The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no ‘gold standard’ reference test. Hence, a task force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in the light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on: clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of PCD; also to provide advice when the diagnosis is not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for PCD.

Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.

SUMMARY Dyx1c1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deletion of Dyx1c1 exons 2–4 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder characterized by chronic airway disease, laterality defects, and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1c.T2A start codon mutation recovered from an ENU mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also created laterality and ciliary motility defects. In humans, recessive loss-of-function DYX1C1 mutations were identified in twelve PCD individuals. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans revealed disruptions of outer and inner dynein arms (ODA/IDA). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA/IDA assembly factor DNAAF2/KTU. Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

Reduced generation of multiple motile cilia (RGMC) is a rare mucociliary clearance disorder. Affected persons suffer from recurrent infections of upper and lower airways because of highly reduced numbers of multiple motile respiratory cilia. Here we report recessive loss-of-function and missense mutations in MCIDAS-encoding Multicilin, which was shown to promote the early steps of multiciliated cell differentiation in Xenopus. MCIDAS mutant respiratory epithelial cells carry only one or two cilia per cell, which lack ciliary motility-related proteins (DNAH5; CCDC39) as seen in primary ciliary dyskinesia. Consistent with this finding, FOXJ1-regulating axonemal motor protein expression is absent in respiratory cells of MCIDAS mutant individuals. CCNO, when mutated known to cause RGMC, is also absent in MCIDAS mutant respiratory cells, consistent with its downstream activity. Thus, our findings identify Multicilin as a key regulator of CCNO/FOXJ1 for human multiciliated cell differentiation, and highlight the 5q11 region containing CCNO and MCIDAS as a locus underlying RGMC.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307(∗)), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus.