Kim G. Nielsen

Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children.

Primary ciliary dyskinesia (PCD) is associated with abnormal ciliary structure and function, which results in retention of mucus and bacteria in the respiratory tract, leading to chronic oto-sino-pulmonary disease, situs abnormalities and abnormal sperm motility. The diagnosis of PCD requires the presence of the characteristic clinical phenotype and either specific ultrastructural ciliary defects identified by transmission electron microscopy or evidence of abnormal ciliary function. Although the management of children affected with PCD remains uncertain and evidence is limited, it remains important to follow-up these patients with an adequate and shared care system in order to prevent future lung damage. This European Respiratory Society consensus statement on the management of children with PCD formulates recommendations regarding diagnostic and therapeutic approaches in order to permit a more accurate approach in these patients. Large well-designed randomised controlled trials, with clear description of patients, are required in order to improve these recommendations on diagnostic and treatment approaches in this disease.

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartageners syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40 is expressed in tissues that contain motile cilia, and mutations in Ccdc40 result in cilia with reduced ranges of motility. We further show that CCDC40 mutations in humans result in a variant of PCD characterized by misplacement of the central pair of microtubules and defective assembly of inner dynein arms and dynein regulatory complexes. CCDC40 localizes to motile cilia and the apical cytoplasm and is required for axonemal recruitment of CCDC39, disruption of which underlies a similar variant of PCD.

Recessive HYDIN mutations cause primary ciliary dyskinesia without randomization of left-right body asymmetry.

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.

Factors influencing age at diagnosis of primary ciliary dyskinesia in European children.

Primary ciliary dyskinesia (PCD) is a hereditary disorder of mucociliary clearance causing chronic upper and lower airways disease. We determined the number of patients with diagnosed PCD across Europe, described age at diagnosis and determined risk factors for late diagnosis. Centres treating children with PCD in Europe answered questionnaires and provided anonymous patient lists. In total, 223 centres from 26 countries reported 1,009 patients aged <20 yrs. Reported cases per million children (for 5–14 yr olds) were highest in Cyprus (111), Switzerland (47) and Denmark (46). Overall, 57% were males and 48% had situs inversus. Median age at diagnosis was 5.3 yrs, lower in children with situs inversus (3.5 versus 5.8 yrs; p<0.001) and in children treated in large centres (4.1 versus 4.8 yrs; p = 0.002). Adjusted age at diagnosis was 5.0 yrs in Western Europe, 4.8 yrs in the British Isles, 5.5 yrs in Northern Europe, 6.8 yrs in Eastern Europe and 6.5 yrs in Southern Europe (p<0.001). This strongly correlated with general government expenditures on health (p<0.001). This European survey suggests that PCD in children is under-diagnosed and diagnosed late, particularly in countries with low health expenditures. Prospective studies should assess the impact this delay might have on patient prognosis and on health economic costs across Europe.

Lung function in patients with primary ciliary dyskinesia: a cross-sectional and 3-decade longitudinal study.

RATIONALE Early diagnosis and treatment is considered important to prevent lung damage in primary ciliary dyskinesia (PCD). OBJECTIVES Few studies have addressed long-term evolution of lung function after PCD diagnosis. We investigated whether long-term lung function was dependent on age or level of lung function at PCD diagnosis. METHODS An observational, single-center, cross-sectional, and three-decade longitudinal study of FEV(1) and FVC related to age at diagnosis until current age was performed. Linear regression was used to describe the relation between first measured lung function values and age at diagnosis across the cohort. Courses of lung function after diagnosis and the according slopes were used to group patients into increasing, stable, or decreasing courses. Additionally, slopes from courses of 10 years of follow-up were related to age at diagnosis and initial level of lung function, respectively, using linear regression. MEASUREMENTS AND MAIN RESULTS Seventy-four children and adults with PCD were observed for median 9.5 (range, 1.5-30.2) years during which 2,937 lung function measurements were performed. First measured FEV(1) was less than 80% of predicted in one-third of preschool-diagnosed children. During observation, 34% of patients lost more than 10 percentage points, 57% were stable, and 10% improved more than 10 percentage points in FEV(1). Courses of lung function after diagnosis were related to neither age at diagnosis nor initial level. CONCLUSIONS Our study strongly suggests that PCD is a disease of serious threat to lung function already at preschool age, and with a high degree of variation in courses of lung function after diagnosis that was not linked to either age or level of lung function at diagnosis. Early diagnosis did not protect against decline in lung function.

Flow-dependent effect of formoterol dry-powder inhaled from the Aerolizer

The output and size distribution of aerosols from dry powder inhalers are dependent on the flow rate through the device. Therefore, in an in vivo study, we examined the flow-dependency of the effect of formoterol when delivered from a dry powder inhaler, the Aerolizer, in a flow range relevant to schoolchildren. In a preliminary study comprising 126 asthmatic children aged 3-10 yrs, the relationship between age and peak inspiratory flow (PIF) rate through the Aerolizer was determined. Mean PIF was 104 L.min-1 and all children aged > 5 yrs performed a PIF > 60 L.min-1. Sixteen children aged 8-15 yrs with exercise-induced asthma (EIA) took part in the main trial comparing the protective effect of 12 micrograms formoterol inhaled at 60 and 120 L.min-1. The effect from high and low inspiratory flow was judged from the protective effect against EIA 12 h after drug administration. The decrease in forced expiratory volume in one second (FEV1) after exercise was 34% on the placebo day, but only 15% when formoterol was inhaled at the high flow rate. This difference was statistically significant. The decrease in FEV1 was 23% after treatment with formoterol inhaled at the low flow rate, that was not significantly different from placebo or from high-flow formoterol treatment. These clinical findings correspond with the in vitro findings of flow-dependent fine particle mass from the Aerolizer, and corroborate the relationship between fine particle mass of aerosol and clinical effect. The results indicate a flow-dependent effect of formoterol dry powder inhaled from the Aerolizer, within the range of inspiratory flow rate obtainable by school-children. This questions its applicability in children with asthma.

European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia

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 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 primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia. International ERS guidelines recommend a combination of tests to diagnose primary ciliary dyskinesia http://ow.ly/sJhH304InBN

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Mutations in ZMYND10, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms in Humans and Flies, Cause Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.