In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.

SUMMARY Dyx1c1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deletion of Dyx1c1 exons 2–4 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder characterized by chronic airway disease, laterality defects, and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1c.T2A start codon mutation recovered from an ENU mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also created laterality and ciliary motility defects. In humans, recessive loss-of-function DYX1C1 mutations were identified in twelve PCD individuals. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans revealed disruptions of outer and inner dynein arms (ODA/IDA). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA/IDA assembly factor DNAAF2/KTU. Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

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 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’.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder (prevalence 1:10 000 to 1:40 000 births) characterised by impaired mucociliary clearance because of abnormal motile ciliary function [1, 2]. Five main ultrastructural PCD phenotypes have been described. Most result from a lack of dynein arms (DAs): no outer and inner DAs (2DAs), outer DAs alone (ODA) or inner DAs with microtubular disorganisation (IDA/MTD); or defects yielding an abnormal central complex (CC). Some patients with genetically confirmed PCD have apparently normal ciliary structure on electron microscopy (nEM). More than 30 genes encoding proteins involved in the structure or assembly of the axoneme, the ciliary internal cytoskeleton, are implicated in PCD [3]; their analysis enables identification of bi-allelic disease-causing mutations in 50-75% of patients. Approximately half of PCD cases are associated with situs inversus, thereby defining Kartagener's syndrome. Moreover, because motile cilia and sperm flagella share common axonemal structures, most PCD-affected males are thought to be infertile [4]. According to the literature, male infertility is caused by severe or total asthenozoospermia and is currently treated by recourse to in vitro fertilisation or intracytoplasmic sperm injection [5, 6]. However, spontaneous fatherhood of PCD patients has been reported.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head.

BackgroundPrimary ciliary dyskinesia (PCD) is a rare disorder with variable disease progression. To date, mutations in more than 20 different genes have been found. At present, PCD subtypes are described according to the ultrastructural defect on transmission electron microscopy (TEM) of the motile cilia. PCD with normal ultrastructure (NU) is rarely reported because it requires additional testing. Biallelic mutations in DNAH11 have been described as one cause of PCD with NU.The aim of our study was to describe the clinical characteristics of a large population of patients with PCD, in relation to the ultrastructural defect. Additionally, we aimed to demonstrate the need for biopsy and cell culture to reliably diagnose PCD, especially the NU subtype.MethodsWe retrospectively analyzed data from 206 patients with PCD. We compared the clinical characteristics, lung function, microbiology and imaging results of 68 patients with PCD and NU to those of 90 patients with dynein deficiencies and 41 patients with central pair abnormalities. In addition, we aimed to demonstrate the robustness of the diagnosis of the NU subtype in cell culture by data from genetic analysis.ResultsPCD with NU comprised 33% (68/206) of all patients with PCD. Compared to other subtypes, patients with PCD and NU had a similar frequency of upper and lower respiratory tract problems, as well as similar lung function and imaging. With the currently widely applied approach, without cell culture, the diagnosis would have been missed in 16% (11/68) of patients with NU. Genetic analysis was performed in 29/68 patients with PCD and NU, and biallelic mutations were found in 79% (23/29) of tested patients.ConclusionsWe reported on the clinical characteristics of a large population of patients with PCD and NU. We have shown that systematic performance of biopsy and cell culture increases sensitivity to detect PCD, especially the subtype with NU.PCD with NU has similar clinical characteristics as other PCD types and requires biopsy plus ciliogenesis in culture for optimal diagnostic yield.