Loss-of-Function Mutations in LRRC6, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms, Cause Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a group of autosomal-recessive disorders resulting from cilia and sperm-flagella defects, which lead to respiratory infections and male infertility. Most implicated genes encode structural proteins that participate in the composition of axonemal components, such as dynein arms (DAs), that are essential for ciliary and flagellar movements; they explain the pathology in fewer than half of the affected individuals. We undertook this study to further understand the pathogenesis of PCD due to the absence of both DAs. We identified, via homozygosity mapping, an early frameshift in LRRC6, a gene that encodes a leucine-rich-repeat (LRR)-containing protein. Subsequent analyses of this gene mainly expressed in testis and respiratory cells identified biallelic mutations in several independent individuals. The situs inversus observed in two of them supports a key role for LRRC6 in embryonic nodal cilia. Study of native LRRC6 in airway epithelial cells revealed that it localizes to the cytoplasm and within cilia, whereas it is absent from cells with loss-of-function mutations, in which DA protein markers are also missing. These results are consistent with the transmission-electron-microscopy data showing the absence of both DAs in cilia or flagella from individuals with LRRC6 mutations. In spite of structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein involved in the same PCD phenotype, the two proteins are not redundant. The evolutionarily conserved LRRC6, therefore, emerges as an additional player in DA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than was previously thought

Ultra-structural cell distribution of the melanoma marker iodobenzamide: improved potentiality of SIMS imaging in life sciences

BACKGROUND: Analytical imaging by secondary ion mass spectrometry (SIMS) provides images representative of the distribution of a specific ion within a sample surface. For the last fifteen years, concerted collaborative research to design a new ion microprobe with high technical standards in both mass and lateral resolution as well as in sensitivity has led to the CAMECA NanoSims 50, recently introduced onto the market. This instrument has decisive capabilities, which allow biological applications of SIMS microscopy at a level previously inaccessible. Its potential is illustrated here by the demonstration of the specific affinity of a melanoma marker for melanin. This finding is of great importance for the diagnosis and/or treatment of malignant melanoma, a tumour whose worldwide incidence is continuously growing. METHODS: The characteristics of the instrument are briefly described and an example of application is given. This example deals with the intracellular localization of an iodo-benzamide used as a diagnostic tool for the scintigraphic detection of melanic cells (e.g. metastasis of malignant melanoma). B16 melanoma cells were injected intravenously to C(57)BL(6)/J(1)/co mice. Multiple B16 melanoma colonies developed in the lungs of treated animals within three weeks. Iodobenzamide was injected intravenously in tumour bearing mice six hours before sacrifice. Small pieces of lung were prepared for SIMS analysis. RESULTS: Mouse lung B16 melanoma colonies were observed with high lateral resolution. Cyanide ions gave "histological" images of the cell, representative of the distribution of C and N containing molecules (e.g. proteins, nucleic acids, melanin, etc.) while phosphorus ions are mainly produced by nucleic acids. Iodine was detected only in melanosomes, confirming the specific affinity of the drug for melanin. No drug was found in normal lung tissue. CONCLUSION: This study demonstrates the potential of SIMS microscopy, which allows the study of ultra structural distribution of a drug within a cell. On the basis of our observations, drug internalization via membrane sigma receptors can be excluded

Standardised clinical data from patients with primary ciliary dyskinesia:FOLLOW-PCD

Clinical data on primary ciliary dyskinesia (PCD) are limited, heterogeneous and mostly derived from retrospective chart reviews, leading to missing data and unreliable symptoms and results of physical examinations. We need standardised prospective data collection to study phenotypes, severity and prognosis and improve standards of care.A large, international and multidisciplinary group of PCD experts developed FOLLOW-PCD, a standardised clinical PCD form and patient questionnaire. We identified existing forms for clinical data collection via the Better Experimental Approaches to Treat PCD (BEAT-PCD) COST Action network and a literature review. We selected and revised the content items with the working group and patient representatives. We then revised several drafts in an adapted Delphi process, refining the content and structure.FOLLOW-PCD has a modular structure, to allow flexible use based on local practice and research focus. It includes patient-completed versions for the modules on symptoms and lifestyle. The form allows a comprehensive standardised clinical assessment at baseline and for annual reviews and a short documentation for routine follow-up. It can either be completed using printable paper forms or using an online REDCap database.Data collected in FOLLOW-PCD version 1.0 is available in real-time for national and international monitoring and research. The form will be adapted in the future after extensive piloting in different settings and we encourage the translation of the patient questionnaires to multiple languages. FOLLOW-PCD will facilitate quality research based on prospective standardised data from routine care, which can be pooled between centres, to provide first-line and real-time evidence for clinical decision-making

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins

Local consequences of sinonasal diseases on tissular and cellular morphology

L’obstruction nasale chronique (ONC) est un symptôme banal qui révèle de nombreuses pathologies rhinologiques ostéo-cartilagineuses et/ou muqueuses. Les explorations de l’ONC aident à la démarche diagnostique et permettent d’évaluer le retentissement de l’ONC sur la muqueuse nasale.Afin de répondre aux problèmes que je rencontre dans ma pratique clinique en rhinologie, j’ai orienté mes travaux de recherche vers le développement de nouveaux outils d’exploration des pathologies nasales obstructives :1. A l’échelle tissulaire, dans le cadre des rhinites chroniques, en particulier le dysfonctionnement nasal neurovégétatif qui restait un diagnostic d’élimination. J’ai montré que la compliance nasale, mesurée par rhinométrie acoustique, est perturbée dans les deux causes les plus fréquentes de rhinite chronique, d’une part, et est utile au diagnostic de dysfonctionnement nasal neurovégétatif, d’autre part.2. A l’échelle cellulaire, en m’intéressant à la cellule ciliée de l’épithélium respiratoire. J’ai étudié les avantages et les limites de l’étude ciliaire par microscopie électronique à transmission pour le diagnostic de dyskinésie ciliaire primitive. A l’aide de la vidéomicroscopie numérique à haute vitesse, j’ai développé des paramètres objectifs permettant de caractériser précisément le battement ciliaire. J’ai montré que ces paramètres étaient utiles pour le diagnostic de dyskinésie ciliaire primitive, pour étudier les cils respiratoires au cours de l’amaurose congénitale de Leber ou pour étudier le rôle de la protéine BUG22 dans le battement ciliaire de la paramécie.Chronic nasal obstruction (CNO) is a common symptom and corresponds to diseases affecting bones, cartilages and/or mucosa of the nose. Objective monitoring of nasal patency is useful for identifying the etiology of CNO and for evaluating its effects on the mucosa.My research is guided by the numerous challenges encountered through my clinical practice of rhinology. I have developed new objective tools for exploring obstructive nasal diseases:1. At tissue level, I focused on chronic rhinitis, especially the neurovegetative nasal dysfunction which remained a diagnosis of exclusion so far. Using acoustic rhinometry, I have shown that the nasal compliance was abnormal in the two most common causes of chronic rhinitis and was a useful measure for the diagnosis of neurovegetative nasal dysfunction.2. At cell level, I focused on the ciliated cell of the respiratory epithelium. I studied the advantages and limits of transmission electron microscopy analysis of cilia for the diagnosis of primary ciliary dyskinesia. Using digital high-speed videomicroscopy, I have established objective parameters that precisely characterize ciliary beating. I have shown that these parameters were useful for diagnosing primary ciliary dyskinesia, for studying respiratory cilia of patients affected by Leber congenital amaurosis or for studying the role of BUG22 protein in ciliary beating of paramecia

Les balles fongiques sinusiennes chez l'immunodéprimé

Les balles fongiques sinusiennes (BFS) sont une forme bénigne d infection fongique, décrite chez des sujets immunocompétents (IC). A l inverse, chez les patients immunodéprimés (ID), les infections fongiques sinusiennes sont graves en raison de l invasion tissulaire par les agents fongiques. Chez les ID, les BFS ont un risque supposé d évolution vers une forme invasive. Le but de ce travail original était d étudier les BFS dans une population ID afin de préciser les caractéristiques cliniques, para-cliniques et thérapeutiques des BFS sur ce terrain. Il s agit d une étude rétrospective de 19 patients ID opérés de BFS entre 1997 et 2007. Les caractéristiques des BFS ont été comparées à celles des BFS de 162 patients IC opérés dans notre institution pendant la même période. Comme chez les IC, la symptomatologie clinique des BFS chez les ID n était pas spécifique. Les localisations sinusiennes à risque de complication (éthmoïdales et sphénoïdales) étaient significativement plus fréquentes chez les ID. Quatre cas de BFS destructive sans invasion tissulaire ont été observés exclusivement chez les ID. Dans les deux population, le traitement était réalisé par chirurgie sinusienne endoscopique, associée pour les BFS destructives, à un traitement antifongique systémique. Ce travail montre pour la première fois que les BFS ont un risque de complication chez les ID. En conséquence, compte tenu du caractère aspécifique des symptômes et de la localisation souvent profonde des BFS de l ID, un scanner des sinus de dépistage systématique peut se discuter en cas d immunodépression avérée ou programmée. En cas de suspicion de BFS chez un ID, l exérèse chirurgicale ne doit pas être différée.PARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Effets du stress sur la perméabilité nasale (étude prospective sur 12 sujets sains)

L obstruction nasale est un symptôme fréquent qui altère la qualité de vie. Une des principales causes est la rhinite.Le stress est physiologique mais peut participer au développement de maladies psychosomatiques.Deux études récentes ont montré que le stress participe à la physiopathologie des rhinites allergiques. Ace jour l effet du stress sur la perméabilité nasale n a jamais été étudié chez le sujet sain.L objectif principal de notre étude était d évaluer l effet du stress sur la perméabilité nasale chez dessujets sains. L objectif secondaire était de corréler cet effet au caractère anxieux des sujets.Il s agissait d une étude prospective portant sur 12 adultes sains. Les sujets étaient soumis à un test destress, le Trier Social Stress Test. Une mesure des aires et des volumes des cavités nasales parrhinométrie acoustique était réalisée en pré, per et post-test. Une évaluation de l anxiété par unquestionnaire d anxiété (STAI) était réalisée en début et en fin d épreuve. La fréquence cardiaque étaitmesurée en continu.Les sujets étudiés ont eu une élévation de la fréquence cardiaque et une élévation du score d anxiétéentre le début et la fin de l épreuve. Ils ont donc été stressés. Les aires et les volumes des cavitésnasales ont varié durant l épreuve de manière hétérogène. Chez les plus anxieux, il existait unediminution significative de la perméabilité nasale en phase de relaxation, en post-test.Cette étude décrit pour la première fois les effets que peuvent avoir le stress sur la perméabilité nasale.Lors de la phase de relaxation, il pourrait exister un effet différent en fonction du caractère anxieux.Ceci pourrait expliquer en pathologie le rôle du stress et du caractère anxieux dans l étiopathogénie desmaladies rhino-sinusiennesNasal obstruction is a common symptom that affects quality of life. Rhinitis is one of theprincipal cause.Stress is physiological but can be involved in the onset of psychosomatic disease. Two recentstudies have shown that stress is involved in the pathophysiology of allergic rhinitis. Untilnow, there is no study about the effect of stress on nasal airway of healthy subjects.The main objective of our study was to assess the effect of stress on nasal airway of healthysubjects. The secondary objective was to correlate this potential effect to the trait anxiety ofthe subjects.The study was prospective and included 12 young healthy adults. All subjects underwent theTrier Social Stress Test. Nasal airways were based on measures of acoustic rhinometry andwere investigated at 3 times points: before, during and after the test. Before and after the test,the subjects underwent the State-Trait Anxiety Inventory (STAI) to assess anxiety. Heart ratewas measured during all the protocol.The subjects had an increase in heart rate and in anxiety score between the beginning and theend of test. Therefore, they were all stressed. Areas and volumes of nasal cavity fluctuatedduring the protocol but variations were heterogeneous. The most anxious subjects had asignificant decrease in volume of nasal cavity after the test, during the relaxation response.This study is the first that shows the potential effects of stress on nasal airway. During therelaxation response, the effect could be different depending on trait anxiety. This couldexplain the role of stress and trait anxiety in the pathophysiology of rhinolologic and sinonasal diseasesPARIS12-CRETEIL BU Médecine (940282101) / SudocSudocFranceF

Ciliary defects and genetics of primary ciliary dyskinesia.

International audienceCilia are evolutionarily conserved structures that play key roles in diverse cell types. Motile cilia are involved in the most prominent ciliopathy called primary ciliary dyskinesia (PCD) that combines respiratory symptoms, male infertility, and, in nearly 50% cases, situs inversus. The diagnosis of PCD relies on the identification of ciliary abnormalities that mainly concern outer and/or inner dynein arms (ODA, IDA). PCD is a genetic condition, usually inherited as an autosomal recessive trait. To date, six genes have been clearly implicated in PCD. Two "major" genes, DNAI1 and DNAH5, underlie PCD in nearly half of the patients with ODA defects, whereas RPGR, DNAH11 and TXNDC3 are implicated in rare families with specific phenotypes (retinitis pigmentosa, abnormal beating of structurally normal cilia, and situs ambiguous, respectively). The relative contribution of DNAI2 is currently being assessed. In all the other patients with ODA or other ultrastructural defects, the causative genes remain to be identified