At least 6.3% of patients with PCD have heterotaxy, and most of those have cardiovascular abnormalities. The prevalence of congenital heart disease with heterotaxy is 200-fold higher in PCD than in the general population (1:50 versus 1:10 000); thus, patients with PCD should have cardiac evaluation. Conversely, mutations in genes that adversely affect both respiratory and embryological nodal cilia are a significant cause of heterotaxy and congenital heart disease, and screening for PCD is indicated in those patients.

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...

Rationale: Primary ciliary dyskinesia (PCD) is characterized by recurrent airway infections and randomization of left-right body asymmetry. To date, autosomal recessive mutations have only been identified in a small number of patients involving DNAI1 and DNAH5, which encode outer dynein arm components. Methods: We screened 109 white PCD families originating from Europe and North America for presence of DNAH5 mutations by haplotype analyses and/or sequencing. Results: Haplotype analyses excluded linkage in 26 families. In 30 PCD families, we identified 33 novel (12 nonsense, 8 frameshift, 5 splicing, and 8 missense mutations) and two known DNAH5 mutations. We observed clustering of mutations within five exons harboring 27 mutant alleles (52%) of the 52 detected mutant alleles. Interestingly, 6 (32%) of 19 PCD families with DNAH5 mutations from North America carry the novel founder mutation 10815delT. Electron microscopic analyses in 22 patients with PCD with mutations invariably detected outer dynein arm ciliary defects. Highresolution immunofluorescence imaging of respiratory epithelial cells from eight patients with DNAH5 mutations showed mislocalization of mutant DNAH5 and accumulation at the microtubule organizing centers. Mutant DNAH5 was absent throughout the ciliary axoneme in seven patients and remained detectable in the proximal ciliary axoneme in one patient carrying compound heterozygous splicing mutations at the 3-end (IVS75-2AϾT, IVS76ϩ5GϾA). In a preselected subpopulation with documented outer dynein arm defects (n ϭ 47), DNAH5 mutations were identified in 53% of patients. Conclusions: DNAH5 is frequently mutated in patients with PCD exhibiting outer dynein arm defects and mutations cluster in five exons.

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