Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects.

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.

Cell and cytokine profile in nasal secretions in cystic fibrosis.

BACKGROUND: Nasal polyposis (NP) frequently complicates the course of cystic fibrosis (CF). The aim of this study was to determine the pattern of inflammatory cells and mediators in nasal secretions from patients with or without NP compared to patients with idiopathic NP and healthy controls. METHODS: Eighteen CF patients with NP (NP+ group: 6 untreated, 12 treated with nasal steroids), and 15 without NP (NP- group) were included in this prospective study and compared to 9 patients with idiopathic NP and 12 healthy controls. Differential cell count eosinophil cationic protein (ECP), interleukin-5 (IL-5) and IL-8 were determined in nasal lavage fluids. RESULTS: The total cell count, the number and the percentage of neutrophils and eosinophils, the levels of IL-8, IL-5 and ECP were significantly higher in nasal secretions from both NP+ and NP- as compared with controls. No difference was found between untreated and treated CF patients with NP. No difference was found between NP+ and NP- groups. Compared to idiopathic NP group, both NP+ and NP- groups had higher percentage of neutrophils and lower percentage of eosinophils. There were no differences according to the use of topical steroids, systemic antibiotherapy, or the type of mutation. CF patients with positive nasal culture had a higher percentage of neutrophils than those with negative culture. CF patients with atopy had a higher percentage of eosinophils than non-atopic patients. CONCLUSION: Our results demonstrate that nasal inflammation is a prominent feature in patients with CF and does not differ according to the presence of NP. IL-8 and IL-5 may play crucial roles in recruitment and activation of neutrophils and eosinophils in upper airways of CF patients.