Abstract
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.
Highlights
Primary ciliary dyskinesia (PCD) (OMIM# 244400) is a genetic disorder characterized by impaired ciliary function, leading to diverse clinical manifestations that include chronic sinopulmonary disease, persistent middle ear effusions, laterality defects, and infertility
These genes can be classified into three groups: genes encoding proteins that are components of the outer or inner dynein arms including DNAH5, DNAI1, DNAL1, DNAI2, TXNDC3 and DNAH11; genes encoding proteins implicated in axonemal organization of the central pair microtubules, radial spokes and nexin-dynein regulatory complex (N-DRC) including CCDC39, CCDC40, CCDC164, CCDC103, CCDC114, RSPH9, RSPH4A, and HYDIN; and genes encoding proteins that are cytoplasmic and do not localize to the ciliary axoneme, including HEATR2, DNAAF1, DNAAF2, DNAAF3 and LRRC6 [11,12,18,21,23,24,25,27]
Mutations in other genes such as CCDC39 (MIM 613798) and CCDC40 (MIM 613799) produce inconsistent ultrastructural abnormalities characterized by disordered microtubules in only some respiratory cells [14,19,20], which underscores the limitations of electron microscopy (EM) as a diagnostic test for PCD [15]
Summary
Genetic 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
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