Analyses of 1,236 genotyped primary ciliary dyskinesia individuals identify regional clusters of distinct DNA variants and significant genotype-phenotype correlations.

BACKGROUND: Primary ciliary dyskinesia (PCD) represents a group of rare hereditary disorders characterized by deficient ciliary airway clearance that can be associated with laterality defects. We aimed to describe the underlying gene defects, geographical differences in genotypes and their relationship to diagnostic findings and clinical phenotypes. METHODS: Genetic variants and clinical findings (age, sex, body mass index, laterality defects, FEV1) were collected from 19 countries using the ERN LUNG International PCD Registry. Genetic data were evaluated according to ACMG guidelines. We assessed regional distribution of implicated genes and genetic variants as well as genotype correlations with laterality defects and FEV1. RESULTS: 1236 individuals carried 908 distinct pathogenic DNA variants in 46 PCD genes. We found considerable variation in the distribution of PCD genotypes across countries due to the presence of distinct founder variants. The prevalence of PCD genotypes associated with pathognomonic ultrastructural defects (mean 72%; 47-100%) and laterality defects (mean 42%; 28-69%) varied widely among the countries. The prevalence of laterality defects was significantly lower in PCD individuals without pathognomonic ciliary ultrastructure defects (18%). The PCD cohort had a reduced median FEV1 z-score (-1.66). In the group of individuals with CCNO (-3.26), CCDC39 (-2.49), and CCDC40 (-2.96) variants, FEV1 z-scores were significantly lower, while the group of DNAH11 (-0.83) and ODAD1 (-0.85) variant individuals had significantly milder FEV1 z-score reductions compared to the whole PCD cohort. CONCLUSION: This unprecedented multinational dataset of DNA variants and information on their distribution across countries facilitates interpretation of genetic epidemiology of PCD and provides prediction of diagnostic and phenotypic features such as the course of lung function.

Nasal nitric oxide measurement in children for the diagnosis of primary ciliary dyskinesia: European Respiratory Society technical standard.

Nasal nitric oxide (nNO) is extremely low in most people with primary ciliary dyskinesia (PCD) and its measurement is an important contributor to making the diagnosis. Existing guidelines and technical standards focus on nNO measurements in older, cooperative children using chemiluminescence analysers. However, measurements of nNO in pre-school-age children (age 2-5 years) may facilitate early diagnosis and electrochemical rather than chemiluminescence analysers are widely used. Pre-schoolers often need different methods to be employed when measuring nNO. Hence, a European Respiratory Society Task Force has developed this technical standard as the first step towards standardising sampling, analysis and reporting of nNO measured as part of the diagnostic testing for PCD in all age groups, including pre-school-age children. Furthermore, we considered both chemiluminescence and electrochemical analysers that are in use worldwide. There was a paucity of quality evidence for electrochemical analysers and sampling methods used in young children, and the Task Force proposes future research priorities to allow updates of this technical standard.

De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry.

Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.

Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

C11orf70 Mutations Disrupting the Intraflagellar Transport-Dependent Assembly of Multiple Axonemal Dyneins Cause Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomized left-right body asymmetry. PCD is mostly caused by mutations affecting the core axoneme structure of motile cilia that is essential for movement. Genes that cause PCD when mutated include a group that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified two unrelated families (three affected children) with mutations in the uncharacterized C11orf70 gene (official gene name CFAP300). The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Tagged C11orf70 in Paramecium and Chlamydomonas localizes mainly in the cytoplasm with a small amount in the ciliary component. IFT139/TTC21B (IFT-A protein) and FLA10 (IFT kinesin) depletion experiments show that its transport within cilia is IFT dependent. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.

Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus.

Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.

Topological data analysis reveals genotype-phenotype relationships in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a heterogeneous inherited disorder caused by mutations in approximately 50 cilia-related genes. PCD genotype-phenotype relationships have mostly arisen from small case series because existing statistical approaches to investigating relationships have been unsuitable for rare diseases. METHODS: We applied a topological data analysis (TDA) approach to investigate genotype-phenotype relationships in PCD. Data from separate training and validation cohorts included 396 genetically defined individuals carrying pathogenic variants in PCD genes. To develop the TDA models, 12 clinical and diagnostic variables were included. TDA-driven hypotheses were subsequently tested using traditional statistics. RESULTS: Disease severity at diagnosis, measured by forced expiratory volume in 1 s (FEV1) z-score, was significantly worse in individuals with CCDC39 mutations (compared to other gene mutations) and better in those with DNAH11 mutations; the latter also reported less neonatal respiratory distress. Patients without neonatal respiratory distress had better preserved FEV1 at diagnosis. Individuals with DNAH5 mutations were phenotypically diverse. Cilia ultrastructure and beat pattern defects correlated closely to specific causative gene groups, confirming these tests can be used to support a genetic diagnosis. CONCLUSIONS: This large scale, multi-national study presents PCD as a syndrome with overlapping symptoms and variations in phenotype according to genotype. TDA modelling confirmed genotype-phenotype relationships reported by smaller studies (e.g. FEV1 worse with CCDC39 mutation) and identified new relationships, including FEV1 preservation with DNAH11 mutations and diversity of severity with DNAH5 mutations.

Clinical utility of NGS diagnosis and disease stratification in a multiethnic primary ciliary dyskinesia cohort.

BACKGROUND: Primary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests. METHODS: The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries. RESULTS: Most (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results. CONCLUSIONS: This study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia.

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left-right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.

PCD Detect: enhancing ciliary features through image averaging and classification.

Primary ciliary dyskinesia (PCD) is an inherited disorder of the motile cilia. Early accurate diagnosis is important to help prevent lung damage in childhood and to preserve lung function. Confirmation of a diagnosis traditionally relied on assessment of ciliary ultrastructure by transmission electron microscopy (TEM); however, >50 known PCD genes have made the identification of biallelic mutations a viable alternative to confirm diagnosis. TEM and genotyping lack sensitivity, and research to improve accuracy of both is required. TEM can be challenging when a subtle or partial ciliary defect is present or affected cilia structures are difficult to identify due to poor contrast. Here, we demonstrate software to enhance TEM ciliary images and reduce background by averaging ciliary features. This includes an option to classify features into groups based on their appearance, to generate multiple averages when a nonhomogeneous abnormality is present. We validated this software on images taken from subjects with well-characterized PCD caused by variants in the outer dynein arm (ODA) heavy chain gene DNAH5. Examining more difficult to diagnose cases, we detected 1) regionally restricted absence of the ODAs away from the ciliary base, in a subject carrying mutations in DNAH9; 2) loss of the typically poorly contrasted inner dynein arms; and 3) sporadic absence of part of the central pair complex in subjects carrying mutations in HYDIN, including one case with an unverified genetic diagnosis. We show that this easy-to-use software can assist in detailing relationships between genotype and ultrastructural phenotype, improving diagnosis of PCD.

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

Risk factors for situs defects and congenital heart disease in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype-phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects.

High prevalence of CCDC103 p.His154Pro mutation causing primary ciliary dyskinesia disrupts protein oligomerisation and is associated with normal diagnostic investigations.

RATIONALE: Primary ciliary dyskinesia is a genetically heterogeneous inherited condition characterised by progressive lung disease arising from abnormal cilia function. Approximately half of patients have situs inversus. The estimated prevalence of primary ciliary dyskinesia in the UK South Asian population is 1:2265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal. OBJECTIVES: To determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive. METHODS: Next-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with primary ciliary dyskinesia. The effect of a CCDC103 p.His154Pro missense variant compared with other dynein arm-associated gene mutations on diagnostic/phenotypic variability was tested. CCDC103 p.His154Pro variant pathogenicity was assessed by oligomerisation assay. RESULTS: Sixteen of 86 (19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels, normal ciliary beat pattern and frequency and a spectrum of partial and normal dynein arm retention. Fifteen (94%) patients or their sibling(s) had situs inversus suggesting CCDC103 p.His154Pro patients without situs inversus are missed. CONCLUSIONS: The CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes primary ciliary dyskinesia that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive standard diagnostic tests.

Primary ciliary dyskinesia with normal ultrastructure: three-dimensional tomography detects absence of DNAH11.

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software.DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal.

Lung function in patients with primary ciliary dyskinesia: an iPCD Cohort study.

Primary ciliary dyskinesia (PCD) has been considered a relatively mild disease, especially compared to cystic fibrosis (CF), but studies on lung function in PCD patients have been few and small.This study compared lung function from spirometry of PCD patients to normal reference values and to published data from CF patients. We calculated z-scores and % predicted values for forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) using the Global Lung Function Initiative 2012 values for 991 patients from the international PCD Cohort. We then assessed associations with age, sex, country, diagnostic certainty, organ laterality, body mass index and age at diagnosis in linear regression models. Lung function in PCD patients was reduced compared to reference values in both sexes and all age groups. Children aged 6-9 years had the smallest impairment (FEV1 z-score -0.84 (-1.03 to -0.65), FVC z-score -0.31 (-0.51 to -0.11)). Compared to CF patients, FEV1 was similarly reduced in children (age 6-9 years PCD 91% (88-93%); CF 90% (88-91%)), but less impaired in young adults (age 18-21 years PCD 79% (76-82%); CF 66% (65-68%)). The results suggest that PCD affects lung function from early in life, which emphasises the importance of early standardised care for all patients.

Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia.

RATIONALE: The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the United Kingdom 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 biallelic disease-causing mutations is also diagnostic, but many disease-causing genes are unknown, and testing is not widely available outside the United States. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. OBJECTIVES: To determine utility of a panel of six fluorescent labeled antibodies as a diagnostic tool for PCD. METHODS: The study used immunofluorescent labeling 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 with diagnostic outcome. MEASUREMENTS AND MAIN RESULTS: Immunofluorescence correctly identified mislocalized 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. In addition, immunofluorescence provided a result in 55% (39) of cases that were previously inconclusive. Immunofluorescence results were available within 14 days, costing $187 per sample compared with electron microscopy (27 days; cost $1,452). CONCLUSIONS: Immunofluorescence is a highly specific diagnostic test for PCD, and it improves the speed and availability of diagnostic testing. However, sensitivity is limited and immunofluorescence is not suitable as a stand-alone test.

European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia.

The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no "gold standard" reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia.

The international primary ciliary dyskinesia cohort (iPCD Cohort): methods and first results.

Data on primary ciliary dyskinesia (PCD) epidemiology is scarce and published studies are characterised by low numbers. In the framework of the European Union project BESTCILIA we aimed to combine all available datasets in a retrospective international PCD cohort (iPCD Cohort).We identified eligible datasets by performing a systematic review of published studies containing clinical information on PCD, and by contacting members of past and current European Respiratory Society Task Forces on PCD. We compared the contents of the datasets, clarified definitions and pooled them in a standardised format.As of April 2016 the iPCD Cohort includes data on 3013 patients from 18 countries. It includes data on diagnostic evaluations, symptoms, lung function, growth and treatments. Longitudinal data are currently available for 542 patients. The extent of clinical details per patient varies between centres. More than 50% of patients have a definite PCD diagnosis based on recent guidelines. Children aged 10-19 years are the largest age group, followed by younger children (≤9 years) and young adults (20-29 years).This is the largest observational PCD dataset available to date. It will allow us to answer pertinent questions on clinical phenotype, disease severity, prognosis and effect of treatments, and to investigate genotype-phenotype correlations.

Scoliosis convexity and organ anatomy are related.

PURPOSE: Primary ciliary dyskinesia (PCD) is a respiratory syndrome in which 'random' organ orientation can occur; with approximately 46% of patients developing situs inversus totalis at organogenesis. The aim of this study was to explore the relationship between organ anatomy and curve convexity by studying the prevalence and convexity of idiopathic scoliosis in PCD patients with and without situs inversus. METHODS: Chest radiographs of PCD patients were systematically screened for existence of significant lateral spinal deviation using the Cobb angle. Positive values represented right-sided convexity. Curve convexity and Cobb angles were compared between PCD patients with situs inversus and normal anatomy. RESULTS: A total of 198 PCD patients were screened. The prevalence of scoliosis (Cobb >10°) and significant spinal asymmetry (Cobb 5-10°) was 8 and 23%, respectively. Curve convexity and Cobb angle were significantly different within both groups between situs inversus patients and patients with normal anatomy (P ≤ 0.009). Moreover, curve convexity correlated significantly with organ orientation (P < 0.001; ϕ = 0.882): In 16 PCD patients with scoliosis (8 situs inversus and 8 normal anatomy), except for one case, matching of curve convexity and orientation of organ anatomy was observed: convexity of the curve was opposite to organ orientation. CONCLUSIONS: This study supports our hypothesis on the correlation between organ anatomy and curve convexity in scoliosis: the convexity of the thoracic curve is predominantly to the right in PCD patients that were 'randomized' to normal organ anatomy and to the left in patients with situs inversus totalis.

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.