Do Ambient Ozone or Other Pollutants Modify Effects of Controlled Ozone Exposure on Pulmonary Function?

Rationale: In a previous trial (MOSES [Multicenter Ozone Study of oldEr Subjects]), 3 hours of controlled ozone (O3) exposure caused concentration-related reductions in lung function with evidence of airway inflammation and injury, but without convincing evidence of effects on cardiovascular function. However, the subjects' exposures to indoor and outdoor air pollution in the hours and days before each controlled O3 exposure may have modified biomarker responses to the controlled O3 exposures.Objectives: We sought to determine whether personal measures of nitrogen dioxide (NO2) and O3, or ambient concentrations of O3, particulate matter ≤2.5 µm in aerodynamic diameter, NO2, carbon monoxide (CO), and sulfur dioxide (SO2) in the 72 and 96 hours before the exposure visit modified biomarker responses to controlled O3 exposure.Methods: MOSES subjects were exposed for 3 hours in random order to clean air containing 0 ppb O3, 70 ppb O3, or 120 ppm O3, alternating 15 minutes of moderate exercise with 15 minutes of rest. Cardiovascular and pulmonary endpoints (biomarkers of autonomic function, repolarization, ST segment change, arrhythmia, prothrombotic vascular status, systemic inflammation, vascular function, pulmonary function, oxidative stress, and lung injury) were measured on the day before, the day of, and up to 22 hours after each exposure. We evaluated whether ambient pollutant concentrations in the 96 hours before the pre-exposure visit modified pre- to post-exposure lung function biomarker responses to the controlled O3 exposures, using tertiles of passive personal exposure samplers (PES) of O3 and NO2, ambient air pollutant concentrations, and mixed effects linear regression. We also similarly explored the effect modification of controlled O3 effects on biomarkers of other MOSES outcome groups in the same way. Although we used P < 0.01 to define statistical significance, we did not formally correct for multiple comparisons.Results: The effects of MOSES controlled O3 exposures on forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were modified by ambient NO2 and CO, and PES NO2. Reductions in FEV1 and FVC were observed only when these concentrations were in the "medium" or "high" tertile in the 72 hours before the pre-exposure visit. There was no such modification of the effect of controlled O3 exposure on any other cardiopulmonary outcome group.Conclusions: Reductions in markers of lung function, but not other pathways, by the MOSES controlled O3 exposure were modified by ambient NO2 and CO, and PES NO2, and these reductions were observed only when these pollutant concentrations were elevated in the hours and days before the pre-exposure visit.Clinical trial registered with ClinicalTrials.gov (NCT01487005).

Nasal Nitric Oxide Measurement in Primary Ciliary Dyskinesia. A Technical Paper on Standardized Testing Protocols.

Nasal nitric oxide concentrations are extremely low in primary ciliary dyskinesia (PCD), and measurement of this nasal gas is recommended as a PCD diagnostic test in cooperative patients aged 5 years and older. However, nasal nitric oxide measurements must be performed with chemiluminescence analyzers using a standardized protocol to ensure proper results, because nasal nitric oxide values can be influenced by various internal and external factors. Repeat nasal nitric oxide testing on separate visits is required to ensure that low diagnostic values are persistent and consistent with PCD. This technical paper presents the standard operating procedures for nasal nitric oxide measurement used by the PCD Foundation Clinical and Research Centers Network at various specialty centers across North America. Adherence to this document ensures reliable nasal nitric oxide testing and high diagnostic accuracy when employed in a population with appropriate clinical phenotypes for PCD.

Ozone effects on blood biomarkers of systemic inflammation, oxidative stress, endothelial function, and thrombosis: The Multicenter Ozone Study in oldEr Subjects (MOSES).

The evidence that exposure to ozone air pollution causes acute cardiovascular effects is mixed. We postulated that exposure to ambient levels of ozone would increase blood markers of systemic inflammation, prothrombotic state, oxidative stress, and vascular dysfunction in healthy older subjects, and that absence of the glutathione S-transferase Mu 1 (GSTM1) gene would confer increased susceptibility. This double-blind, randomized, crossover study of 87 healthy volunteers 55-70 years of age was conducted at three sites using a common protocol. Subjects were exposed for 3 h in random order to 0 parts per billion (ppb) (filtered air), 70 ppb, and 120 ppb ozone, alternating 15 min of moderate exercise and rest. Blood was obtained the day before, approximately 4 h after, and approximately 22 h after each exposure. Linear mixed effect and logistic regression models evaluated the impact of exposure to ozone on pre-specified primary and secondary outcomes. The definition of statistical significance was p<0.01. There were no effects of ozone on the three primary markers of systemic inflammation and a prothrombotic state: C-reactive protein, monocyte-platelet conjugates, and microparticle-associated tissue factor activity. However, among the secondary endpoints, endothelin-1, a potent vasoconstrictor, increased from pre- to post-exposure with ozone concentration (120 vs 0 ppb: 0.07 pg/mL, 95% confidence interval [CI] 0.01, 0.14; 70 vs 0 ppb: -0.03 pg/mL, CI -0.09, 0.04; p = 0.008). Nitrotyrosine, a marker of oxidative and nitrosative stress, decreased with increasing ozone concentrations, with marginal significance (120 vs 0 ppb: -41.5, CI -70.1, -12.8; 70 vs 0 ppb: -14.2, CI -42.7, 14.2; p = 0.017). GSTM1 status did not modify the effect of ozone exposure on any of the outcomes. These findings from healthy older adults fail to identify any mechanistic basis for the epidemiologically described cardiovascular effects of exposure to ozone. The findings, however, may not be applicable to adults with cardiovascular disease.

Cardiovascular function and ozone exposure: The Multicenter Ozone Study in oldEr Subjects (MOSES).

BACKGROUND: To date, there have been relatively few studies of acute cardiovascular responses to controlled ozone inhalation, although a number of observational studies have reported significant positive associations between both ambient ozone levels and acute cardiovascular events and long-term ozone exposure and cardiovascular mortality. OBJECTIVES: We hypothesized that short-term controlled exposure to low levels of ozone in filtered air would induce autonomic imbalance, repolarization abnormalities, arrhythmia, and vascular dysfunction. METHODS: This randomized crossover study of 87 healthy volunteers 55-70 years of age was conducted at three sites using a common protocol, from June 2012 to April 2015. Subjects were exposed for 3 h in random order to 0 ppb (filtered air), 70 ppb ozone, and 120 ppb ozone, alternating 15 min of moderate exercise with 15 min of rest. A suite of cardiovascular endpoints was measured the day before, the day of, and up to 22 h after each exposure. Mixed effect linear and logit models evaluated the impact of exposure to ozone on pre-specified primary and secondary outcomes. Site and time were included in the models. RESULTS: We found no significant effects of ozone exposure on any of the primary or secondary measures of autonomic function, repolarization, ST segment change, arrhythmia, or vascular function (systolic blood pressure and flow-mediated dilation). CONCLUSIONS: In this multicenter study of older healthy women and men, there was no convincing evidence for acute effects of 3-h, relatively low-level ozone exposures on cardiovascular function. However, we cannot exclude the possibility of effects with higher ozone concentrations, more prolonged exposure, or in subjects with underlying cardiovascular disease. Further, we cannot exclude the possibility that exposure to ambient ozone and other pollutants in the days before the experimental exposures obscured or blunted cardiovascular biomarker response to the controlled ozone exposures.

Interleukin-13 stimulates production of nitric oxide in cultured human nasal epithelium.

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.

Respiratory Responses to Ozone Exposure. MOSES (The Multicenter Ozone Study in Older Subjects).

RATIONALE: Acute respiratory effects of low-level ozone exposure are not well defined in older adults. OBJECTIVES: MOSES (The Multicenter Ozone Study in Older Subjects), although primarily focused on acute cardiovascular effects, provided an opportunity to assess respiratory responses to low concentrations of ozone in older healthy adults. METHODS: We performed a randomized crossover, controlled exposure study of 87 healthy adults (59.9 ± 4.5 yr old; 60% female) to 0, 70, and 120 ppb ozone for 3 hours with intermittent exercise. Outcome measures included spirometry, sputum markers of airway inflammation, and plasma club cell protein-16 (CC16), a marker of airway epithelial injury. The effects of ozone exposure on these outcomes were evaluated with mixed-effect linear models. A P value less than 0.01 was chosen a priori to define statistical significance. MEASUREMENTS AND MAIN RESULTS: The mean (95% confidence interval) FEV1 and FVC increased from preexposure values by 2.7% (2.0-3.4) and 2.1% (1.3-2.9), respectively, 15 minutes after exposure to filtered air (0 ppb). Exposure to ozone reduced these increases in a concentration-dependent manner. After 120-ppb exposure, FEV1 and FVC decreased by 1.7% (1.1-2.3) and 0.8% (0.3-1.3), respectively. A similar concentration-dependent pattern was still discernible 22 hours after exposure. At 4 hours after exposure, plasma CC16 increased from preexposure levels in an ozone concentration-dependent manner. Sputum neutrophils obtained 22 hours after exposure showed a marginally significant increase in a concentration-dependent manner (P = 0.012), but proinflammatory cytokines (IL-6, IL-8, and tumor necrosis factor-α) were not significantly affected. CONCLUSIONS: Exposure to ozone at near ambient levels induced lung function effects, airway injury, and airway inflammation in older healthy adults. Clinical trial registered with www.clinicaltrials.gov (NCT01487005).

Clinical Features and Associated Likelihood of Primary Ciliary Dyskinesia in Children and Adolescents.

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).

Clinical features of childhood primary ciliary dyskinesia by genotype and ultrastructural phenotype.

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 + 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. 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.

Laterality defects other than situs inversus totalis in primary ciliary dyskinesia: insights into situs ambiguus and heterotaxy.

BACKGROUND: Motile cilia dysfunction causes primary ciliary dyskinesia (PCD), situs inversus totalis (SI), and a spectrum of laterality defects, yet the prevalence of laterality defects other than SI in PCD has not been prospectively studied. METHODS: In this prospective study, participants with suspected PCD were referred to our multisite consortium. We measured nasal nitric oxide (nNO) level, examined cilia with electron microscopy, and analyzed PCD-causing gene mutations. Situs was classified as (1) situs solitus (SS), (2) SI, or (3) situs ambiguus (SA), including heterotaxy. Participants with hallmark electron microscopic defects, biallelic gene mutations, or both were considered to have classic PCD. RESULTS: Of 767 participants (median age, 8.1 years, range, 0.1-58 years), classic PCD was defined in 305, including 143 (46.9%), 125 (41.0%), and 37 (12.1%) with SS, SI, and SA, respectively. A spectrum of laterality defects was identified with classic PCD, including 2.6% and 2.3% with SA plus complex or simple cardiac defects, respectively; 4.6% with SA but no cardiac defect; and 2.6% with an isolated possible laterality defect. Participants with SA and classic PCD had a higher prevalence of PCD-associated respiratory symptoms vs SA control participants (year-round wet cough, P < .001; year-round nasal congestion, P = .015; neonatal respiratory distress, P = .009; digital clubbing, P = .021) and lower nNO levels (median, 12 nL/min vs 252 nL/min; P < .001). CONCLUSIONS: At least 12.1% of patients with classic PCD have SA and laterality defects ranging from classic heterotaxy to subtle laterality defects. Specific clinical features of PCD and low nNO levels help to identify PCD in patients with laterality defects. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00323167; URL: www.clinicaltrials.gov.

Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype.

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.

Pulmonary responses in current smokers and ex-smokers following a two hour exposure at rest to clean air and fine ambient air particles.

BACKGROUND: Increased susceptibility of smokers to ambient PM may potentially promote development of COPD and accelerate already present disease. OBJECTIVES: To characterize the acute and subacute lung function response and inflammatory effects of controlled chamber exposure to concentrated ambient fine particles (CAFP) with MMAD ≤ 2.5 microns in ex-smokers and lifetime smokers. METHODS: Eleven subjects, aged 35-74 years, came to the laboratory 5 times; a training day and two exposure days separated by at least 3 weeks, each with a post-exposure visit 22 h later. Double-blind and counterbalanced exposures to "clean air" (mean 1.5 ± 0.6 µg/m3) or CAFP (mean 108.7 ± 24.8 µg/m3 ) lasted 2 h with subjects at rest. RESULTS: At 3 h post-exposure subjects' DTPA clearance half-time significantly increased by 6.3 min per 100 µg/m3 of CAFP relative to "clean air". At 22 h post-exposure they showed significant reduction of 4.3% per 100 µg/m3 in FEV1 and a significant DLCO decrease by 11.1% per 100 µg/m3 of CAFP relative to "clean air". At both 3 h and 22 h the HDL cholesterol level significantly decreased by 4.5% and 4.1%, respectively. Other blood chemistries and markers of lung injury, inflammation and procoagulant activity were within the normal range of values at any condition. CONCLUSIONS: The results suggest that an acute 2 h resting exposure of smokers and ex-smokers to fine ambient particulate matter may transiently affect pulmonary function (spirometry and DLCO) and increase DTPA clearance half-time. Except for a post exposure decrease in HDL no other markers of pulmonary inflammation, prothrombotic activity and lung injury were significantly affected under the conditions of exposure.

Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia.

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 disease-specific 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 ± 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. 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.

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.

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 of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure.

RATIONALE: Primary 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). OBJECTIVES: To 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. METHODS: 82 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. RESULTS: Of 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. CONCLUSIONS: Mutations 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.

Lung function and inflammatory responses in healthy young adults exposed to 0.06 ppm ozone for 6.6 hours.

RATIONALE: Exposure to ozone causes a decrease in spirometric lung function and an increase in airway inflammation in healthy young adults at concentrations as low as 0.08 ppm, close to the National Ambient Air Quality Standard for ground level ozone. OBJECTIVES: To test whether airway effects occur below the current ozone standard and if they are more pronounced in potentially susceptible individuals, such as those deficient in the antioxidant gene glutathione S-transferase mu 1 (GSTM1). METHODS: Pulmonary function and subjective symptoms were measured in 59 healthy young adults (19-35 yr) immediately before and after exposure to 0.0 (clean air, CA) and 0.06 ppm ozone for 6.6 hours in a chamber while undergoing intermittent moderate exercise. The polymorphonuclear neutrophil (PMN) influx was measured in 24 subjects 16 to 18 hours postexposure. MEASUREMENTS AND MAIN RESULTS: Subjects experienced a significantly greater (P = 0.008) change in FEV(1) (± SE) immediately after exposure to 0.06 ppm ozone compared with CA (-1.71 ± 0.50% vs. -0.002 ± 0.46%). The decrement in FVC was also greater (P = 0.02) after ozone versus CA (-2.32 ± 0.41% vs. -1.13 ± 0.34%). Similarly, changes in %PMN were greater after ozone (54.0 ± 4.6%) than CA (38.3 ± 3.7%) exposure (P < 0.001). Symptom scores were not different between ozone versus CA. There were no significant differences in changes in FEV(1), FVC, and %PMN between subjects with GSTM1-positive and GSTM1-null genotypes. CONCLUSIONS: Exposure of healthy young adults to 0.06 ppm ozone for 6.6 hours causes a significant decrement of FEV(1) and an increase in neutrophilic inflammation in the airways. GSTM1 genotype alone appears to have no significant role in modifying the effects.

An alternative form and level of the human health ozone standard.

Controlled human laboratory studies have shown that there is a disproportionately greater pulmonary function response from higher hourly average ozone (O3) concentrations than from lower hourly average values and thus, a nonlinear relationship exists between O3 dose and pulmonary function (FEV1) response. The nonlinear dose-response relationship affects the efficacy of the current 8-h O3 standard to describe adequately the observed spirometric response to typical diurnal O3 exposure patterns. We have reanalyzed data from five controlled human response to O3 health laboratory experiments as reported by Hazucha et al. (1992), Adams (2003, 2006a, 2006b), and Schelegle et al. (2009). These investigators exposed subjects to multi-hour variable/stepwise O3 concentration profiles that mimicked typical diurnal patterns of ambient O3 concentrations. Our findings indicate a common response pattern across most of the studies that provides valuable information for the development of a lung function (FEV1)-based alternate form for the O3 standard. Based on our reanalysis of the realistic exposure profiles used in these experiments, we suggest that an alternative form of the human health standard, similar to the proposed secondary (i.e., vegetation) standard form, be considered. The suggested form is an adjusted 5-h cumulative concentration weighted O3 exposure index, which addresses both the delay associated with the onset of response (FEV1 decrement) and the nonlinearity of response (i.e., the greater effect of higher concentrations over the mid- and low-range values) on an hourly basis.

Primary ciliary dyskinesia in Amish communities.

Primary ciliary dyskinesia is an autosomal recessive multigenic disease that results in impaired mucociliary clearance. We have diagnosed 9 subjects with primary ciliary dyskinesia from geographically dispersed Amish communities, on the basis of clinical characteristics and ciliary ultrastructural defects. Despite consanguinity, affected individuals had evidence of genetic heterogeneity.

Concentrated ambient ultrafine particle exposure induces cardiac changes in young healthy volunteers.

RATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis. OBJECTIVES: To characterize the effects of acute exposure to ambient ultrafine particles in young healthy humans. METHODS: Nineteen healthy nonsmoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 microm) particles were concentrated by a factor of up to 20-fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (ultrafine concentrated ambient particles [UFCAPs]). Subjects underwent bronchoalveolar lavage 18 hours after each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry, and hematological parameters, as well as heart rate variability and repolarization indices. MEASUREMENTS AND MAIN RESULTS: Exposure to UFCAPs was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid. CONCLUSIONS: These findings show mild inflammatory and prothrombic responses and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.