Search

Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous condition where dysfunction of motile cilia results in chronic respiratory disease. Ciliary ultrastructure examined by Transmission Electron Microscopy (TEM) or biallelic mutations in a known PCD gene are usually used to confirm the diagnosis. However, 15-30% cases of PCD have apparently normal ciliary ultrastructure or no known genetic cause, making diagnosis difficult. Electron tomography, an extension of TEM, produces high resolution 3D ultrastructural reconstructions. We hypothesise that electron tomography can be performed on existing diagnostic material to detect ultrastructural abnormalities in patients with PCD and ‘normal ultrastructure’ and/or novel gene variants. Longitudinal and transverse sections of proximal cilia were examined from araldite embedded nasal brush biopsies. 14 patients with PCD and 6 healthy controls were studied. Dual axis tomograms were collected on a Jeol 1400+ TEM. The data were analysed and averaged using IMOD software. Electron tomography indicated deficiency in the outer dynein arm volume (n=7), absence of central pair projections (n=3) and partial absence of nexin link structures (n=3) in patients with PCD and previously reported ‘normal ultrastructure’. A subset of the inner dynein arms were shown to be missing in a patient previously reported as an IDA defect with a variant in the novel gene PIH1D3. Electron tomography performed on diagnostic samples is effective in visualising defects which are difficult to identify using conventional TEM. Tomography could be used with genotyping to confirm a diagnosis and to investigate new PCD genes.

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software.DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal.

Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous condition. characterised by impaired mucociliary clearance and chronic sino-pulmonary disease. Over 30 causative gene mutations resulting in dysfunctional ciliary motility and structure have been identified. Ciliary ultrastructure examined by Transmission Electron Microscopy (TEM) is currently the diagnostic gold standard. Patients with mutations in the gene DNAH11 have a hyperfrequent ciliary beat and clinical symptoms of PCD. Diagnosis of a DNAH11 defect by TEM is difficult due to apparently normal ciliary ultrastructure. The electron tomography technique, an extension of TEM, produces 3D models of cilia with superior resolution. The aim of this study was to determine if electron tomography can detect ultrastructural abnormalities in patients with DNAH11 defects. Immunofluorescence by specific antibodies for DNAH11 showed localisation to the proximal portion of cilia. Proximal cilia cross sections from araldite embedded nasal brush biopsies were examined. Dual axis tomograms were collected on a Philips CM200 electron microscope. The data were analysed using IMOD software and averaged using PEET. Electron tomography and averaging of cilia cross sections indicated a deficiency in the outer dynein arm, consistent across all patients with a DNAH11 defect (n = 5) compared to healthy controls (n = 3) and patients with PCD due to a defect of the central pair or nexin link (n = 3) (Figure 1). A reduction in outer dynein arm volume of 30% was identified compared to healthy and PCD controls. In conclusion, a mutation in DNAH11 results in a subtle abnormality in ultrastructure. The defects are specific to the ‘forearm’ of the outer dynein arm and only detectable at the base of the cilium where DNAH11 is located. Electron tomography is highly effective in visualising this defect.