Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, "9+2"-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.

Reduced generation of multiple motile cilia (RGMC) is a rare mucociliary clearance disorder. Affected persons suffer from recurrent infections of upper and lower airways because of highly reduced numbers of multiple motile respiratory cilia. Here we report recessive loss-of-function and missense mutations in MCIDAS-encoding Multicilin, which was shown to promote the early steps of multiciliated cell differentiation in Xenopus. MCIDAS mutant respiratory epithelial cells carry only one or two cilia per cell, which lack ciliary motility-related proteins (DNAH5; CCDC39) as seen in primary ciliary dyskinesia. Consistent with this finding, FOXJ1-regulating axonemal motor protein expression is absent in respiratory cells of MCIDAS mutant individuals. CCNO, when mutated known to cause RGMC, is also absent in MCIDAS mutant respiratory cells, consistent with its downstream activity. Thus, our findings identify Multicilin as a key regulator of CCNO/FOXJ1 for human multiciliated cell differentiation, and highlight the 5q11 region containing CCNO and MCIDAS as a locus underlying RGMC.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic destructive airway disease and randomization of left/right body asymmetry. Males often have reduced fertility due to impaired sperm tail function. The complex PCD phenotype results from dysfunction of cilia of the airways and the embryonic node and the structurally related motile sperm flagella. This is associated with underlying ultrastructural defects that frequently involve the outer dynein arm (ODA) complexes that generate cilia and flagella movement. Applying a positional and functional candidate-gene approach, we identified homozygous loss-of-function DNAI2 mutations (IVS11+1G> A) in four individuals from a family with PCD and ODA defects. Further mutational screening of 105 unrelated PCD families detected two distinct homozygous mutations, including a nonsense (c.787C > T) and a splicing mutation (IVS3-3T > G) resulting in out-of-frame transcripts. Analysis of protein expression of the ODA intermediate chain DNAI2 showed sublocalization throughout respiratory cilia. Electron microscopy showed that mutant respiratory cells from these patients lacked DNAI2 protein expression and exhibited ODA defects. High-resolution immunofluorescence imaging demonstrated absence of the ODA heavy chains DNAH5 and DNAH9 from all DNAI2 mutant ciliary axonemes. In addition, we demonstrated complete or distal absence of DNAI2 from ciliary axonemes in respiratory cells of patients with mutations in genes encoding the ODA chains DNAH5 and DNAI1, respectively. Thus, DNAI2 and DNAH5 mutations affect assembly of proximal and distal ODA complexes, whereas DNAI1 mutations mainly disrupt assembly of proximal ODA complexes.

Different ultrastructural defects responsible for PCD result in predictable beat patterns. Recognition of these might help in the diagnostic evaluation of patients suspected of having PCD.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed ‘radial spoke defect’. We sequenced CCDC39 and CCDC40 in 54 ‘radial spoke defect’ families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice and frameshift predicting early protein truncation, which suggests this defect is caused by ‘null’ alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganisation and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as ‘IDA and nexin-dynein regulatory complex (N-DRC) defect’, rather than ‘radial spoke defect’.

The effects of coronavirus on human nasal ciliated respiratory epithelium. M.A. Chilvers, M. McKean, A. Rutman, B.S. Myint, M. Silverman, C. O9Callaghan. #ERS Journals Ltd 2001. ABSTRACT: Human coronavirus (HCoV) accounts for 15-30% of common colds, but only one case report has described the effect of a coronavirus infection, that was asymptomatic, on human respiratory epithelium.The authors examined the effects of infection with HCoV on ciliary structure and function in healthy volunteers infected by intranasal inoculation with HCoV 229E. A further four volunteers were sham infected with ultraviolet-inactivated virus. Immediately before inoculation (day 0) and 3 days later (day 3), ciliated epithelium was obtained by brushing the inferior nasal turbinate. Ciliary beat frequency was determined and beat pattern analysed for evidence of dyskinesia (0=normal, 3=severely dyskinetic) using digital high-speed video photography. Ciliary ultrastructure was examined by transmission electron microscopy. Symptom diaries were kept for the duration of the study.All subjects inoculated with HCoV, including the three who did not develop symptoms of an upper respiratory tract infection, had disruption of their respiratory epithelium on day 3. Although there was no difference in the mean ciliary beat frequency between day 0 (11.3 Hz (95% confidence interval (CI): 8.6-14.0) and day 3 (9.4 Hz (95% CI 7.2-11.6)), there was a significant increase (pv0.05) in the ciliary dyskinesia score between day 0 (0.2 (95% CI 0-0.5)) and day 3 (1.1 (95% CI 0.5-1.7). In sham-infected subjects, no differences in epithelial integrity, or ciliary structure and function were found between day 0 and day 3.Inoculation of healthy volunteers with human coronavirus caused disruption of the ciliated epithelium and ciliary dyskinesia. This is likely to impair mucociliary clearance. Damage to the respiratory epithelium, due to human coronavirus infection, may occur without overt clinical symptoms. Eur Respir J 2001; 18: 965-970.

Background: There are very few data on normal ciliary beat frequency, beat pattern, and ultrastructure in healthy children and adults. A study was undertaken to define ciliary structure, beat frequency and beat pattern in a healthy paediatric and young adult population. Methods: Ciliated epithelial samples were obtained from 76 children and adult volunteers aged 6 months to 43 years by brushing the inferior nasal turbinate. Beating cilia were recorded using a digital high speed video camera which allowed analysis of ciliary beat pattern and beat frequency. Tissue was fixed for transmission electron microscopy. Results: The mean ciliary beat frequency for the paediatric population (12.8 Hz (95% CI 12.3 to 13.3)) was higher than for the adult group (11.5 Hz (95% CI 10.3 to 12.7 Hz), p<0.01, t test); 10% (range 6-24%) of ciliated edges were found to have areas of dyskinetically beating cilia. All samples had evidence of mild epithelial damage. This reflected changes found in all measurements used for assessment of epithelial damage. Ciliary ultrastructural defects were found in less than 5% of cilia. Conclusion: Normal age related reference ranges have been established for ciliary structure and beat frequency. In a healthy population localised epithelial damage may be present causing areas of ciliary dyskinesia.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.