SummaryPrimary ciliary dyskinesia (PCD) is a genetically heterogeneous, rare lung disease resulting in chronic oto‐sino‐pulmonary disease in both children and adults. Many physicians incorrectly diagnose PCD or eliminate PCD from their differential diagnosis due to inexperience with diagnostic testing methods. Thus far, all therapies used for PCD are unproven through large clinical trials. This review article outlines consensus recommendations from PCD physicians in North America who have been engaged in a PCD centered research consortium for the last 10 years. These recommendations have been adopted by the governing board of the PCD Foundation to provide guidance for PCD clinical centers for diagnostic testing, monitoring, and appropriate short and long‐term therapeutics in PCD patients. Pediatr Pulmonol. 2016;51:115–132. © 2015 The Authors. Pediatric Pulmonology Published by Wiley Periodicals, Inc.

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be ␤-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (DNAH5) or intermediate (DNAI1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for PCD is available for the most common mutations. The respiratory manifestations of PCD (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of PCD patients have laterality defects (including situs inversus totalis and, less commonly, heterotaxy and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most PCD patients have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with PCD.

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.

The Forum of International Respiratory Societies has released a report entitled Respiratory Disease in the World: Realities of Today-Opportunities for Tomorrow. The report identifies five conditions that primarily contribute to the global burden of respiratory disease (asthma, chronic obstructive pulmonary disease, acute respiratory infections, tuberculosis, and lung cancer), and offers an action plan to prevent and treat those diseases. It describes the staggering magnitude of the global burden of lung disease: hundreds of millions of people suffer and four million people die prematurely from respiratory diseases each year. The situation is not hopeless, because most major respiratory illnesses are avoidable. Much of the disease burden can be mitigated by reducing exposure to indoor and outdoor air pollution, restraining tobacco use, and relieving urban overcrowding. Implementation of the strategies described in the Forum of International Respiratory Societies respiratory diseases report would have a profound effect on respiratory health, reduce economic costs, and enhance health equality in the world.

Rationale: 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.Objectives: To use a standard protocol for measuring nNO to establish a diseasespecific cutoff value at one site, and then validate at six other sites.Methods: 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. Measurements and Main Results:At the lead site, nNO values in PCD (mean 6 standard deviation, 20.7 6 24.1 nl/min; range, 1.5-207.3 nl/min) only rarely overlapped with the nNO values of healthy control subjects (304.6 6 118.8; 125.5-867.0 nl/min), asthma (267.8 6 103.2; 125.0-589.7 nl/min), or chronic obstructive pulmonary disease (223.7 6 87.1; 109.7-449.1 nl/min); however, there was overlap with cystic fibrosis (134.0 6 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.Conclusions: 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.

Rationale: The relationship between clinical phenotype of childhood primary ciliary dyskinesia (PCD) and ultrastructural defects and genotype is poorly defined.Objectives: To delineate clinical features of childhood PCD and their associations with ultrastructural defects and genotype.Methods: 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.Measurements and Main Results: 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 1 IDA; n = 18) versus subjects with IDA and central apparatus defects with microtubular disorganization (IDA/ CA/MTD; n = 40). Median FEV 1 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.Conclusions: 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.

Receptor-mediated gene transfer has been used to introduce genes into tissues ofanimals in vivo. The genes introduced by this approach have been transiently expressed at low levels in animal tissues. Hi levels ofexpression, for longer periods, have been attaine by the induction of cell division (i.e., partial hepatectomy) or disruption of lysosomal degradation of the DNA. We have studied the correlation of specific structural features on the DNA/ligand complexes with their ability to efficiently introduce DNA into the livers of intact animals. A chimeric gene contain the phosphoenolpyruvate carboxykinase gene promoter (nucleotides -460 to +73) linked to the structural gene for human factor IX (PEPCK-hFIX gene) was condensed with galctlated poly(L-lysine) by titration with NaCl, resulting in complexes of defined size (10-12 nm in diameter) and shape. The PEPCK-hFIX gene complex was injected into the caudal vena cava ofadult rats and the conjugated DNA was specifically targeted to the livers ofthe animals; no detectable DNA was noted in other tissues. The plaid containing the PEPCK-hFIX gene was found as an episome in the livers of the rats 32 days after inection of the DNA complex. Human factor IX DNA, mRNA, and functional protein were deed up to 140 days after administration ofthe DNA complex (the duration of the experiment). Transcription from the PEPCK promoter could be induced over the entire course of the experiment by feeding the rats a high-protein, carbohydrate-free diet. We conclude that the structure of the DNA/ligand complexes is of key importance for the successful introduction of genes into the tissues of animals by receptormediated endocytosis.