Mislocalization of DNAH5 and DNAH9 in Respiratory Cells from Patients with Primary Ciliary Dyskinesia

Abstract

Rationale: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by recurrent infections of the airways and situs inversus in half of the affected offspring. The most frequent genetic defects comprise recessive mutations of DNAH5 and DNAI1, which encode outer dynein arm (ODA) components. Diagnosis of PCD usually relies on electron microscopy, which is technically demanding and sometimes difficult to interpret. Methods: Using specific antibodies, we determined the subcellular localization of the ODA heavy chains DNAH5 and DNAH9 in human respiratory epithelial and sperm cells of patients with PCD and control subjects by high-resolution immunofluorescence imaging. We also assessed cilia and sperm tail function by high-speed video microscopy. Results: In normal ciliated airway epithelium, DNAH5 and DNAH9 show a specific regional distribution along the ciliary axoneme, indicating the existence of at least two distinct ODA types. DNAH5 was completely or only distally absent from the respiratory ciliary axoneme in patients with PCD with DNAH5Ϫ (n ϭ 3) or DNAI1Ϫ (n ϭ 1) mutations, respectively, and instead accumulated at the microtubuleorganizing centers. In contrast to respiratory cilia, sperm tails from a patient with DNAH5 mutations had normal ODA heavy chain distribution, suggesting different modes of ODA generation in these cell types. Blinded investigation of a large cohort of patients with PCD and control subjects identified DNAH5 mislocalization in all patients diagnosed with ODA defects by electron microscopy (n ϭ 16). Cilia with complete axonemal DNAH5 deficiency were immotile, whereas cilia with distal DNAH5 deficiency showed residual motility. Conclusions: Immunofluorescence staining can detect ODA defects, which will possibly aid PCD diagnosis. Keywords: fluorescent antibody technique; genetics; respiratory tract diseasesPrimary ciliary dyskinesia (PCD; OMIM 242650) represents a rare autosomal recessive disorder affecting 1 in 20,000 individuals (1). Clinical manifestations include recurrent infections of the upper and lower respiratory tract, caused by reduced mucociliary clearance; sperm immobility; and situs inversus in half of the affected offspring, caused by randomization of left-right asymmetry (2). Chronic airway disease often leads to bronchiectasis and can progress to respiratory failure. The PCD phenotype results from axonemal abnormalities and dysfunction of motile cilia and flagella. Cilia and flagella are hairlike organelles extending from the cell surface. In vertebrates, multiple motile cilia or motile monocilia are located on various epithelial cells and move extracellular fluid (3). These include the respiratory epithelium of the airways (respiratory cilia), the embryonic node (primary cilia), and the ependyma of the brain ventricles (ependymal cilia). The sperm tail (flagellum) propels the sperm cell through liquid. The involvement of motile cilia in diverse processes such as left-right axis pattern formation, cerebrospinal fluid flow, and mucociliary clea...

Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children

“…It is the human orthologue of the Chlamydomonas c-heavy chain gene, the mutants of which are slow-swimming algae with ultrastructural ODA defects [18,33]. Mutations in human DNAH5 are regularly associated with ODA defects, random left-right asymmetry and male infertility [18,34,35]. The mutational prevalence of DNAH5 is currently known in a total of 134 PCD families [18,35].…”

“…IDA defects, in particular, are difficult to determine since they are less electron dense and less frequent along the ciliary axoneme. In addition, it has been shown that dynein motor protein composition varies along the ciliary length, meaning that ultrastructural defects can be missed using EM, depending upon the site of the ciliary cross-section [34]. Obtention of samples that do not have significant secondary damage can be difficult.…”

“…This method has been developed and is routinely used at the University Hospital Freiburg (Freiburg, Germany) [34,35,88]. It has been shown that IFM can be used to identify ODA defects, including regional defects within the ciliary shaft [34,35].…”

Standardizing Nasal Nitric Oxide Measurement as a Test for Primary Ciliary Dyskinesia

“…For nNO validation studies at six (non-UNC) sites, PCD was confirmed by PCD-specific ciliary EM defects and, by the presence of biallelic mutations in PCD genes (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Informed consent was obtained at the University of North Carolina at Chapel Hill and collaborating institutions under the auspices of Committees on the Protection of the Rights of Human Subjects.…”

Sequence analysis of 21 genes located in the Kartagener syndrome linkage region on chromosome 15q

“…2,3 Recently, it has been shown that immunofluorescence staining of ciliated epithelium with antibodies targeting DNAH5 can detect outer dynein arms defects and therefore aid diagnosis of PCD. 4 Inheritance of PCD is autosomal recessive in most cases, although pedigrees with an X-linked mode of inheritance have also been described. 5,6 To date, mutations in four genes have been found as causative for PCD, exclusively in patients with outer dynein arm defects.…”