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1.  Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganisation and absent inner dynein arms 
Human mutation  2013;34(3):462-472.
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’.
PMCID: PMC3630464  PMID: 23255504
primary ciliary dyskinesia; cilia; CCDC39; CCDC40; radial spoke; dynein regulatory complex; nexin link
2.  Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure 
Primary 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.
We 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.
PCD 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.
We 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.
PMCID: PMC4016480  PMID: 24450482
Primary cell culture; Ultrastructure; Population characteristics; Primary ciliary dyskinesia; Transmission electron microscopy; DNAH11
7.  CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs 
Nature genetics  2010;43(1):72-78.
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 asymmetry1. 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.
PMCID: PMC3509786  PMID: 21131972
8.  Rhinoplasty from a rhinologist's perspective: Need for recognition of associated sinonasal conditions 
Facial plastic surgeons may primarily focus on esthetic improvement of the nasal shape in patients seeking rhinoplasty (RP). However, medical conditions inside the nasal cavity should not be neglected because they may lead to unresolved sinonasal problems and, hence, dissatisfaction after esthetic RP. This observational study investigated the prevalence of sinonasal symptoms and endonasal pathology in patients requesting esthetic RP.
Patients seeking RP (n = 269) were given a questionnaire evaluating nasal obstruction and sinonasal symptoms using visual analog scales and the 22-item Sino-Nasal Outcome Test. In addition, patients underwent nasal endoscopy to evaluate anatomic and/or mucosal disease and skin-prick testing in case of clinical suspicion of allergy. Two control groups consisted of patients with an otological or general ear/nose/throat problem (n = 65) and patients who planned for endoscopic sinus surgery (ESS; n = 90).
The general appraisal of nasal breathing on a scale from 0–10 in patients seeking RP was as low as 4.3 ± 3.1. Structural pathology was found in 62% of RP patients, with septal deviation being the most frequent problem encountered (54%), followed by internal nasal valve dysfunction (14%). Mucosal disease was present in 28% of RP patients. The mean SNOT-22 score of RP patients (31.8 ± 23.3) was significantly higher than the control group (11.6 ± 7.9; p < 0.001), but lower than the ESS patients (48.5 ± 22.0; p < 0.001).
The prevalence of endonasal structural or mucosal pathology in patients seeking RP is high and should not be overlooked at the time of planning surgery.
PMCID: PMC3903105  PMID: 23232202
ESS; esthetic; facial; functional; mucosal disease; nasal breathing; nasal valve; revision; rhinoplasty; sinonasal
9.  Sensitization rate and clinical profile of Congolese patients with rhinitis 
Allergy & Rhinology  2012;3(1):e16-e24.
In the African continent, the sensitization pattern and clinical profile are unknown in patients with rhinitis/rhinosinusitis attending the outpatient ear, nose, and throat (ENT) clinics. We therefore aimed to analyze the clinical characteristics of rhinitis/rhinosinusitis patients in Democratic Republic of Congo (DRC), classify allergic rhinitis (AR) according to the Allergic Rhinitis and Its Impact on Asthma criteria, and evaluate the sensitization profile and its associated factors. From January to May 2009, 423 patients with rhinitis symptoms attending the Outpatient ENT clinic of the University Hospital and Saint Joseph Hospital of Kinshasa were evaluated for allergy symptoms, severity, and duration of symptoms and underwent skin-prick tests (SPTs) for a panel of 15 allergens. Of 423 patients 35.2% had positive SPT results, with 40.9% showing polysensitization. Dermatophagoides pteronyssinus (DPT) (68.5%) and cockroach (36.2%) were the most common allergens among sensitized patients. Patients with rhinitis/rhinosinusitis mainly presented in decreasing order with sneezing, facial pain/pressure, nasal obstruction, postnasal discharge, nose itching, clear nasal discharge, and eye itching. Persistent and moderate/severe AR represented 61.4 and 69.3%, respectively. Sensitization was independently associated with younger age, rhinoconjunctivitis, and reaction to nonspecific trigger factors. In conclusion, 35.2% of patients attending the ENT Outpatient Clinic in DRC for rhinitis problems had a positive SPT to at least one allergen, with mainly DPT and cockroach allergens being involved; and a substantial portion showed persistent and moderate/severe AR. Therefore, allergy should not be neglected as an etiologic factor in rhinologic disease in the African continent.
PMCID: PMC3404473  PMID: 22852125
Congo; rhinitis; rhinosinusitis; skin-prick testing; symptoms
10.  Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy 
PLoS Genetics  2009;5(3):e1000422.
Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease.
Author Summary
This manuscript reports on a successful gene therapy attempt on human airway epithelial cells of a patient suffering from Primary Ciliary Dyskinesia. In this autosomal recessive disease, cilia of the epithelial cells that border the upper and lower respiratory tracks are not functioning. As a result, patients suffer from recurrent airway infections leading progressively to respiratory insufficiency. There is no treatment as of today that could restore normal ciliary beating. In this report, we showed that it is feasible to transfer a therapeutic gene to human airway epithelial cells with a lentivirus. This transferred gene is transcribed and expressed. Moreover, defective cells that had immotile cilia due to compound heterozygous mutations in the DNAI1 gene recovered ciliary beating after treatment with a lentivirus containing a normal DNAI1 gene. This is the first report on gene therapy in Primary Ciliary Dyskinesia. Since lentivirus is able to insert therapeutic genes into the cell genome, this result may have impact on in vivo gene therapy in this disease and in diseases related to human epithelial airway cells such as cystic fibrosis.
PMCID: PMC2650261  PMID: 19300481
11.  IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells 
Journal of Clinical Investigation  2001;108(12):1817-1824.
In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor α subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13’s effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.
PMCID: PMC209466  PMID: 11748265
12.  Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis 
Genome Biology  2001;2(7):research0026.1-research0026.9.
Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.
Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.
We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.
PMCID: PMC55323  PMID: 11516339

Results 1-12 (12)