Novel Gene Discovery in Primary Ciliary Dyskinesia

Primary Ciliary Dyskinesia (PCD) is one of the ‘ciliopathies’, genetic disorders affecting either cilia structure or function. PCD is a rare recessive disease caused by defective motile cilia. Affected individuals manifest with neonatal respiratory distress, chronic wet cough, upper respiratory tract problems, progressive lung disease resulting in bronchiectasis, laterality problems including heart defects and adult infertility. Early diagnosis and management are essential for better respiratory disease prognosis. PCD is a highly genetically heterogeneous disorder with causal mutations identified in 36 genes that account for the disease in about 70% of PCD cases, suggesting that additional genes remain to be discovered. Targeted next generation sequencing was used for genetic screening of a cohort of patients with confirmed or suggestive PCD diagnosis. The use of multi-gene panel sequencing yielded a high diagnostic output (> 70%) with mutations identified in known PCD genes. Over half of these mutations were novel alleles, expanding the mutation spectrum in PCD genes. The inclusion of patients from various ethnic backgrounds revealed a striking impact of ethnicity on the composition of disease alleles uncovering a significant genetic stratification of PCD in different populations. Pathogenic mutations were also identified in several new candidate genes not previously linked to PCD. Molecular and cell biology techniques were coupled with model organism studies to characterize the involvement of the new candidate genes in cilia motility and PCD. Paramecium was proven to be a good model for functional characterization of PCD potential candidate genes. The previously uncharacterized C11orf70 was identified to play a highly conserved role in dynein assembly and intraflagellar transport (IFT)-related cilia cargo trafficking. Mutations identified in DNAH9 resulted in a distinct motile cilia defect with mild respiratory symptoms, unusual in PCD. Mutations identified in two intraflagellar transport genes, IFT74 and WDR19, linked together primary and motile ciliopathy phenotypes observed in the affected individuals

Proceedings of the 3rd BEAT-PCD Conference and 4th PCD Training School

Abstract Primary ciliary dyskinesia (PCD) is a chronic suppurative airways disease that is usually recessively inherited and has marked clinical phenotypic heterogeneity. Classic symptoms include neonatal respiratory distress, chronic rhinitis since early childhood, chronic otitis media, recurrent airway infections leading to bronchiectasis, chronic sinusitis, laterality defects with and without congenital heart disease including abnormal situs in approximately 50% of the cases, and male infertility. Lung function deteriorates progressively from childhood throughout life. ‘Better Experimental Approaches to Treat Primary Ciliary Dyskinesia’ (BEAT-PCD) is a network of scientists and clinicians coordinating research from basic science through to clinical care with the intention of developing treatments and diagnostics that lead to improved long-term outcomes for patients. BEAT-PCD activities are supported by EU funded COST Action (BM1407). The third BEAT-PCD conference and fourth PCD training school were held jointly in February 2018 in Lisbon, Portugal. Presentations and workshops focussed on advancing the knowledge and skills relating to PCD in: basic science, epidemiology, diagnostic testing, clinical management and clinical trials. The multidisciplinary conference provided an interactive platform for exchanging ideas through a program of lectures, poster presentations, breakout sessions and workshops. Three working groups met to plan consensus statements. Progress with BEAT-PCD projects was shared and new collaborations were fostered. In this report, we summarize the meeting, highlighting developments made during the meeting

Primary Ciliary Dyskinesia Due to Microtubular Defects is Associated with Worse Lung Clearance Index

PURPOSE: Primary ciliary dyskinesia (PCD) is characterised by repeated upper and lower respiratory tract infections, neutrophilic airway inflammation and obstructive airway disease. Different ultrastructural ciliary defects may affect lung function decline to different degrees. Lung clearance index (LCI) is a marker of ventilation inhomogeneity that is raised in some but not all patients with PCD. We hypothesised that PCD patients with microtubular defects would have worse (higher) LCI than other PCD patients. METHODS: Spirometry and LCI were measured in 69 stable patients with PCD. Age at testing, age at diagnosis, ethnicity, ciliary ultrastructure, genetic screening result and any growth of Pseudomonas aeruginosa was recorded. RESULTS: Lung clearance index was more abnormal in PCD patients with microtubular defects (median 10.24) than those with dynein arm defects (median 8.3, p = 0.004) or normal ultrastructure (median 7.63, p = 0.0004). Age is correlated with LCI, with older patients having worse LCI values (p = 0.03, r = 0.3). CONCLUSION: This study shows that cilia microtubular defects are associated with worse LCI in PCD than dynein arm defects or normal ultrastructure. The patient's age at testing is also associated with a higher LCI. Patients at greater risk of obstructive lung disease should be considered for more aggressive management. Differences between patient groups may potentially open avenues for novel treatments

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

Clinical and genetic spectrum in 33 Egyptian families with suspected primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare genetic disorder of motile cilia dysfunction generally inherited as an autosomal recessive disease. Genetic testing is increasingly considered an early step in the PCD diagnostic workflow. We used targeted panel next generation sequencing (NGS) for genetic screening of 33 Egyptian families with highly clinically suspected PCD. All variants prioritized were Sanger confirmed in the affected individuals and correctly segregated within the family. Targeted NGS yielded a high diagnostic output (70%) with bi‐allelic mutations identified in known PCD genes. Mutations were identified in 13 genes overall, with CCDC40 and CCDC39 the most frequently mutated genes among Egyptian patients. Most identified mutations were predicted null effect variants (79%) and not reported before (85%). This study reveals that the genetic landscape of PCD among Egyptians is highly heterogeneous, indicating that a targeted NGS approach covering multiple genes will provide a superior diagnostic yield than Sanger sequencing for genetic diagnosis. The high diagnostic output achieved here highlights the potential of placing genetic testing early within the diagnostic workflow for PCD, in particular in developing countries where other diagnostic tests can be less available

HYDIN variants cause primary ciliary dyskinesia in the Finnish population

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare genetic disorder 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. Thus far, disease causing variants 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 highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics and management. METHODS: Whole exome sequencing was used to identify disease causing variants in Finnish PCD cohort. The effect of the identified HYDIN variants on cilia structure and function was confirmed by high-speed video analysis, immunofluorescence and electron tomography. RESULTS: In this study, we identified three Finnish PCD patients carrying homozygous loss-of-function variants and one patient with compound heterozygous variants within HYDIN. The functional studies showed defects in the axonemal central pair complex. All patients had clinical PCD symptoms including chronic wet cough and recurrent airway infections, associated with mostly static airway cilia. CONCLUSION: Our results are consistent with the previously identified important role of HYDIN in the axonemal central pair complex and improve specific diagnostics of PCD in different national populations

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

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

Hydrocephalus and diffuse choroid plexus hyperplasia in primary ciliary dyskinesia-related MCIDAS mutation

OBJECTIVE: To report a neuroradiologic phenotype associated with reduced generation of multiple motile cilia (RGMC) and mutations in the multicilin gene. We hypothesize that the observed phenotype may reflect the emerging role that ependymal cilia play in regulating CSF production. METHOD: Clinical and radiologic records were retrospectively reviewed for 7 consecutive patients diagnosed by the Leicester UK national primary ciliary dyskinesia (PCD) diagnostic laboratory. RESULTS: On MRI scanning, all patients demonstrated hydrocephalus, choroid plexus hyperplasia (CPH), and arachnoid cysts. No patient had any sign of neurologic deficit. All patients had significant lung disease. CONCLUSIONS: We conclude that there is a high incidence of hydrocephalus, arachnoid cysts, and CPH in MCIDAS-associated RGMC. In all cases, the observed hydrocephalus seems arrested in childhood without progression or adverse neurologic sequelae. Our new observation of CPH, which is associated with CSF overproduction, is the first macroscopic evidence that ependymal cilia may be involved in the regulation of CSF production and flow. We suggest that brain imaging should be performed in all cases of RGMC and that a diagnosis of PCD or RGMC be strongly considered in patients with unexplained hydrocephalus and a lifelong “wet”-sounding cough

Novel FAM126A mutations in hypomyelination and congenital cataract disease

n/