Gerard W. Dougherty

Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia

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.

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.

ARMC4 mutations cause primary ciliary dyskinesia with randomization of left/right body asymmetry.

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.

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

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

CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151ts272a and mouse Ccdc151Snbl mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.

Mutations in PIH1D3 Cause X-Linked Primary Ciliary Dyskinesia with Outer and Inner Dynein Arm Defects

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance. Here we report maternally inherited and de novo mutations in PIH1D3 in four men affected with PCD. PIH1D3 is located on the X chromosome and is involved in the preassembly of both outer (ODA) and inner (IDA) dynein arms of cilia and sperm flagella. Loss-of-function mutations in PIH1D3 lead to absent ODAs and reduced to absent IDAs, causing ciliary and flagellar immotility. Further, PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4. This result has clinical and genetic counseling implications for genetically unsolved male case subjects with a classic PCD phenotype that lack additional phenotypes such as intellectual disability or retinitis pigmentosa.

DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

TTC25 deficiency results in defects of the outer dynein arm docking machinery and primary ciliary dyskinesia with left-right body asymmetry randomization

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Ciliary function and motor protein composition of human fallopian tubes

STUDY QUESTION What is the motor protein composition and function of human fallopian tube (FT) cilia? SUMMARY ANSWER Although the motor protein composition and function of human FT cilia resemble that of respiratory cilia, females with primary ciliary dyskinesia (PCD) are not necessarily infertile. WHAT IS KNOWN ALREADY FTs are lined with multiple motile cilia, which show a 9 + 2 ultrastructure by transmission electron microscopy. Case reports suggest an increased incidence of subfertility and ectopic pregnancy in women with PCD, a disease characterized by dysfunction of motile cilia and flagella. STUDY DESIGN, SIZE, DURATION This study consisted of an observational laboratory study on human FT specimens from five healthy females recruited from April 2012 to December 2013 and a descriptive observational retrospective analysis of a clinical PCD database. PARTICIPANTS/MATERIALS, SETTING, METHODS Human FT tissue was obtained from five healthy females after tubal ligation during caesarean delivery. Motor protein composition was assessed by immunofluorescence microscopy using antibodies against dynein arms and nexin-dynein regulatory complex subunits. Ciliary motility was analysed by high-speed video microscopy. A retrospective search of our database of PCD individuals was performed for information on conception and childbirth. MAIN RESULTS AND THE ROLE OF CHANCE The motor protein composition of human FT cilia was identical to that of respiratory cilia. FT cilia showed coordinated beating, resulting in a directed fluid flow towards the uterine cavity. We identified nine PCD individuals with severe dysfunction of respiratory cilia who gave birth to children after spontaneous conception. This suggests that ciliary beating is not the key motor of ovum transport. LIMITATIONS, REASON FOR CAUTION FT cilia of affected PCD females were not available for analysis. Thus, it remains to be proven that FT cilia indeed show the same defects as respiratory cilia in PCD individuals. Comprehensive epidemiological studies are needed to determine the extent of female (sub-) fertility in PCD. WIDER IMPLICATIONS OF THE FINDINGS Knowledge of the exact protein composition and function of FT cilia will contribute to a better understanding of cilia-generated fluid flow in female reproduction. These findings are important for subsequent studies of function and protein composition of FT cilia in PCD patients.