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Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia

Johanna Raidt, Julia Wallmeier, Rim Hjeij, Jörg Große Onnebrink, Petra Pennekamp, Niki T. Loges, Heike Olbrich, Karsten Häffner, Gerard W. Dougherty, Heymut Omran, Claudius Werner
European Respiratory Journal 2014 44: 1579-1588; DOI: 10.1183/09031936.00052014
Johanna Raidt
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Julia Wallmeier
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Rim Hjeij
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Jörg Große Onnebrink
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Petra Pennekamp
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Niki T. Loges
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Heike Olbrich
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Karsten Häffner
2Dept of Pediatrics, University Hospital Freiburg, Freiburg, Germany
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Gerard W. Dougherty
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Heymut Omran
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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Claudius Werner
1University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany
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  • For correspondence: claudius.werner@ukmuenster.de
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Abstract

Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory tract infections. High-speed video-microscopy analysis (HVMA) of ciliary beating, currently the first-line diagnostic tool for PCD in most centres, is challenging because recent studies have expanded the spectrum of HVMA findings in PCD from grossly abnormal to very subtle. The objective of this study was to describe the diversity of HVMA findings in genetically confirmed PCD individuals.

HVMA was performed as part of the routine work-up of individuals with suspected PCD. Subsequent molecular analysis identified biallelic mutations in the PCD-related genes of 66 individuals. 1072 videos of these subjects were assessed for correlation with the genotype.

Biallelic mutations (19 novel) were found in 17 genes: DNAI1, DNAI2, DNAH5, DNAH11, CCDC103, ARMC4, KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, DYX1C1, ZMYND10, CCDC39, CCDC40, CCDC164, HYDIN, RSPH4A and RSPH1. Ciliary beat pattern variations correlated well with the genetic findings, allowing the classification of typical HVMA findings for different genetic groups. In contrast, analysis of ciliary beat frequency did not result in additional diagnostic impact.

In conclusion, this study provides detailed knowledge about the diversity of HVMA findings in PCD and may therefore be seen as a guide to the improvement of PCD diagnostics.

Abstract

PCD is associated with a variety of ciliary beat pattern abnormalities which correlate with genetic subtypes http://ow.ly/zh5jP

Introduction

Primary ciliary dyskinesia (PCD) (MIM 244400) is a rare genetic disorder of ciliary motility resulting in neonatal respiratory distress, chronic upper and lower respiratory tract disease, male infertility, and organ laterality defects in almost 50% of cases (Kartagener's Syndrome) [1].

PCD is both under-diagnosed and diagnosed too late [2]. Direct visualisation of ciliary beat pattern (CBP) and frequency (CBF) by high-speed video-microscopy analysis (HVMA) has been recommended as a first-line diagnostic test, along with transmission electron microscopy (TEM) [3]. Further methods that complete the armamentarium for PCD diagnosis include immunofluorescence (IF) microscopy, genetics and measurement of nasal nitric oxide (nNO) production [4].

Although HVMA is increasingly applied, it is a challenging method for various reasons: 1) chronic infection and inflammation often result in additional secondary ciliary damage, which obscures the primary beat abnormality [1, 5]; 2) standardised procedures between centres are lacking; 3) there is a shortage of quantitative data for the interpretation of CBPs; and 4) HVMA abnormalities may be subtle in recently identified variants.

Mutations in multiple genes have been described as being a cause of autosomal recessive, nonsyndromic PCD. Mutations in nine genes affect outer dynein arm (ODA) components: DNAI1, DNAI2, DNAH5, DNAL1, CCDC103, NME8/TXNDC3, CCDC114 and ARMC4 [1, 6, 7] result in ODA defects, whereas DNAH11 mutations result in PCD with normal ultrastructure [8]. Nine genes encode the cytoplasmic proteins required for the assembly of both ODA and inner dynein arm (IDA) complexes: KTU/DNAAF1, LRRC50/DNAAF2, C19orf51/DNAAF3, LRRC6, HEATR2, DYX1C1/DNAAF4, ZMYND10, SPAG1 and C21orf59 [1, 9–12]. Mutations in CCDC39 and CCDC40 result in an IDA and microtubular disorganisation defect [13, 14]. Mutations in CCDC164 and CCDC65 result in nexin link defects [15, 16]. Mutations in HYDIN cause a deficiency of the C2b projection of the central pair (CP) microtubules [17]. RSPH9, RSPH4A [18] and RSPH1 [19] affect radial spoke protein components. Mutations in genes affecting flagellar motility, such as DNAH1 (IDA component) [20], have not yet been linked to PCD.

In this study, we correlated the findings of the HVMA with a diverse set of genetic PCD variants using Sisson-Ammons Video Analysis (SAVA) (Ammons Engineering, Mt Morris, MI, USA) [21], a widely-accepted system for ciliary beat analysis. These videos were acquired during clinical routine PCD work-up. It was not the aim of the study to systematically re-evaluate SAVA or to compare it to other systems.

Methods

Subjects

Subjects were referred to our centre for PCD work-up. PCD diagnosis was based on typical clinical features and PCD-specific abnormal findings in at least two of the following methods: nNO measurement, HVMA, TEM or IF microscopy. Videos from 10 individuals without acute or chronic respiratory symptoms (healthy controls (HCs)) and 10 individuals with respiratory symptoms but in whom PCD was excluded (disease controls (DCs)) were included for comparison. Although DCs were referred for PCD work-up, none displayed key PCD characteristics (situs abnormality, neonatal respiratory distress, both upper and lower airways problems). Beyond that, PCD was ruled out by at least one additional finding: high nNO values, normal TEM or IF. As ultrastructural analysis using TEM or IF microscopy may be unremarkable in PCD [1, 8, 15–17] or even misleading [22], we only analysed individuals with confirmed biallelic mutations. The study was approved by the Ethics Committee of the Westphalian Wilhelms University of Münster (Münster, Germany). Written informed consent to participate in this study was obtained from each individual or by the legal guardian of subjects under 18 years or age.

Nasal brush biopsy and high-speed video-microscopy analysis

Ciliated respiratory epithelial cells were obtained by nasal brush biopsy using a cytology brush, and were suspended in RPMI 1640 medium. HVMA was carried out directly after brushing. Videos were recorded using a Basler scA640-120 fm digital high-speed video camera (Basler AG, Ahrensburg, Germany) attached to an inverted phase-contrast microscope (Zeiss Axio Vert. A1; Carl Zeiss AG, Göttingen, Germany) equipped with a 40× objective. Digital image sampling was performed at 120–150 frames per second (fps) and a 640×480 pixel resolution. Image processing was performed using SAVA [21]. The temperature was maintained at 25°C by a Pecon Temp Controller 2000-2 (PeCon GmbH, Erbach, Germany). The ciliary beat was evaluated by views from both the side and the top in real time and in slow motion playback. CBP was described using the following terms: recognisability of regular forward and recovery strokes; static cilia; almost static cilia with minimal movements; stiff beating due to a reduced bending capacity/amplitude; abnormal circular beating. The quality of the video samples was scored using an adapted classification system [5]: excellent (ciliated cell bundle without projections), good (ciliated cell bundle with minor projections), acceptable (ciliated cell bundle with major projections), insufficient (isolated ciliated cell within an epithelial strip or single cell). Insufficient videos were excluded from the analysis. HVMA was performed independently by two investigators: J. Raidt or J. Wallmeier, and C. Werner or H. Omran. In all cases, HVMA was performed prior to genetic testing. Results from the HVMA were not modified by knowledge of the genetic result. Samples were only taken in individuals who were not receiving nasal steroids or decongestants, and who did not show signs of acute respiratory infection for at least 3 weeks in order to minimise secondary ciliary dyskinesia.

Mutational analysis

Mutational analysis was performed after whole genome linkage analysis as described elsewhere [6] or by using a direct candidate gene approach. DNA was isolated from blood samples or from lymphocyte cultures standard methods. Amplification of coding exons, including the exon–intron boundaries, was performed using a volume of 50 μL containing 30 ng of DNA, 50 pmol of each primer, 2 mM dNTPs and 1.0 U GoTaq DNA polymerase (Promega Corporation, Madison, WI, USA). PCR conditions and primer details are available upon request. PCR products were verified by agarose gel electrophoresis, purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and sequenced bidirectionally using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Life Technologies Ltd, Paisley, UK). Samples were separated and analysed on an Applied Biosystems 3730xl DNA Analyser. Sequence data were evaluated using CodonCode software (www.codoncode.com). Only biallelic mutations that could be clearly confirmed as causing PCD were scored.

Statistics

Statistical analysis was performed using Microsoft Excel (www.microsoft.com) and SPSS 22 (www.ibm.com). The mean CBF of each video was assessed using SAVA [21], utilising representative regions of interest (ROI). For each genetic PCD variant, the median CBF, lower/upper quartile and range were determined.

Results

Genetics

Biallelic mutations were identified in 17 genes from 66 PCD subjects of 55 families. 29 subjects exhibited homozygous mutations and 37 individuals exhibited compound heterozygous mutations. Genetic results from 49 individuals have been published previously (see table 1 for references). 19 mutations have not been previously published in the literature or in Ensembl (www.ensembl.org). All mutations resulted in loss-of-function of the respective proteins. All mutations and their functional consequences are shown in table 1.

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Table 1– Detailed genetic findings in 66 subjects with primary ciliary dyskinesia

High-speed video-microscopy analysis

1072 out of 1527 videos from affected individuals (92 out of 130 videos from DCs; 104 out of 125 from HCs) fulfilled the quality control criteria and were therefore analysed. The number of videos available for each gene is presented in table 2. Real-time videos of ciliated nasal epithelial cells are shown in the online supplementary material (videos S1-S19).

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Table 2– Mean ciliary beat frequencies in Hz for genetic primary ciliary dyskinesia variants, disease controls (DCs) and healthy controls (HCs)

Ciliary beat frequency

CBF measurements are summarised in figure 1 and table 2. In the DC group, CBFs ranged 3.95–8.44 Hz with a median frequency of 5.32 Hz; in the HC group, CBFs ranged 4.25–11.63 Hz with a median frequency of 6.36 Hz. CBF in subjects carrying mutations leading to ODA defects (DNAH5, DNAI1, DNAI2, ARMC4 and CCDC103) was severely reduced, with minimal residual ciliary movements in most videos. As an exception, cilia from individual OP-1193 II2 carrying biallelic CCDC103 mutations were largely completely immotile. In subjects with mutations in KTU/DNAAF2, LRRC50/DNAAF1, LRRC6 and ZMYND10, which encode assembly factors of ODA/IDA complexes, immotility was invariably observed. By contrast, HVMA yielded heterogeneous findings in individuals carrying DYX1C1 mutations: in patient F-648 II1, a slightly decreased CBF of 3.45 Hz with maximum values of 7.07 Hz was present, whereas cilia in patient OP-86 II2 were immotile.

Figure 1–
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Figure 1–

Boxplot illustrating ciliary beat frequency (CBF) measured using high-speed video-microscopy analysis in individuals with genetically confirmed primary ciliary dyskinesia sorted according to gene. The bars indicate median CBF values. The boxes respresent 25–75th percentiles. The whiskers indicate maximum and minimum values. DC: disease control. HC: healthy control. #: CBF measurements in CCDC39 or CCDC40 mutant cilia yielded implausibly low values using Sisson-Ammens Video Analysis (Ammons Engineering, Mt Morris, MI, USA).

In subjects with mutations in DNAH11, CBF tended to be increased (median 8.7 Hz (25–75th percentile 6.92–11.31 Hz)). There was a considerable CBF variability (range 1.44–24.86 Hz) with hyperkinetic edges and almost static cilia within the same specimen.

In CCDC39 and CCDC40 mutants, SAVA proved unreliable in measuring CBF: median CBF was 2.58 Hz in the CCDC39 patient and 4.94 Hz for CCDC40. In order to further clarify this discrepancy with the obvious visual appearance of an extremely increased frequency, we compared the videos with those from individuals carrying DNAH11 mutations (increased CBF) and DCs (normal CBF). We found that the CBF was higher in CCDC39 and CCDC40 (videos S13–S14). Determination of CBF by timing 10 ciliary beat cycles from slow-motion playback of the video files did indeed result in values that were clearly above 15 Hz.

CBF of CCDC164 mutant cilia was slightly increased with a broad overlap to DCs. In individuals with HYDIN mutations, CBF was normal. In individuals with mutations in radial spoke head components, CBF was slightly decreased (RSPH4A) or normal (RSPH1).

Taken together, CBF values were clearly abnormal in individuals with ODA and ODA/IDA defects. In the other groups, CBF measurement could not distinguish PCD individuals from DCs.

Ciliary beat pattern

A normal beat cycle is characterised by a strong beating stroke followed by a recovery stroke (fig. 2; video S1). Whereas cilia are in a straight position during the beating stroke, the recovery stroke is initiated by bending of the proximal axoneme. Top-view analysis revealed that cilia generally beat within a plane with only small deviations from the longitudinal axis.

Figure 2–
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Figure 2–

Diagram of normal ciliary beat cycle. A normal ciliary beat pattern is characterised by a strong beating stroke (black) followed by a recovery stroke (grey). Whereas the cilia are in a straight position during the beating stroke, the recovery stroke is initiated by a bending of the proximal axoneme.

Most videos from subjects with ODA defects derived from individuals with DNAH5 mutations. Typically, DNAH5 mutant cilia were in a bent position and showed minimal residual movements. Beating of neighbouring cilia was highly uncoordinated (video S2). A similar pattern was observed in other ODA-defect variants (DNAI1, DNAI2, ARMC4 and CCDC103) (videos S3–S6).

Mutations resulting in combined ODA/IDA defects (KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, ZMYND10 and DYX1C1) resulted in complete ciliary immotility (videos S7–S11). As an exception, individual F-648 II1, who had a homozygous stop mutation in DYX1C1, displayed a subtle reduction of the beating amplitude due to an impaired recovery stroke in most videos (video S12), whereas static cilia were present only occasionally.

CBP of subjects with CCDC39 and CCDC40 mutations was extremely stiff. A distinction between beating and recovery strokes was not possible (videos S13–S14). CCDC164 mutant cilia beat with reduced amplitude (video S15) that was much less pronounced than observed in CCDC39 and CCDC40.

Cilia of subjects with DNAH11 mutations showed a reduced proximal bending (video S16). The degree of stiffness was less pronounced than in CCDC39/CCDC40 cilia but higher than in CCDC164 cilia.

HYDIN mutant cilia lacked coordinated beating activity. The bending capacity along the ciliary axoneme was reduced, resulting in decreased beating amplitudes (video S17). Cilia did not generate distinct effective or recovery strokes. In few regions, immotile cilia were also found. A rotatory ciliary movement was observed only occasionally.

In individuals with mutations in radial spoke head components, we found a stiff CBP in a subject with RSPH4A mutations (video S18) and in a subject with RSPH1 mutations (video S19). Circular motion was not seen in RSPH4A mutant cilia. In RSPH1 mutant cilia, circular motion was seen only in a minority of cell clusters.

Discussion

Diagnosing PCD requires a combined approach of complementary methods, all of which have limitations in sensitivity and specificity [1, 3, 4, 30]. Although high-throughput genetic technologies may overcome this in future, many genetic PCD variants remain undiscovered (currently 60–70% of cases can be identified genetically [1]) and this methodology is not yet implemented for routine diagnostics. Because the TEM of certain PCD variants is unremarkable [8, 15, 17, 30], HVMA has become the first-line diagnostic test for establishing PCD diagnosis.

HVMA protocols differ among centres in many respects: sampling techniques, microscopes and cameras, temperature during analysis, software, and evaluation criteria. In our study, we used a high-speed video camera at 120–150 fps for capturing video, and SAVA for HVMA analysis. SAVA conveniently allows the determination of CBF and slow-motion video-pattern analysis [21]. We analysed cilia using a total magnification of 400× (resulting from a 40× objective and a 10× ocular lens magnification) at 25°C. Potential disadvantages of this approach include a reduced resolution of cilia as opposed to systems with a higher magnification (1000×). However, using a higher magnification also has potential limitations. Use of 100× objectives (numeric aperture 1.46) results in a markedly reduced depth of focus in the z plane (0.23 μm). Consequently, cilia not recorded strictly from the side may beat outside the range of the focal z plane as shown in video S20, which was captured with a 100× objective. Thus, the limited depth of focus when using a 100× objective bears the risk of misinterpretation of CBPs when the entire ciliary beating cycle is not visualised. This can be avoided by using a 40× objective (numeric aperture 0.56), which has a larger depth of focus of 1.5 μm. In our system, we captured videos at 120 fps, whereas others use cameras with a higher speed of up to 500 fps. Using 120 fps might hinder analyses of very fast ciliary beat variants, a limitation we tried to overcome by analysing CBP at 25°C. This lowers CBF to a point that sufficient images are available for CBP analysis. The ideal temperature for HVMA is also a matter of debate: whereas some centres cool the specimen (because CBP does not change at low temperatures [31] and analyses are facilitated due to the reduced CBF) others perform HVMA at 37°C [32]. However, the temperature in the upper airways increases from about 25°C in the nasal vestibule to 34°C in the nasopharynx [33]. Thus, one might argue that 37°C represents a supra-physiological temperature for nasal-derived cells. We have not systematically compared our protocol with other HVMA systems [31, 34, 35] so it is unclear whether the potential differences are of practical significance. However, our approach enabled us to describe most known subtle PCD variants, such as CP defects [17], isolated nexin link defects [15] and functional ODA defects [8].

SAVA calculates CBF by determination of relative light intensity changes of each pixel within a ROI (as used in this study) or the whole field (whole-field analysis). SAVA only proved inappropriate in samples from individuals with CCDC39 or CCDC40 mutations as values were implausibly low. We interpreted this using the extremely stiff CBP. This resulted in only small changes in pixel light intensity that could not be detected accurately using SAVA. Therefore, we recommend a critical visual validation of CBF measurements. Another limitation of HVMA for CBP analysis is the lack of quantitative parameters that describe beat pattern anomalies. Thus, results are dependent on visual interpretation by experienced personnel. Whereas SAVA and other systems are able to quantify CBF, development of quantitative CBP analysis is still at a preliminary stage [36].

In our study, CBF measurement identified PCD variants with severely reduced CBF. However, in these variants, CBP was equally severely altered and discrimination of PCD subjects from unaffected DCs was readily possible by visual analysis alone. In PCD variants with detectable ciliary motility: 1) mean CBF tended to be increased in DNAH11 mutant individuals but displayed an overlap with DCs; 2) mean CBF was within the normal range; or 3) CBF measurement was inaccurate (CCDC39/CCDC40).

In DCs, mean CBF was slightly lower than in HCs, possibly due to the higher degree of secondary dyskinesia. Our values are in line with CBFs measured at 24°C in sinonasal epithelial cells from individuals with chronic sinusitis, where values ranged 3.37–9.13 Hz [37], whereas others have found higher values (7.9 Hz) [34]. These differences might reflect the heterogeneity of HVMA protocols and highlight the need for each centre to carefully determine reference values.

Unlike CBF, CBP analysis nicely correlated with genetic findings and allowed HVMA findings to be classified into different groups, as follows. 1) Combined ODA/IDA defects almost invariably showed complete ciliary immotility. 2) Isolated ODA defects exhibited minimal residual, highly disorganised beating. Completely immotile cilia were seen only occasionally. 3) CCDC39 and CCDC40 mutations resulted in an extremely stiff CBP with hardly any amplitude and an increased CBF. 4) DNAH11 mutations resulted in a hyperkinetic CBP that was less stiff than in CCDC39 and CCDC40 mutations. CBF tended to be increased compared to controls. Notably, we also found videos with almost static cilia from DNAH11 mutants, confirming recent findings in mice [38]. 5) Mutations in CCDC164 resulted in very subtle HVMA abnormalities. CBP appeared rigid due to reduced amplitude. This phenotype was very mild and could easily be interpreted as normal. 6) Mutations affecting the central pair or radial spoke apparatus have been described as resulting in a circular motion [18, 39] or in a low-amplitude movement [19]. In our PCD subjects, HYDIN mutations resulted in rotatory movements only in a minority of ciliary fields. This variant was characterised by a mild reduction of the ciliary bending capacity making it difficult to establish the diagnosis with HVMA alone. The two individuals with RSPH4A and RSPH1 mutations exhibited a stiff CBP with a reduced beating amplitude, a pattern described in RSPH1 mutations [19]. Circular movements were present only in a minority of cilia in the RSPH1 subject. Thus, circular ciliary motion is not a compulsory finding in CP or radial spoke defects.

Although typical HVMA findings can be attributed to genetic PCD variants, it should be stressed that phenotypic variations can be found even within one gene, as evidenced in individuals with DYX1C1 mutations. Similarly, discrepant phenotypes have even been reported in patients with identical genotypes [19], stressing that ciliary beating is not only determined by the molecular defect. Taken together, these findings emphasise that HVMA findings cannot be easily translated to ultrastructural or genetic findings. Thus, additional assays are still necessary to elucidate the underlying mechanisms leading to PCD [6, 9, 15].

Currently, many institutions implement HVMA in order to provide state-of-the-art PCD diagnostics. We hope that our HVMA findings in distinct genetic PCD variants will help disseminate the valuable knowledge necessary for diagnosing PCD. Awareness of the variability of abnormal findings associated with PCD will avoid false-positive as well as false-negative results, and will therefore improve PCD care. Because the interpretation of HVMA findings requires a sufficient sample size of diverse variants, we refer readers to our website pcd.uni-muenster.de, where an extensive, continuously updated collection of HVMA videos of different genetic PCD variants is accessible.

Acknowledgments

We are grateful to the PCD-affected individuals and their families for their participation. We also gratefully acknowledge the German patient support group Kartagener Syndrom und Primaere Ciliaere Dyskinesie e.V. Finally, we thank M. Herting, D. Ernst, K. Vorspohl and S. Helms (all University Children's Hospital Münster, Dept of General Pediatrics, Pediatric Pulmonology Unit, Münster, Germany) for their excellent technical work.

Footnotes

  • For editorial comments see page 1418.

  • This article has supplementary material available from erj.ersjournals.com

  • Support statement: This work was supported by European Commission FP7 (Seventh Framework Programme for Research) grant no. 305404 (BESTCILIA) to Heymut Omran, European Commission FP7 grant no. 241955 (SYSCILIA) to Heymut Omran, the “Deutsche Forschungsgemeinschaft” (DFG OM 6/4, OM 6/5) and the IZKF Münster (Om2/009/12).

  • Conflict of interest: None declared.

  • Received January 16, 2014.
  • Accepted July 2, 2014.
  • ©ERS 2014

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Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia
Johanna Raidt, Julia Wallmeier, Rim Hjeij, Jörg Große Onnebrink, Petra Pennekamp, Niki T. Loges, Heike Olbrich, Karsten Häffner, Gerard W. Dougherty, Heymut Omran, Claudius Werner
European Respiratory Journal Dec 2014, 44 (6) 1579-1588; DOI: 10.1183/09031936.00052014

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Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia
Johanna Raidt, Julia Wallmeier, Rim Hjeij, Jörg Große Onnebrink, Petra Pennekamp, Niki T. Loges, Heike Olbrich, Karsten Häffner, Gerard W. Dougherty, Heymut Omran, Claudius Werner
European Respiratory Journal Dec 2014, 44 (6) 1579-1588; DOI: 10.1183/09031936.00052014
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