Scott D. Sagel

Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation

BACKGROUND A new approach in the treatment of cystic fibrosis involves improving the function of mutant cystic fibrosis transmembrane conductance regulator (CFTR). VX-770, a CFTR potentiator, has been shown to increase the activity of wild-type and defective cell-surface CFTR in vitro. METHODS We randomly assigned 39 adults with cystic fibrosis and at least one G551D-CFTR allele to receive oral VX-770 every 12 hours at a dose of 25, 75, or 150 mg or placebo for 14 days (in part 1 of the study) or VX-770 every 12 hours at a dose of 150 or 250 mg or placebo for 28 days (in part 2 of the study). RESULTS At day 28, in the group of subjects who received 150 mg of VX-770, the median change in the nasal potential difference (in response to the administration of a chloride-free isoproterenol solution) from baseline was -3.5 mV (range, -8.3 to 0.5; P=0.02 for the within-subject comparison, P=0.13 vs. placebo), and the median change in the level of sweat chloride was -59.5 mmol per liter (range, -66.0 to -19.0; P=0.008 within-subject, P=0.02 vs. placebo). The median change from baseline in the percent of predicted forced expiratory volume in 1 second was 8.7% (range, 2.3 to 31.3; P=0.008 for the within-subject comparison, P=0.56 vs. placebo). None of the subjects withdrew from the study. Six severe adverse events occurred in two subjects (diffuse macular rash in one subject and five incidents of elevated blood and urine glucose levels in one subject with diabetes). All severe adverse events resolved without the discontinuation of VX-770. CONCLUSIONS This study to evaluate the safety and adverse-event profile of VX-770 showed that VX-770 was associated with within-subject improvements in CFTR and lung function. These findings provide support for further studies of pharmacologic potentiation of CFTR as a means to treat cystic fibrosis. (Funded by Vertex Pharmaceuticals and others; ClinicalTrials.gov number, NCT00457821.).

Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis.

Culture of bronchoalveolar lavage fluid (BALF) is the gold standard for detection of pathogens in the lower airways in cystic fibrosis (CF). However, current culture results do not explain all clinical observations in CF, including negative culture results during pulmonary exacerbation and inflammation in the absence of pathogens. We hypothesize that organisms not routinely identified by culture occur in the CF airway and may contribute to disease. To test this hypothesis we used a culture-independent molecular approach, based on use of rRNA sequence analysis, to assess the bacterial composition of BALF from children with CF and disease controls (DC). Specimens from 42 subjects (28 CF) were examined, and ≈6,600 total clones were screened to identify 121 species of bacteria. In general, a single rRNA type dominated clone libraries from CF specimens, but not DC. Thirteen CF subjects contained bacteria that are not routinely assessed by culture. In four CF subjects, candidate pathogens were identified and include the anaerobe Prevotella denticola, a Lysobacter sp., and members of the Rickettsiales. The presumptive pathogens Tropheryma whipplei and Granulicatella elegans were identified in cases from the DC group. The presence of unexpected bacteria in CF may explain inflammation without documented pathogens and consequent failure to respond to standard treatment. These results show that molecular techniques provide a broader perspective on airway bacteria than do routine clinical cultures and thus can identify targets for further clinical evaluation.

Clinical Mechanism of the Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor in G551D-mediated Cystic Fibrosis

RATIONALE Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator recently approved for patients with CF age 6 and older with the G551D mutation. OBJECTIVES To evaluate ivacaftor in a postapproval setting and determine mechanism of action and response of clinically relevant markers. METHODS We conducted a longitudinal cohort study in 2012-2013 in G551D CF patients age 6 and older with no prior exposure to ivacaftor. Study assessments were performed at baseline, 1, 3, and 6 months after ivacaftor initiation. Substudies evaluated mucociliary clearance, β-adrenergic sweat secretion rate, gastrointestinal pH, and sputum inflammation and microbiology Measurements and Main Results: A total of 151 of 153 subjects were prescribed ivacaftor and 88% completed the study through 6 months. FEV1 % predicted improved from baseline to 6 months (mean absolute change, 6.7%; P < 0.001). Similarly, body mass index improved from baseline to 6 months (mean change, 0.8 kg/m(2); P < 0.001). Sweat chloride decreased from baseline to 6 months (mean change, -53.8 mmol/L; 95% confidence interval, -57.7 to -49.9; P < 0.001), reflecting augmented CFTR function. There was significant improvement in hospitalization rate (P < 0.001) and Pseudomonas aeruginosa burden (P < 0.01). Significant improvements in mucociliary clearance (P < 0.001), gastrointestinal pH (P = 0.001), and microbiome were also observed, providing clinical mechanisms underlying the therapeutic benefit of ivacaftor. CONCLUSIONS Significant clinical and physiologic improvements were observed on initiation of ivacaftor in a broad patient population, including reduced infection with P. aeruginosa. Biomarker studies substantially improve the understanding of the mechanistic consequences of CFTR modulation on pulmonary and gastrointestinal physiology.

Impact of Pseudomonas and Staphylococcus infection on inflammation and clinical status in young children with cystic fibrosis.

OBJECTIVE To assess the effects of Pseudomonas aeruginosa and Staphylococcus aureus infection on lower airway inflammation and clinical status in young children with cystic fibrosis (CF). STUDY DESIGN We studied 111 children age < 6 years who had 2 P aeruginosa-positive oropharyngeal cultures within 12 months. We examined bronchoalveolar lavage fluid (BALF) inflammatory markers (ie, cell count, differential, interleukin [IL]-8, IL-6, neutrophil elastase), CF-related bacterial pathogens, exotoxin A serology, and clinical indicators of disease severity. RESULTS Young children with CF with both upper and lower airway P aeruginosa infection had higher neutrophil counts, higher IL-8 and free neutrophil elastase levels, increased likelihood of positive exotoxin A titers, and lower Shwachman scores compared with those with positive upper airway cultures only. S aureus was associated with increased lower airway inflammation, and the presence of both P aeruginosa and S aureus had an additive effect on concentrations of lower airway inflammatory markers. BALF markers of inflammation were increased with the number of different bacterial pathogens detected. CONCLUSIONS Young children with CF who have upper and lower airway P aeruginosa infection have increased endobronchial inflammation and poorer clinical status compared with those with only upper airway P aeruginosa infection. The independent and additive effects of S aureus on inflammation support the significance of polymicrobial infection in early CF lung disease.

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 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.

Inflammation and Airway Microbiota during Cystic Fibrosis Pulmonary Exacerbations

Background Pulmonary exacerbations (PEx), frequently associated with airway infection and inflammation, are the leading cause of morbidity in cystic fibrosis (CF). Molecular microbiologic approaches detect complex microbiota from CF airway samples taken during PEx. The relationship between airway microbiota, inflammation, and lung function during CF PEx is not well understood. Objective To determine the relationships between airway microbiota, inflammation, and lung function in CF subjects treated for PEx. Methods Expectorated sputum and blood were collected and lung function testing performed in CF subjects during early (0–3d.) and late treatment (>7d.) for PEx. Sputum was analyzed by culture, pyrosequencing of 16S rRNA amplicons, and quantitative PCR for total and specific bacteria. Sputum IL-8 and neutrophil elastase (NE); and circulating C-reactive protein (CRP) were measured. Results Thirty-seven sputum samples were collected from 21 CF subjects. At early treatment, lower diversity was associated with high relative abundance (RA) of Pseudomonas (r = −0.67, p<0.001), decreased FEV1% predicted (r = 0.49, p = 0.03) and increased CRP (r = −0.58, p = 0.01). In contrast to Pseudomonas, obligate and facultative anaerobic genera were associated with less inflammation and higher FEV1. With treatment, Pseudomonas RA and P. aeruginosa by qPCR decreased while anaerobic genera showed marked variability in response. Change in RA of Prevotella was associated with more variability in FEV1 response to treatment than Pseudomonas or Staphylococcus. Conclusions Anaerobes identified from sputum by sequencing are associated with less inflammation and higher lung function compared to Pseudomonas at early exacerbation. CF PEx treatment results in variable changes of anaerobic genera suggesting the need for larger studies particularly of patients without traditional CF pathogens.

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.

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.

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.