Hereditary Mucin Deficiency Caused by Biallelic Loss of Function of MUC5B.

Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.

Lower Airway Nitrogen Oxide Levels in Children with Primary Ciliary Dyskinesia Is Linked to Neutrophilic Inflammation.

Treatment of primary ciliary dyskinesia pulmonary exacerbations resulted in an increase in sputum nitric oxide (NO) metabolites and decrease in neutrophilic inflammation. The association between the 2 suggests that neutrophilic inflammation contributes to airway NO deficiency in primary ciliary dyskinesia and that reducing inflammation may lead to improved airway NO homeostasis. TRIAL REGISTRY: ClinicalTrials.gov: NCT01155115.

Does early life exposure to exogenous sources of reactive oxygen species (ROS) increase the risk of respiratory and allergic diseases in children? A longitudinal cohort study.

BACKGROUND: Excess reactive oxygen species (ROS) can cause oxidative stress damaging cells and tissues, leading to adverse health effects in the respiratory tract. Yet, few human epidemiological studies have quantified the adverse effect of early life exposure to ROS on child health. Thus, this study aimed to examine the association of levels of ROS exposure at birth and the subsequent risk of developing common respiratory and allergic diseases in children. METHODS: 1,284 Toronto Child Health Evaluation Questionnaire (T-CHEQ) participants were followed from birth (born between 1996 and 2000) until outcome, March 31, 2016 or loss-to-follow-up. Using ROS data from air monitoring campaigns and land use data in Toronto, ROS concentrations generated in the human respiratory tract in response to inhaled pollutants were estimated using a kinetic multi-layer model. These ROS values were assigned to participants' postal codes at birth. Cox proportional hazards regression models, adjusted for confounders, were then used to estimate hazard ratios (HR) with 95% confidence intervals (CI) per unit increase in interquartile range (IQR). RESULTS: After adjusting for confounders, iron (Fe) and copper (Cu) were not significantly associated with the risk of asthma, allergic rhinitis, nor eczema. However, ROS, a measure of the combined impacts of Fe and Cu in PM2.5, was associated with an increased risk of asthma (HR = 1.11, 95% CI: 1.02-1.21, p < 0.02) per IQR. There were no statistically significant associations of ROS with allergic rhinitis (HR = 0.96, 95% CI: 0.88-1.04, p = 0.35) and eczema (HR = 1.03, 95% CI: 0.98-1.09, p = 0.24). CONCLUSION: These findings showed that ROS exposure in early life significantly increased the childhood risk of asthma, but not allergic rhinitis and eczema.

Does exposure to air pollution increase the risk of acute care in young children with asthma? An Ontario, Canada study.

Owing to their greater outdoor activity and ongoing lung development, children are particularly vulnerable to the harmful effects of exposure to fine particulate matter (PM2.5). However, the effects of PM2.5 components are poorly understood. This study aimed to use a longitudinal birth cohort of children with physician-diagnosed incident asthma to investigate the effect of PM2.5 components at birth on morbidity measured by health services utilization. Of 1277 Toronto Child Health Evaluation Questionnaire (T-CHEQ) participants, the study population included 362 children diagnosed with asthma who were followed for a mean of 13 years from birth until March 31, 2016, or loss-to-follow-up. Concentrations of PM2.5 and its components were assigned based on participants' postal codes at birth. Study outcomes included counts of asthma, asthma-related, and all-cause health services use. Poisson regression in single-, two-, and multi-pollutant models was used to estimate rate ratios (RR) per interquartile range (IQR) increase of exposures. Covariates were included in all models to further adjust for potential confounding. The adjusted RR for sulfate (SO4) and all-cause hospitalizations was statistically significant with RR = 2.23 (95% confidence interval [CI]: 1.25-3.96) in a multi-pollutant model with nitrogen dioxide (NO2) and ozone (O3). In multi-pollutant models with oxidants, the adjusted RRs for SO4 of all-cause hospitalizations and emergency department (ED) visits were also statistically significant with RR = 2.31 (95% CI: 1.32-4.03) and RR = 1.39 (95% CI: 1.02-1.90), respectively. While unadjusted single-pollutant RRs for asthma-specific and asthma-related health services use with the SO4 component of PM2.5 were above one, none were statistically significant. This study found significant associations with exposure to SO4 in PM2.5 and all-cause acute care, chiefly for hospitalizations, in children with asthma.

Effect of asthma exacerbation during pregnancy in women with asthma: a population-based cohort study.

The association between asthma exacerbation during pregnancy and adverse maternal and child health outcomes have not been investigated appropriately. Our objective was to determine the short- and long-term intergenerational effect of asthma exacerbation in pregnant women with asthma.A population cohort study was conducted using data from the Ontario asthma surveillance system and population-level health administrative data. Asthma exacerbation in pregnant women with asthma was defined as at least one of the following criteria: at least five physician visits, or one emergency department visit or one hospital admission for asthma during pregnancy. Pregnancy complications, adverse perinatal outcomes and early childhood respiratory disorders were identified using International Classification of Disease codes (9th and 10th revisions).The cohort consisted of 103 424 singleton pregnancies in women with asthma. Asthma exacerbation in pregnant women with asthma was associated with higher odds of pre-eclampsia (OR 1.30, 95% CI 1.12-1.51) and pregnancy-induced hypertension (OR 1.17, 95% CI 1.02-1.33); babies had higher odds of low birthweight (OR 1.14, 95% CI 1.00-1.31), preterm birth (OR 1.14, 95% CI 1.01-1.29) and congenital malformations (OR 1.21, 95% CI 1.05-1.39). Children born to women with asthma exacerbation during pregnancy had elevated risk of asthma (OR 1.23, 95% CI 1.13-1.33) and pneumonia (OR 1.12, 95% CI 1.03-1.22) during the first 5 years of life.Asthma exacerbation during pregnancy in women with asthma showed increased risk of pregnancy complications, adverse perinatal outcomes and early childhood respiratory disorders in their children, indicating that appropriate asthma management may reduce the risk of adverse health outcomes.

Early life exposure to air pollution and incidence of childhood asthma, allergic rhinitis and eczema.

RATIONALE: There is growing evidence that air pollution may contribute to the development of childhood asthma and other allergic diseases. In this follow-up of the Toronto Child Health Evaluation Questionnaire (T-CHEQ) study, we examined associations between early life exposures to air pollution and incidence of asthma, allergic rhinitis and eczema from birth through adolescence. METHODS: 1286 T-CHEQ participants were followed from birth until outcome (March 31, 2016) or loss to follow-up, with a mean of 17 years of follow-up. Concentrations of nitrogen dioxide (NO2), ozone (O3) and particulate matter with a 50% cut-off aerodynamic diameter of 2.5 µm (PM2.5) from January 1, 1999 to December 31, 2012 were assigned to participants based on their postal codes at birth using ground observations, chemical/meteorological models, remote sensing and land-use regression models. Study outcomes included incidence of physician-diagnosed asthma, allergic rhinitis and eczema. Cox proportional hazard regression models were used to estimate hazard ratios per interquartile range of exposures and outcomes, adjusting for potential confounders. RESULTS: Hazard ratios of 1.17 (95% CI 1.05-1.31) for asthma and 1.07 (95% CI 0.99-1.15) for eczema were observed for total oxidants (O3 and NO2) at birth. No significant increase in risk was found for PM2.5. CONCLUSIONS: Exposures to oxidant air pollutants (O3 and NO2) but not PM2.5 were associated with an increased risk of incident asthma and eczema in children. This suggests that improving air quality may contribute to the prevention of asthma and other allergic disease in childhood and adolescence.

Primary Ciliary Dyskinesia: Longitudinal Study of Lung Disease by Ultrastructure Defect and Genotype.

RATIONALE: In primary ciliary dyskinesia, factors leading to disease heterogeneity are poorly understood. OBJECTIVES: To describe early lung disease progression in primary ciliary dyskinesia and identify associations between ultrastructural defects and genotypes with clinical phenotype. METHODS: This was a prospective, longitudinal (5 yr), multicenter, observational study. Inclusion criteria were less than 19 years at enrollment and greater than or equal to two annual study visits. Linear mixed effects models including random slope and random intercept were used to evaluate longitudinal associations between the ciliary defect group (or genotype group) and clinical features (percent predicted FEV1 and weight and height z-scores). MEASUREMENTS AND MAIN RESULTS: A total of 137 participants completed 732 visits. The group with absent inner dynein arm, central apparatus defects, and microtubular disorganization (IDA/CA/MTD) (n = 41) were significantly younger at diagnosis and in mixed effects models had significantly lower percent predicted FEV1 and weight and height z-scores than the isolated outer dynein arm defect (n = 55) group. Participants with CCDC39 or CCDC40 mutations (n = 34) had lower percent predicted FEV1 and weight and height z-scores than those with DNAH5 mutations (n = 36). For the entire cohort, percent predicted FEV1 decline was heterogeneous with a mean (SE) decline of 0.57 (0.25) percent predicted/yr. Rate of decline was different from zero only in the IDA/MTD/CA group (mean [SE], -1.11 [0.48] percent predicted/yr; P = 0.02). CONCLUSIONS: Participants with IDA/MTD/CA defects, which included individuals with CCDC39 or CCDC40 mutations, had worse lung function and growth indices compared with those with outer dynein arm defects and DNAH5 mutations, respectively. The only group with a significant lung function decline over time were participants with IDA/MTD/CA defects.

Procedure volume and mortality after surgical lung biopsy in interstitial lung disease.

Surgical volume-outcome relationships are well established but have not been studied in patients with interstitial lung disease (ILD) undergoing surgical lung biopsy (SLB). Our study objective was to determine if hospital SLB volume is associated with post-operative mortality in patients with ILD.A cohort study using administrative, population-based data from Ontario, Canada was performed in adults with ILD who underwent a SLB between 2001 and 2014. The association between yearly hospital SLB volume and 30-day post-operative mortality was assessed using multilevel logistic regression modelling.3057 surgical lung biopsies for ILD were performed during the study period with a median (interquartile range) yearly hospital volume of 73 (34-143) procedures. 30-day mortality was 7.1%, 20.2% and 1.9% in overall, nonelective and elective patients, respectively. Higher yearly hospital SLB volume was associated with lower odds of 30-day post-operative mortality after adjusting for patient characteristics (OR 0.84, 95% CI 0.73-0.97; p=0.02), with the association appearing stronger for nonelective versus elective procedures (OR 0.84, 95% CI 0.69-1.02; p=0.08 versus OR 0.94, 95% CI 0.74-1.18; p=0.57).Higher yearly hospital SLB volume was associated with lower post-operative mortality in patients with ILD, with the association appearing to be mainly driven by nonelective cases. SLB mortality was significantly higher for nonelective cases.

Pediatric asthma: An unmet need for more effective, focused treatments.

BACKGROUND: Despite remarkable advances in our understanding of asthma, there are still several unmet needs associated with the management of pediatric asthma. METHODS: A two-day, face-to-face meeting was held in London, United Kingdom, on October 28 and 29, 2017, involving a group of international expert clinicians and scientists in asthma management to discuss the challenges and unmet needs that remain to be addressed in pediatric asthma. RESULTS: These unmet needs include a lack of clinical efficacy and safety evidence, and limited availability of non-steroid-based alternative therapies in patients <6 years of age. An increased focus on children is needed in the context of clinical practice guidelines for asthma; current pediatric practice relies mostly on extrapolations from adult recommendations. Furthermore, no uniform definition of pediatric asthma exists, which hampers timely and robust diagnosis of the condition in affected patients. CONCLUSIONS: There is a need for a uniform definition of pediatric asthma, clearly distinguishable from adult asthma. Furthermore, guidelines which provide specific treatment recommendations for the management of pediatric asthma are also needed. Clinical trials and real-world evidence studies assessing anti-immunoglobulin E (IgE) therapies and other monoclonal antibodies in children <6 years of age with asthma may provide further information regarding the most appropriate treatment options in these vulnerable patients. Early intervention with anti-IgE and non-steroid-based alternative therapies may delay disease progression, leading to improved clinical outcomes.

Diagnosis of Primary Ciliary Dyskinesia. An Official American Thoracic Society Clinical Practice Guideline.

BACKGROUND: This document presents the American Thoracic Society clinical practice guidelines for the diagnosis of primary ciliary dyskinesia (PCD). TARGET AUDIENCE: Clinicians investigating adult and pediatric patients for possible PCD. METHODS: Systematic reviews and, when appropriate, meta-analyses were conducted to summarize all available evidence pertinent to our clinical questions. Evidence was assessed using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach for diagnosis and discussed by a multidisciplinary panel with expertise in PCD. Predetermined conflict-of-interest management strategies were applied, and recommendations were formulated, written, and graded exclusively by the nonconflicted panelists. Three conflicted individuals were also prohibited from writing, editing, or providing feedback on the relevant sections of the manuscript. RESULTS: After considering diagnostic test accuracy, confidence in the estimates for each diagnostic test, relative importance of test results studied, desirable and undesirable direct consequences of each diagnostic test, downstream consequences of each diagnostic test result, patient values and preferences, costs, feasibility, acceptability, and implications for health equity, the panel made recommendations for or against the use of specific diagnostic tests as compared with using the current reference standard (transmission electron microscopy and/or genetic testing) for the diagnosis of PCD. CONCLUSIONS: The panel formulated and provided a rationale for the direction as well as for the strength of each recommendation to establish the diagnosis of PCD.

Understanding parent perceptions of healthy physical activity for their child with a chronic medical condition: A cross-sectional study.

OBJECTIVE: We sought to determine the prevalence of, and identify factors associated with, parent questions about physical activity for their child with a chronic cardiac, respiratory, or rheumatologic condition. METHODS: This cross-sectional study of 56 children (32 [57%] female), 3 to 18 years of age, with chronic cardiac (n=21), respiratory (n=18), or rheumatologic (n=17) conditions involved a parent questionnaire about their child's physical activity participation and measurement of the child's activity with an omni-directional accelerometer for 1 week. RESULTS: Parents of 20 (36%) children had at least one question about their child's physical activity participation, and the prevalence of questions did not vary by age (Wald chi square = 0.77, P=0.38), gender (Wald chi square = 0.11, P=0.74), or clinic (Wald chi square = 1.77, P=0.41). Parent questions were associated (P = 0.04) with lower levels of activity for boys (95% confidence interval [CI] for estimated marginal means: With questions: 197, 395; Without questions: 346, 500) and higher levels of activity for girls (95% CI for estimated marginal means: With questions: 268, 448; Without questions: 239, 369). A multivariable logistic regression model found that parents with questions had higher odds of having a child who was less well (odds ratio [OR]=19.9 for unwell, OR=5.6 for generally well with some symptoms versus well and asymptomatic) and had a history of cardiac arrhythmia (OR=7.6). CONCLUSIONS: Over one-third of parents reported having questions about physical activity for their child with a chronic medical condition, suggesting substantial uncertainty even among children reported as active. Presence of parent uncertainty is associated with parent reports of the child being unwell or a history of cardiac arrhythmia. By asking parents if they have questions about their child's activity, health care professionals may be better able to identify inactive boys and overprotective attitudes toward active girls.

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

Type F Infant Botulism: Investigation of Recent Clusters and Overview of This Exceedingly Rare Disease.

From 1976 to 2016, neurotoxigenic Clostridium baratii type F caused 18 (<0.5%) reported US infant botulism cases. Six cases occurred during 2012-2013; no common source was identified. Type F infant botulism mostly occurs in very young infants and typically presents more rapidly and severely than illness caused by types A and B botulinum neurotoxin.

Validation of a health-related quality of life instrument for primary ciliary dyskinesia (QOL-PCD).

BACKGROUND: Quality of life (QOL)-primary ciliary dyskinesia (PCD) is the first disease-specific, health-related QOL instrument for PCD. Psychometric validation of QOL-PCD assesses the performance of this measure in adults, including its reliability, validity and responsiveness to change. METHODS: Seventy-two adults (mean (range) age: 33 years (18-79 years); mean (range) FEV1% predicted: 68 (26-115)) with PCD completed the 49-item QOL-PCD and generic QOL measures: Short-Form 36 Health Survey, Sino-Nasal Outcome Test 20 (SNOT-20) and St George Respiratory Questionnaire (SGRQ)-C. Thirty-five participants repeated QOL-PCD 10-14 days later to measure stability or reproducibility of the measure. RESULTS: Multitrait analysis was used to evaluate how the items loaded on 10 hypothesised scales: physical, emotional, role and social functioning, treatment burden, vitality, health perceptions, upper respiratory symptoms, lower respiratory symptoms and ears and hearing symptoms. This analysis of item-to-total correlations led to 9 items being dropped; the validated measure now comprises 40 items. Each scale had excellent internal consistency (Cronbach's α: 0.74 to 0.94). Two-week test-retest demonstrated stability for all scales (intraclass coefficients 0.73 to 0.96). Significant correlations were obtained between QOL-PCD scores and age and FEV1. Strong relationships were also found between QOL-PCD scales and similar constructs on generic questionnaires, for example, lower respiratory symptoms and SGRQ-C (r=0.72, p<0.001), while weak correlations were found between measures of different constructs. CONCLUSIONS: QOL-PCD has demonstrated good internal consistency, test-retest reliability, convergent and divergent validity. QOL-PCD offers a promising tool for evaluating new therapies and for measuring symptoms, functioning and QOL during routine care.

Growth and nutritional status, and their association with lung function: a study from the international Primary Ciliary Dyskinesia Cohort.

Chronic respiratory disease can affect growth and nutrition, which can influence lung function. We investigated height, body mass index (BMI), and lung function in patients with primary ciliary dyskinesia (PCD).In this study, based on the international PCD (iPCD) Cohort, we calculated z-scores for height and BMI using World Health Organization (WHO) and national growth references, and assessed associations with age, sex, country, diagnostic certainty, age at diagnosis, organ laterality and lung function in multilevel regression models that accounted for repeated measurements.We analysed 6402 measurements from 1609 iPCD Cohort patients. Height was reduced compared to WHO (z-score -0.12, 95% CI -0.17 to -0.06) and national references (z-score -0.27, 95% CI -0.33 to -0.21) in male and female patients in all age groups, with variation between countries. Height and BMI were higher in patients diagnosed earlier in life (p=0.026 and p<0.001, respectively) and closely associated with forced expiratory volume in 1 s and forced vital capacity z-scores (p<0.001).Our study indicates that both growth and nutrition are affected adversely in PCD patients from early life and are both strongly associated with lung function. If supported by longitudinal studies, these findings suggest that early diagnosis with multidisciplinary management and nutritional advice could improve growth and delay disease progression and lung function impairment in PCD.

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.

Exome sequencing identifies mutations in CCDC114 as a cause of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ~60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.

Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.