center for Rare Respiratory Diseases located in Armand-Trousseau Children's Hospital, Paris, France, where, since 1985, ciliary and genetic investigations are part of the PCD diagnostic procedure in patients with recurrent respiratory tract infections. A definitive diagnosis of PCD is established on the association of suggestive clinical symptoms (eg, sinopulmonary syndrome and laterality defect), exclusion of other pathologic conditions such as cystic fibrosis or immunodeficiency, and evidence of abnormal ciliary structure and/or function.
The ethical review board of our institution approved the use of the database of the French National Center for Rare Respiratory Diseases for this study (CCTIRS, n°08.015bis). The patients and/or their parents were informed of the goal of the investigations and gave their written consent.
In the French National Center for Rare Respiratory Diseases, all ciliary investigations are performed when patients are free of airway tract infection or respiratory exacerbation for at least 6 weeks and, if necessary, at the end of an antibiotic course. Ciliary beat frequency (CBF) is evaluated on ciliated cells obtained by nasal or bronchial brushing, as described previously.25 The ciliary ultrastructure is analysed on airway biopsies obtained from either the inferior turbinate or the bronchi, immersed in glutaraldehyde, as described previously.25 Results are expressed as a percentage of abnormal cilia over the total number of analysed cilia (at least 50 per biopsy). Each axonemal abnormality is quantified and expressed as a percentage of each ultrastructural defect over the total number of abnormal cilia. Dynein arms are considered to be absent when missing from at least five of the peripheral microtubules. Since 2002, in case of questionable IDA defects on micrographs obtained by transmission electron microscopy (TEM), computerised analyses of cilia are systematically performed in order to improve IDA visualisation.26 Ciliary orientation is systematically evaluated by comparing the position of the central pairs from adjoining cilia; disorientation is defined as an angle >25°.27 The presence of compound cilia is also noted. For this study, ciliary length was evaluated on each sample (10 measures per patient) by means of optic microscopy with the Image-Pro Express 6.0 software (Media Cybernetics Inc, USA). Nasal nitric oxide is measured when feasible, according to patient's age, using a chemiluminescence analyser (Aerocrine, Solna, Sweden), with a transnasal flow rate of 0.3 l/min, following American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines.28
Mutation analysis of CCDC39 and CCDC40 was performed in 40 unrelated families (43 patients) with a PCD phenotype characterised by IDA defects: 34 families (37 patients) with an absence of IDAs associated with axonemal disorganisation, and six families (six patients) with an isolated absence of IDAs. We recorded and compared the clinical and ciliary phenotypes of patients with mutations identified in CCDC39 (CCDC39 group) or in CCDC40 (CCDC40 group). Fifteen patients from the CCDC39 group (table 1) had previously been reported in the study describing the very first mutations in CCDC3923; these 15 patients were included in the current study given that its main objective was phenotype―genotype correlations based on deep phenotyping. The recorded clinical features included sex, geographic origin, familial consanguinity, laterality defects, age at first symptoms and at phenotype evaluation, history of unexplained neonatal respiratory distress, upper and lower respiratory tract infections, nasal polyposis, otitis media with effusion, bronchiectasis confirmed by computed tomographies, and infertility when appropriate. The ciliary phenotype was assessed through the CBF and qualitative and quantitative analyses of the ciliary ultrastructure. In case of male infertility, the flagellar ultrastructural abnormalities, when available, were compared to the ciliary ones.
Genomic DNA was obtained from whole blood samples by use of a FlexiGene kit (Qiagen, France). All CCDC39 and CCDC40 coding exons and flanking intronic sequences were amplified by PCR. The resulting PCR products were subsequently sequenced (ABI 3730XL, Applied Biosystems, USA) on both strands with primers located outside the known polymorphisms referenced in Ensembl (http://www.ensembl.org/Homo_sapiens/Info/Index) or dbSNP (http://www.ncbi.nlm.nih.gov/snp/) databases. The previously described CCDC39 c.1167+1261A>G mutation located in intron 9 and creating a pseudoexon was also searched.23 The molecular analysis was stopped when two unambiguous molecular defects were identified in the homozygous or compound heterozygous state. Primer sequences are available upon request.
Qualitative data were described with frequencies and quantitative data were described with medians, ranges, and interquartile ranges (IQR). Phenotype, age at first symptoms, clinical features, and ciliary ultrastructure were compared between the two genotypes with tests adapted to small samples. Qualitative data were compared with Fisher's exact test and quantitative data were compared with Wilcoxon's test. Family ties were not taken into account. Statistical analyses were performed with SAS V.9.2 software (SAS Institute, Cary, North Carolina, USA). The R software (R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org) was used for plots. All tests were two-sided, and a value of p<0.05 was considered significant.
CCDC39 and CCDC40 mutation spectrum
Biallelic mutations in CCDC39 and CCDC40 were identified in 30/40 (75%) unrelated families with PCD characterised by IDA defects. The mutations were observed in 30/34 (88.2%) unrelated families exhibiting an absence of IDAs associated, in several ciliary sections, with axonemal disorganisation (figure 1): 22/34 (64.7%) have mutations in CCDC39, and 8/34 (23.5%) have mutations in CCDC40. No mutation was found in the six remaining families with an isolated absence of IDAs. The genotypes of patients with CCDC39 and CCDC40 mutations are displayed in tables 1 and 2. Among the 17 CCDC39 mutations identified (three nonsense, nine frameshift, four splice, and one missense mutations), five are novel (figure 2A). The 11 CCDC40 mutations identified (four nonsense, six frameshift, and one splice mutations) include nine that are novel (figure 2B).
Phenotype:genotype correlation study
The phenotype was compared between the following two groups of patients: one consisting of 26 patients from 22 unrelated families with CCDC39 mutations (CCDC39 group), and the other comprising eight unrelated patients with CCDC40 mutations (CCDC40 group). There was no statistically significant difference in origin, sex ratio, familial consanguinity, laterality defects and infertility between these two groups (tables 1 and 2). The clinical phenotypic features of all the PCD patients from families with identified mutations, which are displayed in table 3, were found to be similar. The age at phenotype evaluation (median age, range) was found to be similar between the CCDC39 group (20.9 years, 2―52 years) and the CCDC40 group (17.4 years, 1―68 years). Nasal nitric oxide measurements, available in eight patients with CCDC39 mutations, were found to be dramatically low for all patients, with a median (range) of 13.5 (2―46.3) parts per billion (ppb).
CBF analysis was performed in 28/34 patients with identified mutations. All cilia were found to be immotile, except in five unrelated patients with CCDC39 mutations (DCP481, DCP640, DCP692, DCP414, DCP323) in whom few beating cilia were observed (CBF ranging from 5―18 Hz).
Ciliary length and orientation, and presence of compound cilia, were found to be similar between CCDC39 and CCDC40 groups (table 4). The various axonemal structures could be precisely quantified in all but two patients (DCP854 from CCDC39 group, DCP815 from CCDC40 group) for whom micrographs were not available. All cilia from patients with identified mutations exhibited IDA defects. Associated axonemal defects are displayed in figure 3: the absence of radial spokes and nexin links, leading to axonemal disorganisation, was documented in all the patients with identified mutations, but was observed only in about half of the analysed axonemal sections; defects of ODA, central complex and peripheral microtubules, which were much less frequent, did not concern all the patients. The ultrastructural analyses showed similar defects in patients with mutations in CCDC39 or CCDC40.
The sperm flagellar ultrastructure was analysed in 2/5 and 2/2 infertile male patients with CCDC39 (DCP749 and DCP801) and CCDC40 mutations (DCP143 and DCP102), respectively. In all of them, the axonemal defects were found to be similar at the ciliary and flagellar levels (figure 1).
In our experience, the PCD phenotype characterised by IDA defects is present in about 16% of PCD patients.8 Among them, 81% display an absence of IDAs associated with axonemal disorganisation and 19% have an isolated absence of IDAs.8 The mutation analysis performed in the 40 unrelated families with IDA defects from our cohort allowed us to identify 30 families with biallelic mutations in CCDC39 or CCDC40, which represent the largest cohort so far reported with mutations in these genes. Noteworthy, all the mutations were found in patients with an absence of IDAs associated, in about half of the examined ciliary sections, with an axonemal disorganisation. Patients with mutations in CCDC39 or CCDC40 are phenotypically indistinguishable, in terms of both clinical features and ciliary structure and function. As discussed below, these data have important consequences for the molecular diagnostic strategy to be followed in patients with IDA defects.
Overall, the screening of CCDC39 and CCDC40 elucidated 88.2% (30/34) of independent PCD cases related to an absence of IDAs associated with axonemal disorganisation. This ultrastructural defect, therefore, represents by far the PCD phenotype with the highest proportion of patients with identified molecular defects. Indeed, this proportion is ∼31–63% for DNAI1 and DNAH5 in PCD related to ODA defects,9 29–32 and 17% for LRRC50 in PCD related to the absence of ODAs and IDAs,17 whereas in the case of central complex abnormalities or of Kartagener syndrome with normal ciliary ultrastructure, there are no quantitative data available so far.
Among the 30 independent patients with identified mutations, 22 carry mutations in CCDC39 (15 of them have been previously reported23), and eight carry mutations in CCDC40. Overall, this study identified 14 new mutations: five in CCDC39 and nine in CCDC40. Four recurrent mutations (c.1072delA, c.2190delA, c.357+1G>C, and c.610-2A>G) that had previously been described were also identified in CCDC39. For each recurrent mutation, the patients originate from the same geographic area, in keeping with founder effects.23 Regarding the c.610-2A>G mutation, newly described as a recurrent one, a founder effect has been confirmed by microsatellite analysis for two patients (DCP640 and DCP692), whereas in one case (DCP274) the mutation could have arisen independently (data not shown). Most families with mutations in CCDC39 (13/22) were native from North Africa, while most of those with mutations in CCDC40 (6/8) originated from Europe (France n=4, Italy n=1, Portugal n=1). Although this difference in the distribution of mutations according to origins was not statistically significant, these findings are in line with the first two studies, in which 8/19 families with CCDC39 mutations were native from North Africa,23 and 12/13 patients with CCDC40 mutations were of European extraction.24
All but one mutation (p.Thr594Ile in CCDC39) most likely result in loss of protein function. These are nonsense, frameshift or splicing mutations that would lead to the absence of protein production (due to mRNA decay) or to the production of truncated proteins. All these mutations were identified in the homozygous or compound heterozygous state in patients born to healthy parents, in keeping with the recessive mode of inheritance of the PCD phenotype. The p.Thr594Ile variation identified in CCDC39, which concerns a residue that is poorly conserved throughout evolution, was found in a patient (DCP481) who was also compound heterozygous for two other variations: the c.1363-3delC mutation that is predicted to severely alter splicing, and the c.1168-32A>G transition that might represent a polymorphism since it does not create a splice site or alter the branch point. This patient is one of the five patients with CCDC39 mutations in whom we detected a residual beating of a few cilia. At first glance, the phenotype could be consistent with compound heterozygosity for an unambiguous molecular defect (c.1363-3delC) and a potentially mild mutation (p.Thr594Ile). However, the four remaining patients with residual beating are homozygous or compound heterozygous for two unambiguous deleterious mutations. In addition, for two of these patients (DCP414 and DCP323) who belonged to two independent families, the affected siblings (DCP413 and DCP759, respectively) displayed a typical phenotype characterised by immotility of all observed cilia.
The clinical phenotypic features described in our cohort are in line with those previously reported in PCD (ie, sinopulmonary syndrome, bronchiectasis, laterality defect, neonatal respiratory distress, male infertility).33 Our phenotype-genotype correlation study revealed that, in the case of PCD related to an absence of IDAs associated with axonemal disorganisation, the ultrastructural phenotype allows the selection of two genes with a very high expected mutation rate. Indeed, according to our data obtained in a large cohort of patients, mutations in CCDC39 or CCDC40 account for 88.2% of cases related to this ultrastructural phenotype. Our detailed analysis revealed that patients with mutations in CCDC39 or CCDC40 are phenotypically indistinguishable, thereby precluding the choice of the first gene to be studied. However, the higher frequency of CCDC39 mutations in our cohort, a persistence of some beating cilia, or a North African origin would rather prompt us to start with CCDC39 analysis.
Given the similarity of the phenotypes of patients with mutations in CCDC39 or CCDC40, it is tempting to hypothesise that the CCDC39 and CCDC40 proteins could participate in the same function and/or structure. As shown previously,23 24 mutations in CCDC39 or CCDC40 result in the mislocalisation of GAS11, one of the subunits of the DRC, which is no longer in the axoneme but accumulates in the apical cytoplasm. On the other hand, in the alga Chlamydomonas reinhardtii, mutations in DRC4, the orthologue of GAS11, result in structural defects involving the DRC and IDAs.34 And as shown for DRC4 in Chlamydomonas, mutations in a single DRC subunit result in the lack of other DRC subunits.34–36 CCDC39 and CCDC40, two proteins containing coiled-coil domains, could therefore play an important role in stabilising the DRC. Overall, even if their respective role in the proper assembly of ciliary axonemes remains unexplained, it is clear that these structurally related proteins are also functionally related. This is further supported by the reported mislocalisation of CCDC39 in respiratory cells from patients with CCDC40 mutations; in those cells, CCDC39 was shown to be absent from the axoneme and enriched in the apical cytoplasm at the ciliary base.24 However, the study of the molecular pathology of CCDC39 and CCDC40 reveals that, in spite of these structural and functional similarities, these two proteins are not redundant.
Ten PCD patients with an absence of IDAs, associated (n=4) or not (n=6) with axonemal disorganisation, did not carry any mutation in CCDC39 or in CCDC40. No obvious difference, in terms of clinical phenotypic features, was evident between those 10 individuals and patients with CCDC39 or CCDC40 mutations (data not shown), raising two non-exclusive hypotheses: the possible mutations in intronic or regulatory regions of CCDC39 or CCDC40, and the existence of at least another gene implicated in PCD related to IDA defects. Even though no mutation has yet been identified in patients with an isolated absence of IDAs, the small number of such patients already studied, together with the absence of a gene implicated in this phenotype, should encourage continuing analysis of CCDC39 and CCDC40 in these patients.
The authors are grateful to the patients and their families who participated in this study, and thank all referring physicians for their help and their confidence. This work was supported by the Assistance Publique-Hôpitaux de Paris (PHRC AOM06053, P060245), the Fondation pour la Recherche Médicale, the Legs Poix from the Chancellerie des Universités, and the Milena Carvajal—ProKartagener Foundation.
SB and ML contributed equally to this work.
Competing interests None.
Ethics approval The ethical review board of the French National Center for Rare Respiratory Diseases (CCTIRS, n°08.015bis).
Provenance and peer review Not commissioned; externally peer reviewed.
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