TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella.

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

Frequency of Cystic Fibrosis Transmembrane Conductance Regulator Variants in Individuals Evaluated for Primary Ciliary Dyskinesia.

OBJECTIVE: To evaluate whether cystic fibrosis transmembrane conductance regulator (CFTR) variants are more common among individuals tested for primary ciliary dyskinesia (PCD) compared with controls. STUDY DESIGN: Data were studied from 1021 individuals with commercial genetic testing for suspected PCD and 91 777 controls with genetic testing at the same company (Invitae) for symptoms/diseases unrelated to PCD or CFTR testing. The prevalence of CFTR variants was compared between controls and each of 3 groups of individuals tested for PCD (PCD-positive, -uncertain, and -negative molecular diagnosis). RESULTS: The prevalence of 1 pathogenic CFTR variant was similar among the individual groups. When combining the PCD-uncertain and PCR-negative molecular diagnosis groups, there was a higher prevalence of single pathogenic CFTR variants compared with controls (P = .03). Importantly, >1% of individuals who had negative genetic testing results for PCD had 2 pathogenic CFTR variants (8 of 723), and the incidence of cystic fibrosis (CF) (2 pathogenic variants) is roughly 1 in 3000 individuals of Caucasian ethnicity (∼0.03%). This incidence was also greater than that of 2 pathogenic CFTR variants in the control population (0.09% [84 of 91 777]; P = 9.60 × 10-16). These variants correlate with mild CFTR-related disease. CONCLUSIONS: Our results suggest that a single pathogenic CFTR variant is not likely to be a PCD-mimetic, but ongoing studies are needed in individuals in whom PCD is suspected and genetic testing results are uncertain or negative. Furthermore, CF may be misdiagnosed as PCD, reflecting phenotypic overlap. Among individuals evaluated for PCD, CF should be considered in the differential even in the CF newborn screening era.

A null allele of Dnaaf2 displays embryonic lethality and mimics human ciliary dyskinesia.

The dynein axonemal assembly factor (Dnaaf) protein family is involved in preassembly and stability of dynein arms before they are transported into the cilia. In humans, mutations in DNAAF genes lead to several diseases related to cilia defects such as primary ciliary dyskinesia (PCD; OMIM: 612518). Patients with PCD experience malfunctions in cilia motility, which can result in inflammation and infection of the respiratory tract among other defects. Previous studies have identified that a mutation in DNAAF2 results in PCD and that 40% of these patients also experience laterality defects. In an outbred genetic background, Dnaaf2 homozygotes die after birth and have left/right defects among other phenotypes. Here we characterize a novel null allele of Dnaaf2 obtained from the International Mouse Phenotyping Consortium. Our data indicate that on a defined C57bl/6NJ genetic background, homozygous Dnaaf2 mouse embryos fail to progress beyond organogenesis stages with many abnormalities including left-right patterning defects. These findings support studies indicating that hypomorphic mutations of human DNAAF2 can result in ciliary dyskinesia and identify Dnaaf2 as an essential component of cilia function in vivo.

Nasal Nitric Oxide Measurement and a Modified PICADAR Score for the Screening of Primary Ciliary Dyskinesia in Adults with Bronchiectasis.

Background Determining the underlying diagnosis is essential for the targeted and specific treatment of bronchiectasis. Primary ciliary dyskinesia (PCD) is a rare genetic disease, which is characterized by abnormalities in ciliary structure and/or function and which may result in bronchiectasis. The disease is probably underestimated among adults with bronchiectasis due to the fact that extensive diagnostic testing is required and that the recognition of PCD is low. Objective To evaluate a feasible screening algorithm for PCD among adults with bronchiectasis. Methods Data from all patients who presented to our bronchiectasis outpatient clinic from June 2010 until July 2016 were retrospectively analysed from our database. Nasal NO (nNO) and a modified PICADAR score (PrImary CiliAry DyskinesiA Rule) were measured and compared in the two groups of PCD-bronchiectasis and non-PCD-bronchiectasis. Results 185 of 365 patients (75 males, 110 females) had a sufficient measurement of nNO concentration and complete clinical data and were eligible for analysis. The mean (SD) nNO concentration in nL/ml was significant lower in the PCD group compared to the non-PCD group (25 [31] and 227 [112] nL/min, respectively; p < 0.001). A nNO level of 77 nL/min had the best discriminative value to differentiate between the two groups. Patients with PCD had a significant higher modified PIDACAR score than patients without PCD (5 2 and 1 1, respectively [p < 0.001]). Using ROC curve analysis, the modified PICADAR score of 2 had the best discriminative value with a sensitivity of 1.00 and a specificity of 0.89. Conclusions Low nNO concentration and the modified PICADAR score are suitable and cheap screening tests for PCD in adults with bronchiectasis.

[The functional conditions of nasal cavity mucosa and paranasal sinuses following radical and minimally invasive surgical interventions].

The data of the literature are presented concerning the state of the mucous membrane of the nasal cavity, nasal turbinates, and maxillary sinus during the postoperative period following various surgical procedures.

MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia.

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.

Ciliary beating is depressed in nasal cilia from chronic obstructive pulmonary disease subjects.

UNLABELLED: COPD is characterized by increased cough, mucus production, and airway inflammation. Beating epithelial cell cilia contribute to mucociliary clearance with ciliary beat frequency (CBF) an important measure of cilia function. However, whether CBF varies with COPD severity is unknown. AIMS: 1) to compare nasal cilia samples and their CBF from healthy non-smokers (Control), COPD and At Risk (cough and sputum production) subjects. 2) to determine the effect of pharmacologic agents that modulate mediators implicated in the pathogenesis of COPD on nasal CBF. Nasal brushings of ciliated cells were obtained from Control, At Risk and COPD subjects. Using high speed digital imaging, we measured baseline CBF ex vivo. Then, CBF was re-measured after 30 min perfusion with pharmacologic agents that modulate mediators implicated in COPD (salmeterol xinafoate, tiotropium bromide, licofelone, luteolin, YM976, Defensin HNP-1) and again after 30 min washout. CBF was significantly depressed in moderate and severe COPD compared to At Risk and Control subjects. There was an evident and persistent rise in CBF with all agents tested in COPD cilia except that YM976 effects persisted only in severe COPD. Only YM976 and tiotropium caused a persistent increase in CBF in At Risk cilia. The reduction of nasal CBF in moderate and severe COPD implies that impaired ciliary function may impact mucociliary clearance in COPD, potentially contributing to retention of secretions and infection. Pharmacologic agents with different mechanisms of action can increase CBF of COPD cilia. Further investigation of the signalling pathways influencing CBF of COPD cilia is needed.

Pulmonary infection defense after lung transplantation: does airway ischemia play a role?

PURPOSE OF REVIEW: During a classical lung transplantation procedure, there is no attempt to restore the bronchial circulation, giving rise to ischemia of the mucosa with initial loss of ciliated function. This might compromise the ciliary function and the infection defense mechanisms of the airways. RECENT FINDINGS: Although initially the airways are characterized by ischemia (epithelial sloughing), later on, there seems to be a restoration of the ciliary function and up to now, there is no clear evidence that there is an increased risk of infectious episodes due to the lack of bronchial circulation, although this has not extensively been investigated. SUMMARY: Local pulmonary defense mechanisms after lung transplantation without bronchial revascularization seem to be preserved at least in a stable condition. Whether the loss of bronchial circulation has a negative impact when complications develop (such as bronchiolitis obliterans syndrome) remains speculative.

The ciliary beat frequency of middle ear mucosa in children with chronic secretory otitis media.

The chronic secretory otitis media (CSOM) is a common disease in children. Its cardinal symptoms are recurrent middle ear effusions and conductive hearing loss. Until today, the pathophysiological mechanism of this disease remains unknown. The correlation with adenoids and tubal dysfunction during childhood seems to be obvious, but the origin of middle ear effusions still has to be clarified. It is known that the CSOM affects the mucociliary system in several ways. In order to find out more about these correlations, the ciliary beat frequency was examined in 123 samples of infantile middle ear mucosa suffering from CSOM. Samples were surveyed using a stroboscopic microscopy method. The results of this study showed a significant decrease of ciliary beat frequency (CBF) to an average of 7.4 s(-1) in children with a CSOM. The healthy group of control showed a frequency of 10.1 s(-1). The measured CBF dataset was correlated with microbiological findings. We found a typical bacterial profile in nearly all the cases that were examined but were unable to find a specific bacterium decreasing CBF. This study provides evidence for the diminution of CBF in cases of CSOM in comparison to a healthy control group. Our findings emphasize the importance of stopping the vicious circle of recurrent effusions by paracentesis or grommet insertion.

Bronchiectasis in children with recurrent pneumonia: an immunopathological damage associated with secondary ciliary dysmotility.

The aim of this study is to assess ciliary motion patterns in children with bronchiectasis unrelated to cystic fibrosis or primary ciliary dyskinesia. In 51 children with recurrent pneumonia, high resolution computed tomography (HRCT) was carried out to detect and score bronchiectasis. Moreover, ciliary ultrastructure, beat frequency and motion pattern were evaluated and compared to those observed in 30 healthy children. Bronchiectasis at HRCT was found in 31/51 children. Ciliary dysmotility was found in 20/31 children with bronchiectasis (64.5%). Overall, ciliary dysmotility was found in 39/51 patients (76.5%). Ciliary dysmotility showed a significant correlation with the HRCT score (p=0.02). Absent motion in some fields was found in 44/51 patients (86.3%) and this also showed significant correlation with the HRCT score (p=0.005). The specificity and sensitivity of ciliary dysmotility as an indicator of bronchiectasis was 74.3% and 83.3% respectively. The positive predictive value was 93.5%, and negative predictive value was 50%. Ciliary dysmotility, in children with recurrent airways infections, correlates with the presence and severity of bronchiectasis. Whether ciliary dysmotility is a cause or a consequence of anatomical lesion is a matter of speculation. Very likely there is an amplification and self-maintaining mechanism between the two events which may lead to more serious disease.

Cilia: tuning in to the cell's antenna.

Cilia are microtubule-based organelles that project like antennae from the surface of most cells in the body. Motile cilia move fluid past cells, for example mucus in the airway. Non-motile primary cilia, however, transduce a multitude of sensory stimuli, including chemical concentrations of growth factors, hormones, odorants, and developmental morphogens, as well as osmolarity, light intensity, and fluid flow. Cilia have evolved a complex ultrastructure to accommodate these diverse functions, and an extensive molecular machinery has developed to support the assembly of these organelles. Defects in the cilia themselves, or the machinery required to assemble them, lead to a broad spectrum of human disease symptoms, including polycystic kidney disease, nephronophthisis, hydrocephalus, polydactyly, situs inversus, retinal degeneration, and obesity. While these diseases highlight the pivotal roles of cilia in physiology and development, the mechanistic link between cilia, physiology, and disease remains unclear.

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function.

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

Nasal ciliary beat after insertion of septo-valvular splints.

OBJECTIVE: At present, endonasal paraseptal splints are devices frequently employed in rhinosurgery. We evaluated the local tolerance of a newly shaped device, the Guastella/Mantovani splint (G/M-SVS), with respect to the physiological mechanism of mucociliary clearance. STUDY DESIGN AND SETTING: The study involved 20 patients who underwent septoplasty and/or turbinoplasty or other nasal surgical procedures. A sample of ciliated cells was obtained by nasal brushing and was examined ex vivo to determine the ciliary beat frequency (CBF) and morphology, before and 15 days after surgery. RESULTS: Before surgery the mean CBF was 10.87 Hz +/- 0.56 Hz and when splints were removed it was 10.25 Hz +/- 1.9 Hz. Morphological evaluation of the ciliary motion after surgery demonstrated a normal, coordinated beat. CONCLUSIONS: The G/M-SVS does not appear to interfere with the physiological mechanism of mucociliary clearance since CBF remain within a normal range. SIGNIFICANCE: This is the first study that demonstrates an optimal tolerability and safety of the septo-valvular splints on nasal mucosa.