Mellisa Dixon

Twenty-year review of quantitative transmission electron microscopy for the diagnosis of primary ciliary dyskinesia

Background The examination of ciliary ultrastructure in a nasal sample remains a definitive diagnostic test for primary ciliary dyskinesia (PCD). Methods The quantitative assessment of ciliary ultrastructure in the diagnosis of PCD over a 20-year period was reviewed. Results During this period, 1182 patients were referred for ciliary ultrastructural analysis, 242 (20%) of whom were confirmed as having the disease. The two main causes of PCD identified were a lack of outer dynein arms (43%) and a lack of both inner and outer dynein arms (24%). Other causes included transposition, radial spoke and inner dynein arm defects. No specific ultrastructural defects were detected in 33 patients (3%) diagnosed as having PCD on the basis of their clinical features and screening tests that included a low nasal nitric oxide concentration or slow saccharine clearance and abnormal ciliary beat frequency or pattern. Conclusions Electron microscopy analysis can confirm but does not always exclude a diagnosis of PCD.

Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the ‘empty’ CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the ‘head’ structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia

Rationale: The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the United Kingdom consists of assessing ciliary function by high‐speed microscopy and ultrastructure by election microscopy, but equipment and expertise is not widely available internationally. The identification of biallelic disease‐causing mutations is also diagnostic, but many disease‐causing genes are unknown, and testing is not widely available outside the United States. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. Objectives: To determine utility of a panel of six fluorescent labeled antibodies as a diagnostic tool for PCD. Methods: The study used immunofluorescent labeling of nasal brushings from a discovery cohort of 35 patients diagnosed with PCD by ciliary ultrastructure, and a diagnostic accuracy cohort of 386 patients referred with symptoms suggestive of disease. The results were compared with diagnostic outcome. Measurements and Main Results: Immunofluorescence correctly identified mislocalized or absent staining in 100% of the discovery cohort. In the diagnostic cohort immunofluorescence successfully identified 22 of 25 patients with PCD and normal staining in all 252 in whom PCD was considered highly unlikely. In addition, immunofluorescence provided a result in 55% (39) of cases that were previously inconclusive. Immunofluorescence results were available within 14 days, costing

Characterizing the ultrastructure of primary ciliary dyskinesia transposition defect using electron tomography.

187 per sample compared with electron microscopy (27 days; cost

Generation of a Three-Dimensional Ultrastructural Model of Human Respiratory Cilia

1,452). Conclusions: Immunofluorescence is a highly specific diagnostic test for PCD, and it improves the speed and availability of diagnostic testing. However, sensitivity is limited and immunofluorescence is not suitable as a stand‐alone test.

Bardet Biedl Syndrome: Motile Ciliary Phenotype

Primary ciliary dyskinesia is an autosomal recessive disorder affecting the motility of cilia. There are a range of ultrastructural ciliary defects that lead to associated clinical symptoms including ineffective mucus clearance, reduced lung function, infertility, and left‐right isomerism. Mutations in radial spoke head proteins are a known cause of primary ciliary dyskinesia. Ultrastructually these defects are identified by a portion of cilia lacking a central pair and transposed outer microtubular doublets. We have repeatedly observed an intermittent loss of the central pair in patients with a transposition defect. To further understand the central pair changes in these radial spoke head mutations we employ electron tomography, a high resolution electron microscope technique, to elucidate in three dimensions the ultrastructural arrangements caused by mutation of the RSPH4A gene. We thereby provide an explanation of the structures observed by conventional electron microscopy studies. We demonstrate that the central pair can be present within the cilium. In some cilia, the central pair rotates at the base of the axoneme. We propose that it is this rotation that gives rise to an intermittent appearance of the central pair when viewed under conventional electron microscopy. We discuss the potential causes and consequences of these findings.

Primary Ciliary Dyskinesia Due to Microtubular Defects is Associated with Worse Lung Clearance Index

The ultrastructures of cilia and flagella are highly similar and well conserved through evolution. Consequently, Chlamydomonas is commonly used as a model organism for the study of human respiratory cilia. Since detailed models of Chlamydomonas axonemes were generated using cryoelectron tomography, disparities among some of the ultrastructural features have become apparent when compared with human cilia. Extrapolating information on human disease from the Chlamydomonas model may lead to discrepancies in translational research. This study aimed to establish the first three-dimensional ultrastructural model of human cilia. Tomograms of transverse sections (n = 6) and longitudinal sections (n = 9) of human nasal respiratory cilia were generated from three healthy volunteers. Key features of the cilium were resolved using subatomic averaging, and were measured. For validation of the method, a model of the well characterized structure of Chlamydomonas reinhardtii was simultaneously generated. Data were combined to create a fully quantified three-dimensional reconstruction of human nasal respiratory cilia. We highlight key differences in the axonemal sheath, microtubular doublets, radial spokes, and dynein arms between the two structures. We show a decreased axial periodicity of the radial spokes, inner dynein arms, and central pair protrusions in the human model. We propose that this first human model will provide a basis for research into the function and structure of human respiratory cilia in health and in disease.

Secondary defects detected by transmission electron microscopy in primary ciliary dyskinesia diagnostics

BACKGROUND Cilia line the surface of the respiratory tract and beat in a coordinated wave to protect the lungs against infection. Bardet Biedl Syndrome (BBS) is a rare condition attributed to cilia dysfunction. Murine models of BBS suggest a respiratory phenotype; however, no reports have studied the translation of these findings in patients. METHODS We assessed the clinical symptoms of motile cilia dysfunction and the histology of ciliated respiratory epithelium in patients with BBS. RESULTS We report an increased prevalence of neonatal respiratory distress at birth (12%), general practitioner-diagnosed asthma (21%), otitis media (33%), and rhinitis (36%) in patients with BBS. These symptoms, however, occurred at a significantly reduced prevalence compared with patients with known motile cilia dysfunction (primary ciliary dyskinesia). Respiratory epithelial assessment revealed cellular damage, significant ciliary depletion (on 60% of ciliated cells), and goblet cell hyperplasia in patients with BBS (50% goblet cells). These findings were quantifiably similar to those of patients with asthma (P > .05). Surprisingly, motile cilia function and ultrastructure were grossly normal with the exception of occasional unique inclusions within the ciliary membrane. CONCLUSIONS In conclusion, motile ciliary structure and function are essentially normal in patients with BBS.

Risk factors for situs defects and congenital heart disease in primary ciliary dyskinesia

PurposePrimary ciliary dyskinesia (PCD) is characterised by repeated upper and lower respiratory tract infections, neutrophilic airway inflammation and obstructive airway disease. Different ultrastructural ciliary defects may affect lung function decline to different degrees. Lung clearance index (LCI) is a marker of ventilation inhomogeneity that is raised in some but not all patients with PCD. We hypothesised that PCD patients with microtubular defects would have worse (higher) LCI than other PCD patients.MethodsSpirometry and LCI were measured in 69 stable patients with PCD. Age at testing, age at diagnosis, ethnicity, ciliary ultrastructure, genetic screening result and any growth of Pseudomonas aeruginosa was recorded.ResultsLung clearance index was more abnormal in PCD patients with microtubular defects (median 10.24) than those with dynein arm defects (median 8.3, p = 0.004) or normal ultrastructure (median 7.63, p = 0.0004). Age is correlated with LCI, with older patients having worse LCI values (p = 0.03, r = 0.3).ConclusionThis study shows that cilia microtubular defects are associated with worse LCI in PCD than dynein arm defects or normal ultrastructure. The patient’s age at testing is also associated with a higher LCI. Patients at greater risk of obstructive lung disease should be considered for more aggressive management. Differences between patient groups may potentially open avenues for novel treatments.