Motile and non-motile cilia in human pathology: from function to phenotypes

Ciliopathies are inherited human disorders caused by both motile and non-motile cilia dysfunction that form an important and rapidly expanding disease category. Ciliopathies are complex conditions to diagnose, being multisystem disorders characterized by extensive genetic heterogeneity and clinical variability with high levels of lethality. There is marked phenotypic overlap among distinct ciliopathy syndromes that presents a major challenge for their recognition, diagnosis, and clinical management, in addition to posing an on-going task to develop the most appropriate family counselling. The impact of next-generation sequencing and high-throughput technologies in the last decade has significantly improved our understanding of the biological basis of ciliopathy disorders, enhancing our ability to determine the possible reasons for the extensive overlap in their symptoms and genetic aetiologies. Here, we review the diverse functions of cilia in human health and disease and discuss a growing shift away from the classical clinical definitions of ciliopathy syndromes to a more functional categorization. This approach arises from our improved understanding of this unique organelle, revealed through new genetic and cell biological insights into the discrete functioning of subcompartments of the cilium (basal body, transition zone, intraflagellar transport, motility). Mutations affecting these distinct ciliary protein modules can confer different genetic diseases and new clinical classifications are possible to define, according to the nature and extent of organ involvement. Copyright (C) 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions

Motile cilia defects in diseases other than primary ciliary dyskinesia:The contemporary diagnostic and research role for transmission electron microscopy

Ultrastructural studies have underpinned the cell biological and clinical investigations of the varied roles of motile cilia in health and disease, with a long history since the 1950s. Recent developments from transmission electron microscopy (TEM; cryo-electron microscopy, electron tomography) have yielded higher resolution and fresh insights into the structure and function of these complex organelles. Microscopy in ciliated organisms, disease models, and in patients with ciliopathy diseases has dramatically expanded our understanding of the ubiquity, multisystem involvement, and importance of cilia in normal human development. Here, we review the importance of motile cilia ultrastructural studies in understanding the basis of diseases other than primary ciliary dyskinesia

Primary ciliary dyskinesia (Siewert's / Kartagener's Syndrome): Respiratory symptoms and psycho-social impact

BACKGROUND: Although the pathophysiological defect in primary ciliary dyskinesia (PCD; Siewert's / Kartagener's syndrome) is now well characterised, there are few studies of the impact of the condition upon health function, particularly in later life. This study assesses the health impact of the condition in a large group of patients. In addition, it assesses the similarity in age of diagnosis, symptoms and problems of those with situs inversus (PCD-SI) and those with situs solitus (PCD-SS). METHODS: Postal questionnaire sent to members of the UK Primary Ciliary Dyskinesia Family Support Group. The questionnaire contained the St. George's Respiratory Questionnaire (SGRQ) and the SF-36 questionnaire for assessing health status. RESULTS: 93 questionnaires were returned, representing a 66% response rate. Replies were received from similar numbers of PCD-SI and PCD-SS. Individuals with PCD-SI did not show a significant tendency to be diagnosed earlier, and neither did they show any difference in their symptoms, or the relationship of symptoms to age. Respiratory symptoms were fairly constant up until the age of about 25, after which there was a slow increase in symptoms, and a decline in health status, patients over the age of 40 being about one and a half standard deviations below the mean on the physical component score of the PCS. Patients diagnosed earlier in life, and hence who had received more treatment for their condition, had better scores on the SGRQ Impact and Activity scores. CONCLUSIONS: PCD is a chronic condition which has a progressively greater impact on health in the second half of life, producing significant morbidity and restriction of life style. Early diagnosis, and hence earlier treatment, may improve symptoms and the impact of the condition

Glial cells are functionally impaired in juvenile neuronal ceroid lipofuscinosis and detrimental to neurons.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions

Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia

RATIONALE The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the UK consists of assessing ciliary function by high-speed-microscopy and ultrastructure by election microscopy, but equipment and expertise is not widely available internationally. The identification of bi-allelic disease causing mutations is also diagnostic, but many disease causing genes are unknown, and testing is not widely available outside the USA. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. OBJECTIVES: Determine utility of a panel of six fluorescent labelled antibodies as a diagnostic tool for PCD. METHODS: Immunofluorescent labelling of nasal brushings from a discovery cohort of 35 patients diagnosed with PCD by ciliary ultrastructure, and a diagnostic accuracy cohort of 386 patients referred with symptoms suggestive of disease. The results were compared to diagnostic outcome. MEASUREMENTS AND MAIN RESULTS: Immunofluorescence correctly identified mislocalised or absent staining in 100% of the discovery cohort. In the diagnostic cohort immunofluorescence successfully identified 22 of 25 patients with PCD and normal staining in all 252 in whom PCD was considered highly unlikely. Immunofluorescence additionally provided a result in 55% (39) of cases which were previously inconclusive. Immunofluorescence results were available within 14 days, costing $187 per sample compared to electron microscopy (27 days, cost$1452). CONCLUSIONS: Immunofluorescence is a highly specific diagnostic test for PCD, and improves the speed and availability of diagnostic testing, however, sensitivity is limited and immunofluorescence is not suitable as a stand-alone test

Axonemal structures reveal mechanoregulatory and disease mechanisms

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3 kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some mis-orientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, that gives rise to a non-lethal primary skeletal ciliopathy combined with defective motile cilia and PCD

CFAP300 mutation causing primary ciliary dyskinesia in Finland

Primary ciliary dyskinesia (PCD) is a rare genetic condition characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair-like organelles protruding out of the cell that are responsible for removal of mucus from the airways and organizing internal organ positioning during embryonic development. PCD is caused by mutations in genes coding for structural or assembly proteins in motile cilia. Thus far mutations in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported, thus highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics. In this study, we identified a recurrent loss-of-function mutation c.198_200delinsCC in CFAP300 causing lack of the protein product. PCD patients homozygous for the identified CFAP300 mutation have immotile airway epithelial cilia associated with missing dynein arms in their ciliary axonemes. Furthermore, using super resolution microscopy we demonstrate that CFAP300 is transported along cilia in normal human airway epithelial cells suggesting a role for CFAP300 in dynein complex transport in addition to preassembly in the cytoplasm. Our results highlight the importance of CFAP300 in dynein arm assembly and improve diagnostics of PCD in Finland.publishedVersionPeer reviewe