Introduction
Ciliary motility plays a number of essential roles in the body.1 Notably, coordinated cilia-based fluid movement across the multiciliated epithelial cell surface of respiratory airways forms the major host-defense mechanism of mucociliary clearance. Cerebrospinal fluid flow in the central nervous system is regulated by cilia lining the ventricles, and in the reproductive system, fallopian tube cilia assist propulsion of eggs toward the uterus. Sperm flagella, highly structurally related to cilia, drive male gamete motility. Motile cilia function in early embryogenesis to create a leftward nodal flow in or across vertebrate left-right organizers, which is necessary for induction of an asymmetric gene expression cascade in the lateral plate mesoderm that determines left-right organ asymmetry (situs).2
Motile cilia and sperm flagella extend from the cell body containing a 9+2 axonemal arrangement of two central microtubules and nine peripheral microtubule doublets, except in nodal monocilia, which lack the central microtubule pair (9+0). Microtubule-attached dynein arm motors, radial spokes, and nexin-dynein regulatory complexes arranged with regular periodicity along the peripheral microtubules provide a rigid structure and the biophysical force for ciliary beating. The beat is generated by coordinated sliding of adjacent microtubule doublets, powered via dynein-arm-driven ATP hydrolysis. This axonemal architecture is highly conserved in evolution and is found in the biflagellate alga Chlamydomonas reinhardtii as well as humans, where flagellar/ciliary dyneins make up two distinct structures, the outer dynein arms (ODAs) and the inner dynein arms (IDAs), each anchored to a specific site on the A-tubule of the doublet microtubules. The ODAs, with a regular spacing of 24 nm along the axonemal microtubules, contribute as much as four-fifths of the sliding force needed for flagellar/ciliary bending.3
Primary ciliary dyskinesia (PCD [MIM 244400])4,5 refers to an autosomal-recessive inherited disorder in which structure and assembly of motile cilia and sperm is deficient, often accompanied by visible ultrastructural defects, resulting in dysmotile or static axonemes. PCD is characterized by lifelong recurrent respiratory infections and irreversible, destructive airway disease (bronchiectasis) of early onset. Otitis media and nasal polyps are common and male infertility may occur, as well as laterality defects affecting approximately half of affected individuals, with around 12% manifesting as complex isomerisms and heterotaxies usually associated with congenital heart defects.6,7 Distinct from ultrastructural ciliary defects, CCNO (MIM 607702) mutations have recently been identified to cause a mucociliary clearance disorder related to, but distinct from, PCD that was previously called ciliary aplasia but is now termed RGMC (reduced generation of multiple motile cilia), because in RGMC a few motile cilia are still detectable at the cell surface.8
An estimated 70%–80% of PCD cases involve deficiency and loss of the ciliary outer dynein arms, with around a quarter of that total also involving inner dynein arm loss.9,10 Of 28 genes previously reported to have causative mutations for PCD,11,12 8 encode proteins of the ODAs or the ODA docking complex system (ODA-DC) (DNAH5 [MIM 603335], DNAH11 [MIM 603339], CCDC114 [MIM 615038], DNAL1 [MIM 610062], DNAI1 [MIM 604366], DNAI2 [MIM 605483], NME8 [MIM 607421], and ARMC4 [MIM 615408]),13–21 mutations of which generally cause isolated outer dynein arm deficiency. Ten genes encode cytoplasmic proteins involved in assembly and transport of the dynein arms into axonemes (SPAG1 [MIM 603395], DNAAF1 [MIM 613190], DNAAF2 [MIM 612517], HEATR2 [MIM 614864], DNAAF3 [MIM 614566], DYX1C1 [MIM 608706], ZMYND10 [MIM 607070], LRRC6 [MIM 614930], C21orf59 [MIM 615494], and CCDC103 [MIM 614677]),22–32 mutations of which cause combined outer and inner dynein arm deficiency. Eight other genes with causal mutations are components or associated factors of the nexin-dynein regulatory complexes (CCDC39 [MIM 613798], CCDC40 [MIM 613799], CCDC65 [MIM 611088], and DRC1 [previously known as CCDC164] [MIM 615288]),31,33–35 radial spokes (RSPH1 [MIM 609314], RSPH4A [MIM 612647], and RSPH9 [MIM 612648]),11,36 or central pair microtubules (HYDIN [MIM 610812]).37 Syndromic PCD with retinitis pigmentosa and developmental disorders can be caused by RPGR (MIM 312610) or OFD1 (MIM 300170) mutations38,39 and is characterized by X-linked transmission.
Although much progress in gene identification for PCD has been achieved, it has been recently estimated that the known genes in which mutations cause PCD account for about 65% of PCD cases.40 Therefore, we employed a next-generation sequencing (NGS) approach for linkage mapping and variant identification in order to identify additional PCD-causing mutations. This analysis revealed loss-of-function mutations in CCDC151 in three unrelated families characterized by PCD with specific loss of the ODAs. By analyzing CCDC151-deficient human cells, mice, and zebrafish, we show a requirement for CCDC151 in the correct establishment of left-right asymmetry because loss of CCDC151 function is associated with the randomization of visceral organ positioning. A severe reduction of CCDC151 occurs in the axonemes of nasal respiratory cilia of individuals carrying CCDC151 nonsense mutations, which disrupts assembly of both the ODAs and the ODA targeting and docking components CCDC114 and ARMC4 into axonemes. These results highlight the essential role of CCDC151 in the specification of ciliary motility during human and vertebrate development.