Israel Amirav

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia

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.

Hypertonic saline or high volume normal saline for viral bronchiolitis: Mechanisms and rationale

In recent years and particularly in the last 5 years, there have been substantial advances in our understanding of the mechanisms governing mucus clearance (MC) in health and disease. These have demonstrated the role of hydration of the airway surface liquid (ASL) and the importance of inhaled hypertonic saline for rehyadration and have been recently reviewed. Briefly, it was suggested that MC failure is a dominant factor not only in CF but in most airway diseases and that hydration is the dominant variable governing MC in all airway diseases. This was also found to be true even in normal subjects and furthermore, it has been noted, that exacerbations in many airway diseases result from intermittent catastrophic failures of MC due to dehydration of ASL often triggered by viral infections. Thus, therapy to maintain ASL hydration is probably important during viral exacerbations not only in CF patients but in all chronic airway diseases. Issues crucial to understanding the postulated mechanism involving hypertonic saline inhalation treatment in RSV bronchiolitis will be described in more detail here.

Sole pathogen in acute bronchiolitis: is there a role for other organisms apart from respiratory syncytial virus?

Background: Acute bronchiolitis (AB) is a common disease of young children with peak incidence during the winter season. Respiratory syncytial virus (RSV) is a major causative organism, yet recent relatively small sized studies have suggested an increased role of other organisms as sole or codetected organisms. The aim of this study was to assess the prevalence of sole- and mixed-organisms infections in hospitalized children with AB, using combined antigen-based and polymerase chain reaction assays (PCR). Methods: Sputum or nasal wash specimens obtained from 490 previously healthy children ≤2 years of age hospitalized with AB between December 1, 2005 and March 31, 2006 were tested: (1) For RSV, by rapid antigen detection test; (2) For RSV, influenza A, B, Parainfluenza 1 to 3, and adenovirus antigens by direct fluorescent assay; (3) For influenza A and B, RSV, Parainfluenza 1 to 3 viruses RNA by reverse transcription (RT) PCR assay; (4) For human metapneumovirus and rhinovirus RNA by RT real-time PCR assay; (5) For adenovirus, and Bordetella pertussis DNA by conventional PCR assays; (6) For human bocavirus DNA by real-tine PCR assays. Results: At least 1 organism was detected in 465 (91%) children. In 283 (61%), 117 (25%), and 23 (5%) children, 1, 2, and 3/4 organisms were detected, respectively. The most commonly detected organism was RSV, detected in 76%, and as a sole organism in 49%. Rhinovirus, human metapneumovirus, influenza virus A, bocavirus, Bordetella pertussis, and adenovirus were detected as a sole organism in 7%, 2.1%, 1%, 0.6%, 0.6%, and 0.2% of the children, respectively. Conclusions: Respiratory organisms were detected in the majority of the children, of whom about one third suffered from mixed organism infection. RSV was the most prevalent sole detected organism. The relevance of all other organisms may be much less than previously suggested.

Nebuliser hood compared to mask in wheezy infants: aerosol therapy without tears!

Background: Small volume nebulisers (SVNs) with masks commonly provide aerosol therapy for infants with lung diseases. However, infants and toddlers are often disturbed by and thus reject masks. Aims: To compare the lung deposition efficiency of the “usual” SVN aerosol mask and a prototype hood attached to an SVN. The advantage of the hood is that no mask is needed and medication can readily be administered during sleep. Methods:99mTc salbutamol solution was administered at random by SVN plus mask or hood to 14 wheezy infants (mean age 8 (SD 5) months). The dose and distribution of salbutamol were evaluated using gamma scintigraphy. Clinical response, tolerability by the infants, and parent preference were also compared. Results: Mean total lung deposition was 2.6% with the hood and 2.4% with the mask (p > 0.05). Variability with the mask was greater than with the hood (coefficient of variation (CoV) 54% v 39%). Both treatments provided similar clinical benefit and side effects as reflected in improved oxygen saturation, reduced respiratory frequency, and increased heart rate. Infants accepted the hood better than the mask and there was a positive correlation between poor acceptance and upper airways and stomach deposition for both treatment modalities. Parents preferred the hood treatments. Conclusions: Aerosol therapy by hood is as efficient as by mask but provides a better therapeutic index. It is much better tolerated by infants and preferred by parents. Hood nebulisation is a simple and patient friendly mode of aerosol therapy in wheezy infants.

CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia

Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.

A Double-Blind, Placebo-Controlled, Randomized Trial of Montelukast for Acute Bronchiolitis

BACKGROUND. Cysteinyl leukotrienes are implicated in the inflammation of bronchiolitis. Recently, a specific cysteinyl leukotriene receptor antagonist, montelukast (Singulair [MSD, Haarlem, Netherlands]), has been approved for infants in granule sachets. OBJECTIVE. Our goal was to evaluate the effect of montelukast on clinical progress and on cytokines in acute bronchiolitis. METHODS. This was a randomized, placebo-controlled, double-blind, parallel-group study in 2 medical centers. Fifty-three infants (mean age: 3.8 ± 3.5 months) with a first episode of acute bronchiolitis were randomly assigned to receive either 4-mg montelukast sachets or placebo, every day, from hospital admission until discharge. The primary outcome was length of stay, and secondary outcomes included clinical severity score (maximum of 12) and changes in type 1 and 2 cytokine levels (including interleukin4/IFN-γ ratio as a surrogate for the T-helper 2/T-helper 1 ratio) in nasal lavage. RESULTS. Both groups were comparable at baseline, and cytokine levels correlated positively with disease severity. There were neither differences in length of stay (4.63 ± 1.88 [placebo group] vs 4.65 ± 1.97 days [montelukast group]) nor in clinical severity score and cytokine levels between the 2 groups. No differences in interleukin 4/IFN-γ ratio between the 2 groups were seen. There was a slight tendency for infants in the montelukast group to recover more slowly than those in the placebo group (clinical severity score at discharge: 6.1 ± 2.4 vs 4.8 ± 2.2, respectively). CONCLUSIONS. Montelukast did not improve the clinical course in acute bronchiolitis. No significant effect of montelukast on the T-helper 2/T-helper 1 cytokine ratio when given in the early acute phase could be demonstrated.

LRRC6 mutation causes primary ciliary dyskinesia with dynein arm defects.

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.

Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects

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.