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The relationship between clinical phenotype of childhood primary ciliary dyskinesia (PCD) and ultrastructural defects and genotype is poorly defined. To delineate clinical features of childhood PCD and their associations with ultrastructural defects and genotype. A total of 118 participants younger than 19 years old with PCD were evaluated prospectively at six centers in North America using standardized procedures for diagnostic testing, spirometry, chest computed tomography, respiratory cultures, and clinical phenotyping. Clinical features included neonatal respiratory distress (82%), chronic cough (99%), and chronic nasal congestion (97%). There were no differences in clinical features or respiratory pathogens in subjects with outer dynein arm (ODA) defects (ODA alone; n = 54) and ODA plus inner dynein arm (IDA) defects (ODA + IDA; n = 18) versus subjects with IDA and central apparatus defects with microtubular disorganization (IDA/CA/MTD; n = 40). Median FEV1 was worse in the IDA/CA/MTD group (72% predicted) versus the combined ODA groups (92% predicted; P = 0.003). Median body mass index was lower in the IDA/CA/MTD group (46th percentile) versus the ODA groups (70th percentile; P = 0.003). For all 118 subjects, median number of lobes with bronchiectasis was three and alveolar consolidation was two. However, the 5- to 11-year-old IDA/CA/MTD group had more lobes of bronchiectasis (median, 5; P = 0.0008) and consolidation (median, 3; P = 0.0001) compared with the ODA groups (median, 3 and 2, respectively). Similar findings were observed when limited to participants with biallelic mutations. Lung disease was heterogeneous across all ultrastructural and genotype groups, but worse in those with IDA/CA/MTD ultrastructural defects, most of whom had biallelic mutations in CCDC39 or CCDC40.

Abstract Rationale Primary ciliary dyskinesia (PCD), a genetically heterogeneous, recessive disorder of motile cilia, is associated with distinct clinical features. Diagnostic tests, including ultrastructural analysis of cilia, nasal nitric oxide measurements, and molecular testing for mutations in PCD genes, have inherent limitations. Objectives To define a statistically valid combination of systematically defined clinical features that strongly associates with PCD in children and adolescents. Methods Investigators at seven North American sites in the Genetic Disorders of Mucociliary Clearance Consortium prospectively and systematically assessed individuals (aged 0–18 yr) referred due to high suspicion for PCD. The investigators defined specific clinical questions for the clinical report form based on expert opinion. Diagnostic testing was performed using standardized protocols and included nasal nitric oxide measurement, ciliary biopsy for ultrastructural analysis of cilia, and molecular genetic testing for PCD-associated genes. Final diagnoses were assigned as “definite PCD” (hallmark ultrastructural defects and/or two mutations in a PCD-associated gene), “probable/possible PCD” (no ultrastructural defect or genetic diagnosis, but compatible clinical features and nasal nitric oxide level in PCD range), and “other diagnosis or undefined.” Criteria were developed to define early childhood clinical features on the basis of responses to multiple specific queries. Each defined feature was tested by logistic regression. Sensitivity and specificity analyses were conducted to define the most robust set of clinical features associated with PCD. Measurements and Main Results: From 534 participants 18 years of age and younger, 205 were identified as having “definite PCD” (including 164 with two mutations in a PCD-associated gene), 187 were categorized as “other diagnosis or undefined,” and 142 were defined as having “probable/possible PCD.” Participants with “definite PCD” were compared with the “other diagnosis or undefined” group. Four criteria-defined clinical features were statistically predictive of PCD: laterality defect; unexplained neonatal respiratory distress; early-onset, year-round nasal congestion; and early-onset, year-round wet cough (adjusted odds ratios of 7.7, 6.6, 3.4, and 3.1, respectively). The sensitivity and specificity based on the number of criteria-defined clinical features were four features, 0.21 and 0.99, respectively; three features, 0.50 and 0.96, respectively; and two features, 0.80 and 0.72, respectively. Conclusions Systematically defined early clinical features could help identify children, including infants, likely to have PCD. Clinical trial registered with ClinicalTrials.gov (NCT00323167).

Several studies suggest that nasal nitric oxide (nNO) measurement could be a test for primary ciliary dyskinesia (PCD), but the procedure and interpretation have not been standardized. To use a standard protocol for measuring nNO to establish a disease-specific cutoff value at one site, and then validate at six other sites. At the lead site, nNO was prospectively measured in individuals later confirmed to have PCD by ciliary ultrastructural defects (n = 143) or DNAH11 mutations (n = 6); and in 78 healthy and 146 disease control subjects, including individuals with asthma (n = 37), cystic fibrosis (n = 77), and chronic obstructive pulmonary disease (n = 32). A disease-specific cutoff value was determined, using generalized estimating equations (GEEs). Six other sites prospectively measured nNO in 155 consecutive individuals enrolled for evaluation for possible PCD. At the lead site, nNO values in PCD (mean ± standard deviation, 20.7 ± 24.1 nl/min; range, 1.5-207.3 nl/min) only rarely overlapped with the nNO values of healthy control subjects (304.6 ± 118.8; 125.5-867.0 nl/min), asthma (267.8 ± 103.2; 125.0-589.7 nl/min), or chronic obstructive pulmonary disease (223.7 ± 87.1; 109.7-449.1 nl/min); however, there was overlap with cystic fibrosis (134.0 ± 73.5; 15.6-386.1 nl/min). The disease-specific nNO cutoff value was defined at 77 nl/minute (sensitivity, 0.98; specificity, >0.999). At six other sites, this cutoff identified 70 of the 71 (98.6%) participants with confirmed PCD. Using a standardized protocol in multicenter studies, nNO measurement accurately identifies individuals with PCD, and supports its usefulness as a test to support the clinical diagnosis of PCD.

Heymut Omran, Joseph LoTurco and colleagues show that mutations in the dyslexia susceptibility candidate gene DYX1C1 cause primary ciliary dyskinesia. Their functional studies suggest that DYX1C1 is required for the cytoplasmic preassembly of axonemal dynein complexes. DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N -ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

RationalePrimary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11).ObjectivesTo test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology.Methods82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease.ResultsOf the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations.ConclusionsMutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.

Abstract Rationale Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD. Objectives To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD. Methods Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis. Measurements and Main Results We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern. Conclusions The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

Abstract Smokers are more susceptible to respiratory viral infections, including influenza virus, but the mechanisms mediating this effect are unknown. To determine how epithelial cells contribute to the enhanced susceptibility seen in smokers, we established an in vitro model of differentiated nasal epithelial cells (NECs) from smokers, which showed enhanced mucin expression. The NECs from smokers responded to influenza infection with greater cytotoxicity, release of interleukin-6, and viral shedding than NECs from nonsmokers. Focusing on type I interferon (IFN) expression, we observed that influenza-infected NECs from smokers produced significantly less IFN-α than NECs from nonsmokers. Similarly, the expression of IRF7, a key transcription factor controlling the expression of IFN-α, was significantly decreased in influenza-infected and IFN-β–stimulated NECs from smokers. Furthermore, our data indicate that the DNA methylation of the IRF7 gene and expression of the DNA (cytosine-5-)-methyltransferase 1 was enhanced in NECs from smokers. To confirm these findings in vivo, we initiated a study in which smoking and nonsmoking healthy volunteers were inoculated nasally with the live-attenuated influenza virus (LAIV) vaccine, and nasal biopsies were obtained before and after the administration of LAIV. The LAIV-induced expression of IRF7 was lower in the nasal epithelium from smokers, supporting our in vitro observations. These data demonstrate that infection with influenza results in the reduced expression of transcription factor IRF7 in NECs from smokers, and that these effects may be mediated by an epigenetic modification of the IRF7 gene, thus providing a potential mechanism rendering smokers more susceptible to respiratory virus infections.

Abstract Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder caused by abnormal ciliary ultrastructure and function, characterized clinically by oto-sino-pulmonary disease. Mutations in an intermediate chain dynein (DNAI1; IC78) have recently been described in PCD patients, with outer dynein arm (ODA) defects. The aims of the current study were to test for novel DNAI1 mutations in 13 PCD patients with ODA defects (from 7 unrelated families) and to assess genotype/phenotype correlations in patients and family members. A previously reported mutation (219 + 3insT) was detected in three PCD patients from two families. The opposite allele had the novel missense mutation G1874C (W568S) in both affected individuals from one family, and a nonsense mutation G1875A (W568X) in an affected individual from another family. The tryptophan at position 568 is a highly conserved residue in the WD-repeat region, and a mutation is predicted to lead to abnormal folding of the protein and loss of function. None of these mutations were found in 32 other PCD patients with miscellaneous ciliary defects. Mutations in DNAI1 are causative for PCD with ODA defects, and are likely the genetic origin of clinical disease in some PCD patients with ultrastructural defects in the ODA.

The diversity and extent of signaling functions of nitric oxide (NO) in cell physiology as well as its presence and influence as a common component of ambient air pollution and tobacco smoke are gaining increasing research attention relative to both health and disease. While cellular NO production is typically associated with inflammatory cells and processes, the airway epithelium particularly of the paranasal sinuses, has been documented to be a rich source of excreted NO. Inasmuch as excreted NO derives from both mucosal and inflammatory cell sources, distinguishing the individual contribution of these compartments to total excreted cellular NO is potentially problematic. We simulated an inflammatory mucosal environment by stimulating human nasal epithelial cultures with interleukin-13 (IL-13), a mediator produced by eosinophils in asthma, allergic rhinitis, and sinusitis. While a consistent baseline of NO excretion in control cultures was documented, widely variable individual responses to IL-13 exposure were observed in companion cultures maintained under identical conditions and tested at the same time. These studies suggest that cellular NO excretion by the healthy epithelial mucosa is subject to considerable individual variability and may be significantly elevated among some individuals in the presence of IL-13 stimulation.

Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive, genetic disorder characterized by ciliary dysfunction, sino-pulmonary disease, and situs inversus. Disease-causing mutations have been reported in DNAI1 and DNAH5 encoding outer dynein arm (ODA) proteins of cilia. We analyzed DNAI1 to identify disease-causing mutations in PCD and to determine if the previously reported IVS1+2_3insT (219+3insT) mutation represents a \"founder\" or \"hot spot\" mutation. Patients with PCD from 179 unrelated families were studied. Exclusion mapping showed no linkage to DNAI1 for 13 families; the entire coding region was sequenced in a patient from the remaining 166 families. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on nasal epithelial RNA in 14 families. Mutations in DNAI1 including 12 novel mutations were identified in 16 of 179 (9%) families; 14 harbored biallelic mutations. Deep intronic splice mutations were not identified by reverse transcriptase-polymerase chain reaction. The prevalence of mutations in families with defined ODA defect was 13%; no mutations were found in patients without a defined ODA defect. The previously reported IVS1+2_3insT mutation accounted for 57% (17/30) of mutant alleles, and marker analysis indicates a common founder for this mutation. Seven mutations occurred in three exons (13, 16, and 17); taken together with previous reports, these three exons are emerging as mutation clusters harboring 29% (12/42) of mutant alleles. A total of 10% of patients with PCD are estimated to harbor mutations in DNAI1; most occur as a common founder IVS1+2_3insT or in exons 13, 16, and 17. This information is useful for establishing a clinical molecular genetic test for PCD.