Dataset Information

Ciliary Feature Counter: A program for the Quantitative Assessment of Cilia to Diagnose Primary Ciliary Dyskinesia.

ABSTRACT: Primary ciliary dyskinesia (PCD) is a disorder that affects motile cilia in the airway that are required for the removal of mucus, debris, and pathogens. It is important to diagnose PCD in early childhood to preserve lung function. The confirmation of a diagnosis relies on the assessment of ciliary ultrastructure by transmission electron microscopy (TEM). TEM involves the quantitative assessment of the ciliary ultrastructure to identify PCD defects as well as abnormalities resulting from infection. Many specialist diagnostic centres still rely on physical counters to tally results and paper notes to summarise findings before transferring the results to computer databases/records. To speed up the diagnostic data collection and increase the protection of patient information, we have developed digital ciliary feature counters that conform to the PCD reporting international consensus guideline. These counters can be used on a computer or tablet, and automatically generate notes regarding sample observations. We show that the digital counters are easy to use and can generate TEM diagnostic reports that will be useful for many PCD diagnostic centres.

SUBMITTER: Pinto AL

PROVIDER: S-EPMC7459509 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Ciliary Feature Counter: A program for the Quantitative Assessment of Cilia to Diagnose Primary Ciliary Dyskinesia.

Pinto Andreia L AL Rai Ranjit K RK Hogg Claire C Burgoyne Thomas T

Diagnostics (Basel, Switzerland) 20200728 8

Primary ciliary dyskinesia (PCD) is a disorder that affects motile cilia in the airway that are required for the removal of mucus, debris, and pathogens. It is important to diagnose PCD in early childhood to preserve lung function. The confirmation of a diagnosis relies on the assessment of ciliary ultrastructure by transmission electron microscopy (TEM). TEM involves the quantitative assessment of the ciliary ultrastructure to identify PCD defects as well as abnormalities resulting from infecti ...[more]

PMID: 32731525

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Project description:BackgroundPrimary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.MethodsGenetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.ResultsA single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.ConclusionMutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.

Project description:BackgroundPrimary ciliary dyskinesia (PCD) is a rare congenital respiratory disorder characterized by abnormal ciliary motility leading to chronic airway infections. Qualitative evaluation of ciliary beat pattern based on digital high-speed videomicroscopy analysis has been proposed in the diagnosis process of PCD. Although this evaluation is easy in typical cases, it becomes difficult when ciliary beating is partially maintained. We postulated that a quantitative analysis of beat pattern would improve PCD diagnosis. We compared quantitative parameters with the qualitative evaluation of ciliary beat pattern in patients in whom the diagnosis of PCD was confirmed or excluded.MethodsNasal nitric oxide measurement, nasal brushings and biopsies were performed prospectively in 34 patients with suspected PCD. In combination with qualitative analysis, 12 quantitative parameters of ciliary beat pattern were determined on high-speed videomicroscopy recordings of beating ciliated edges. The combination of ciliary ultrastructural abnormalities on transmission electron microscopy analysis with low nasal nitric oxide levels was the "gold standard" used to establish the diagnosis of PCD.ResultsThis "gold standard" excluded PCD in 15 patients (non-PCD patients), confirmed PCD in 10 patients (PCD patients) and was inconclusive in 9 patients. Among the 12 parameters, the distance traveled by the cilium tip weighted by the percentage of beating ciliated edges presented 96% sensitivity and 95% specificity. Qualitative evaluation and quantitative analysis were concordant in non-PCD patients. In 9/10 PCD patients, quantitative analysis was concordant with the "gold standard", while the qualitative evaluation was discordant with the "gold standard" in 3/10 cases. Among the patients with an inconclusive "gold standard", the use of quantitative parameters supported PCD diagnosis in 4/9 patients (confirmed by the identification of disease-causing mutations in one patient) and PCD exclusion in 2/9 patients.ConclusionsWhen the beat pattern is normal or virtually immotile, the qualitative evaluation is adequate to study ciliary beating in patients suspected for PCD. However, when cilia are still beating but with moderate alterations (more than 40% of patients suspected for PCD), quantitative analysis is required to precise the diagnosis and can be proposed to select patients eligible for TEM.

Dataset Information

Ciliary Feature Counter: A program for the Quantitative Assessment of Cilia to Diagnose Primary Ciliary Dyskinesia.

Publications

Ciliary Feature Counter: A program for the Quantitative Assessment of Cilia to Diagnose Primary Ciliary Dyskinesia.

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