Homozygous missense mutation L673P in adenylate kinase 7 (AK7) leads to primary male infertility and multiple morphological anomalies of the flagella but not to primary ciliary dyskinesia.

Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are well-known to cause primary ciliary dyskinesia (PCD), a disorder characterized by recurrent respiratory tract infections, chronic otitis media, situs inversus, male infertility and in most severe cases, hydrocephalus. Only recently, mutations in genes encoding axonemal proteins with preferential expression in the testis were identified in isolated male infertility; in those cases, individuals displayed severe asthenozoospermia due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF) but not PCD features. In this study, we performed genetic investigation of two siblings presenting MMAF without any respiratory PCD features, and we report the identification of the c.2018T > G (p.Leu673Pro) transversion in AK7, encoding an adenylate kinase, expressed in ciliated tissues and testis. By performing transcript and protein analyses of biological samples from individual carrying the transversion, we demonstrate that this mutation leads to the loss of AK7 protein in sperm cells but not in respiratory ciliated cells, although both cell types carry the mutated transcript and no tissue-specific isoforms were detected. This work therefore, supports the notion that proteins shared by both cilia and sperm flagella may have specific properties and/or function in each organelle, in line with the differences in their mode of assembly and organization. Overall, this work identifies a novel genetic cause of asthenozoospermia due to MMAF and suggests that in humans, more deleterious mutations of AK7 might induce PCD.

Novel transglutaminase-like peptidase and C2 domains elucidate the structure, biogenesis and evolution of the ciliary compartment.

In addition to their role in motility, eukaryotic cilia serve as a distinct compartment for signal transduction and regulatory sequestration of biomolecules. Recent genetic and biochemical studies have revealed an extraordinary diversity of protein complexes involved in the biogenesis of cilia during each cell cycle. Mutations in components of these complexes are at the heart of human ciliopathies such as Nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and Joubert syndrome (JBTS). Despite intense studies, proteins in some of these complexes, such as the NPHP1-4-8 and the MKS, remain poorly understood. Using a combination of computational analyses we studied these complexes to identify novel domains in them which might throw new light on their functions and evolutionary origins. First, we identified both catalytically active and inactive versions of transglutaminase-like (TGL) peptidase domains in key ciliary/centrosomal proteins CC2D2A/MKS6, CC2D2B, CEP76 and CCDC135. These ciliary TGL domains appear to have originated from prokaryotic TGL domains that act as peptidases, either in a prokaryotic protein degradation system with the MoxR AAA+ ATPase, the precursor of eukaryotic dyneins and midasins, or in a peptide-ligase system with an ATP-grasp enzyme comparable to tubulin-modifying TTL proteins. We suggest that active ciliary TGL proteins are part of a cilia-specific peptidase system that might remove tubulin modifications or cleave cilia- localized proteins, while the inactive versions are likely to bind peptides and mediate key interactions during ciliogenesis. Second, we observe a vast radiation of C2 domains, which are key membrane-localization modules, in multiple ciliary proteins, including those from the NPHP1-4-8 and the MKS complexes, such as CC2D2A/MKS6, RPGRIP1, RPGRIP1L, NPHP1, NPHP4, C2CD3, AHI1/Jouberin and CEP76, most of which can be traced back to the last common eukaryotic ancestor. Identification of these TGL and C2 domains aid in the proper reconstruction of the Y-shaped linkers, which are key structures in the transitional zone of cilia, by allowing precise prediction of the multiple membrane-contacting and protein-protein interaction sites in these structures. These findings help decipher key events in the evolutionary separation of the ciliary and nuclear compartments in course of the emergence of the eukaryotic cell.

Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia.

No study has evaluated the correlation between different expression of nitric oxide synthase (NOS) isoforms in nasal epithelial cells and nasal NO (nNO) level in primary ciliary dyskinesia (PCD). Gene expression of endothelial (NOS3) and inducible NOS (NOS2) and their correlation with nNO level, ciliary function and morphology were studied in patients with PCD or secondary ciliary dyskinesia (SCD). NOS3 gene polymorphisms were studied in blood leukocytes. A total of 212 subjects were studied (48 with PCD, 161 with SCD and three normal subjects). nNO level correlated with mean ciliary beat frequency (p = 0.044; r = 0.174). The lower the nNO level the higher was the percentage of immotile cilia (p<0.001; r = -0.375). A significant positive correlation between NOS2 gene expression and nNO levels was demonstrated in all children (p = 0.001; r = 0.428), and this correlation was confirmed in patients with PCD (p = 0.019; r = 0.484). NOS2 gene expression was lower in PCD than in SCD (p = 0.04). The NOS3 isoform correlated with missing central microtubules (p = 0.048; r = 0.447). nNO levels were higher in PCD subjects with the NOS3 thymidine 894 mutation, and this was associated with a higher ciliary beat frequency (p = 0.045). These results demonstrate a relationship between nNO level, NOS mRNA expression and ciliary beat frequency.

Tubulin tyrosine ligase-like 1 deficiency results in chronic rhinosinusitis and abnormal development of spermatid flagella in mice.

Tubulin tyrosine ligase-like 1 (TTLL1) protein is a member of the tubulin tyrosine ligase superfamily of proteins that are involved in the posttranslational polyglutamylation of tubulin in axonemal microtubules within cilia and flagella. To investigate the physiological role of TTLL1, the authors generated mice with a gene trap mutation in the Ttll1 gene that provide confirmation in a mammalian model that polyglutamylation plays an important role in some ciliary and flagellar functions. For the first time, mice homozygous for the Ttll1 mutation exhibited accumulations of exudates in the nasal passages and sinuses, rhinosinusitis, otitis media, and male infertility. In homozygous mutant male mice, abnormal sperm morphology and function were characterized by shortened or absent flagella and immotility. Although homozygous mutant males were infertile, the females were fertile. These findings are consistent with a diagnosis of primary ciliary dyskinesia (PCD) resulting from ciliary dysfunction. They indicate that Ttll1 is essential for normal motility of respiratory cilia and the biogenesis and function of sperm flagella but that the defect does not result in the hydrocephalus or laterality defects often seen in other forms of PCD. The absence of early-onset lethal hydrocephalus in Ttll1-mutant mice may enable studies to evaluate the long-term effects of PCD in the respiratory system of mice. Although no mutations in the orthologous gene have been linked with PCD in humans, investigating the role of TTLL1 and polyglutamylation of tubulin in cilia and flagella should advance an understanding of the biogenesis and function of these organelles in mammals and have potential diagnostic and therapeutic applications.

Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambda-deficient mice: possible implication for the pathogenesis of immotile cilia syndrome.

A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol beta within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.

Dynein arms and spokes after ciliogenesis in cultured respiratory epithelial cells from non-PCD individuals.

Dynein arms and spokes are crucial components of cilia. Reference values for the dynein arms and spokes were calculated based on biopsies from non-PCD patients as well as after ciliogenesis in culture. The mean values in the biopsies (n = 251) were 8.4 +/- 0.5, 2.9 +/- 0.7 and 4.7 +/- 1.0 for the outer dynein arms, inner dynein arms and spokes respectively. After ciliogenesis in culture (n = 462) identical values were found: 8.7 +/- 0.4, 3.0 +/- 0.4 and 5.5 +/- 0.6. The lower limits of normality can be set at 7.0 and 1.2 for the outer and inner dynein arms respectively. The dynein arms and spokes were not influenced by the percentage of secondary abnormalities. In conclusion, dynein arms and spokes are readily identified after ciliogenesis in culture. These parameters are independent of secondary ciliary dyskinesia.