Modulation,microbiota and inflammation in the adult CF gut: A prospective study

BackgroundCystic Fibrosis (CF) has prominent gastrointestinal and pancreatic manifestations. The aim of this study was to determine the effect of Cystic fibrosis transmembrane conductance regulator (CFTR) modulation on, gastrointestinal inflammation, pancreatic function and gut microbiota composition in people with cystic fibrosis (CF) and the G551D-CFTR mutation.MethodsFourteen adult patients with the G551D-CFTR mutation were assessed clinically at baseline and for up to 1 year after treatment with ivacaftor. The change in gut inflammatory markers (calprotectin and lactoferrin), exocrine pancreatic status and gut microbiota composition and structure were assessed in stool samples.ResultsThere was no significant change in faecal calprotectin nor lactoferrin in patients with treatment while all patients remained severely pancreatic insufficient. There was no significant change in gut microbiota diversity and richness following treatment.ConclusionThere was no significant change in gut inflammation after partial restoration of CFTR function with ivacaftor, suggesting that excess gut inflammation in CF is multi-factorial in aetiology. In this adult cohort, exocrine pancreatic function was irreversibly lost. Longer term follow-up may reveal more dynamic changes in the gut microbiota and possible restoration of CFTR function.     

Importance of bacteriology in upper airways of patients with Cystic Fibrosis

BackgroundRecently the influence of the upper airways (UAW) on the general health of a patient with Cystic Fibrosis (CF) has been acknowledged. Surprisingly the microbiology of the upper compartment of the airways receives barely any attention in the treatment of CF. The aim of the present study was to investigate the microbiology of the upper airways in adult patients with CF, to correlate these findings with cultures from the lower airways (LAW) and with clinical characteristics.MethodsIn this cross-sectional study bacteriological and clinical data were gathered from 104 adult patients with CF. UAW samples for culture were collected by nasal lavage and middle meatal swabs; LAW cultures were performed on expectorated sputum or cough swabs. Each patient performed the Rhinosinusitis Outcome Measure (RSOM-31).ResultsIn 72 patients (69.2%) UAW cultures yielded microorganisms other than normal nasal flora and in 50 patients (48.1%) Pseudomonas aeruginosa grew from the UAW cultures. Similarity between UAW and LAW cultures was determined in 50.0% of these 72 patients. In 3 patients P. aeruginosa was cultured from the UAW after successful eradication of P. aeruginosa from the LAW. P. aeruginosa in the UAW did not influence symptoms of sinonasal disease compared to other microorganisms.ConclusionsComparison of UAW and LAW cultures in adult patients with CF showed one or more concordant microorganism in 50.0% of the patients. P. aeruginosa was most frequently cultured from the UAW. P. aeruginosa can be cultured from the UAW after eradication therapy which may suggest persistence of P. aeruginosa in the UAW. We feel this is may be a motive to include the UAW in eradication therapy in Cystic Fibrosis.     

Clinical evidence that V456A is a Cystic Fibrosis causing mutation in South Asians

BackgroundCystic Fibrosis (CF) genotypes in South Asians are variable with a decreased incidence of Delta F508 and an increased incidence of novel mutations. The objective of this study is to provide clinical evidence that V456A, a novel mutation in South Asian Cystic Fibrosis patients, can cause significant lung disease.MethodsWe extracted clinical data from a retrospective chart review of 2 CF patients of South Asian descent.ResultsPatient 1, a 10 year and 11 month old Pakistani female at her initial clinic visit, required multiple hospitalizations for bronchiectasis and pulmonary infections. She was pancreatic sufficient but had slow weight gain. Genetic testing revealed that she is homozygous for the CFTR V456A mutation. Patient 2, an Indian female diagnosed with CF on newborn screening, is compound heterozygous for V456A/R709X. She had slow weight gain with BMI ranging from 12.9 to 13.4 kg/m² from 3 to 5 years of age and was 14.2 kg/m² at 6 years of age. At 6 years of age, pulmonary function tests revealed mild lung disease with FVC of 71%, FEV₁ of 75%, FEF_25–75 of 119%, and FEV₁/FVC of 86% predicted. Sputum cultures were intermittently positive for Staphylococcus aureus and Haemophilus influenza.ConclusionsWe provide evidence that V456A can cause significant pulmonary disease in South Asian Cystic Fibrosis patients.     

Lower exhaled nitric oxide in infants with Cystic Fibrosis compared to healthy controls

Exhaled nitric oxide (FE_NO) is a well-known, non-invasive airway biomarker. In patients with Cystic Fibrosis (CF) FE_NO is decreased. To understand if reduced FE_NO is primary related to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) dysfunction or an epiphenomenon of chronic inflammation, we measured FE_NO in 34 infants with CF prior to clinical symptoms and in 68 healthy controls. FE_NO was lower in CF compared to controls (p = 0.0006) and the effect was more pronounced in CF infants without residual CFTR function (p < 0.0001). This suggests that FE_NO is reduced in CF early in life, possibly associated with underlying CFTR dysfunction.     

Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward

BackgroundNewborn screening for Cystic Fibrosis (CF) is associated with situations where the diagnosis of CF or CFTR related disorders (CFTR-RD) cannot be clearly ruled out.Materials/patients and methodsWe report a case series of 23 children with unconclusive diagnosis after newborn screening for CF and a mean follow-up of 7.7 years (4–13). Comprehensive investigations including whole CFTR gene sequencing, in vivo intestinal current measurement (ICM), nasal potential difference (NPD), and in vitro functional studies of variants of unknown significance, helped to reclassify the patients.ResultsExtensive genetic testing identified, in trans with a CF causing mutation, variants with varying clinical consequences and 3 variants of unknown significance (VUS). Eighteen deep intronic variants were identified by deep resequencing of the whole CFTR gene in 13 patients and were finally considered as non-pathogenic. All patients had normal CFTR dependent chloride transport in ICM. NPD differentiated 3 different profiles: CF-like tracings qualifying the patients as CF, such as F508del/D1152H patients; normal responses, suggesting an extremely low likelihood of developing a CFTR-RD such as F508del/TG11T5 patients; partial CFTR dysfunction above 20% of the normal, highlighting a remaining risk of developing CFTR-RD such as F508del/F1052V patients. The 3 VUS were reclassified as variant with defective maturation (D537N), defective expression (T582I) or with no clinical consequence (M952T).ConclusionThis study demonstrates the usefulness of combining genetic and functional investigations to assess the possibility of evolving to CF or CFTR-RD in babies with inconclusive diagnosis at neonatal screening.     

The study of cystic fibrosis transmembrane conductance regulator gene mutations in a group of patients from Romania

BackgroundCystic fibrosis (CF) is produced by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) gene.MethodsOne hundred twenty eight patients with CF were analysed for mutations in the CFTR gene in order to establish the frequency of CF mutations in the Romanian population. The chief methods of analysis were polymerase chain reaction (PCR) of DNA extracted from blood and electrophoresis of PCR products.ResultsThe frequency of F508del in CF chromosomes from Romania is approximately 56.3%. Other frequent mutations noted are: G542X (3.9%), W1282X (2.3%), and CFTRdele2,3(21 kb)(1.6%); the remaining mutations have frequencies below 1%.ConclusionsWe consider that the frequency of F508del in CF patients from Romania is higher than in previous reports, reaching 56.3%, probably owing to more rigorous selection of patients for genetic testing, allowing improved calculation of mutation frequencies.     

Newborn screening alone insufficient to improve pulmonary outcomes for cystic fibrosis

BackgroundThe Wisconsin Cystic Fibrosis Neonatal Screening Project was a randomized clinical trial (RCT) revealing that children receiving an early diagnosis of CF via newborn screening (NBS) had improved nutritional outcomes but similar lung disease severity compared to those who presented clinically. Because the evaluations of these subjects by protocol ended in 2012, our objective was to assess long-term pulmonary and mortality outcomes.MethodsRetrospective analysis of the RCT cohort utilized longitudinal outcome measures obtained from the Cystic Fibrosis Foundation Patient Registry (CFFPR). Data included screening assignment, clinical characteristics, percent predicted forced expiratory volume in 1 s (ppFEV₁) and mortality. A random intercept model was used to compare the ppFEV₁ decline of subjects between the two groups up to age 26 years. Mortality was analyzed using the Kaplan-Meier method.ResultsOf the 145 subjects who consented to the original study, 104 subjects met inclusion criteria and had adequate data in the CFFPR. Of 57 subjects in the screened group and 47 in the control group, the rates of ppFEV₁ decline were 1.76%/year (95% CI 1.62 to 1.91%) and 1.43%/year (95% CI 1.26 to 1.60%), respectively (p<0.0002). Pseudomonas aeruginosa acquired before 2 years was partially responsible. There was no difference in mortality between the two groups.ConclusionsNBS alone does not improve pulmonary outcomes in CF, particularly when other risk factors supervene. In an era prior to strict infection control and current therapies, NBS for CF may be associated with worse pulmonary outcomes.     

CFTR-function and ventilation inhomogeneity in individuals with cystic fibrosis

BackgroundIncreased (abnormal) ventilation inhomogeneity in individuals with mild Cystic Fibrosis (CF) lung disease may become a treatable trait for small-molecule therapeutics improving Cystic Fibrosis Transmembrane Regulator (CFTR) function. The relationship between CFTR function and ventilation inhomogeneity is unknown. We aimed to identify and quantify increased ventilation inhomogeneity in relation to CFTR function.MethodsThis was an international, multi-center, cross-sectional study. We collated data from individuals aged 3–25 years with minimal (CFTR-MF) or residual (CFTR-RF) function of a variety of CFTR genotypes and FEV₁ ≥ 70% predicted. We measured lung function using nitrogen multiple-breath washout and spirometry. We compared lung clearance index (LCI) and FEV₁ between individuals with CFTR-MF vs CFTR-RF using a mixed effects multi-variable linear regression model to account for study differences and a logistic model based on propensity-score matching to adjust for possible confounding.ResultsWe included 141 with CFTR-MF and 35 with CFTR-RF. LCI (> 1.96 z-score) was elevated in 71.6% individuals with CFTR-MF and in 40.0% with CFTR-RF. FEV₁ (< -1.96 z-score) was reduced in 11.3% individuals with CFTR-MF and in 5.7% with CFTR-RF. The mean difference (95% CI) of LCI and FEV₁ between CFTR-MF and CFTR-RF was 3.71 (1.63 to 5.79) and -0.40 (-0.83 to 0.02) z-score. The LCI differences were similar after adjustment for confounders and in individuals with normal FEV₁.ConclusionIncreased ventilation inhomogeneity is associated with less CFTR function. In individuals with mild CF lung disease, LCI can identify and quantify increased ventilation inhomogeneity, a candidate treatable trait.     

Cystic fibrosis related diabetes in Europe: Prevalence,risk factors and outcome; Olesen et al

BackgroundCystic fibrosis related diabetes (CFRD) has implications for morbidity and mortality with several risk factors identified. We studied the epidemiology of CFRD in the large dataset of the European Cystic Fibrosis Society Patient registry.MethodsData on CF patients were investigated for the prevalence of CFRD as well as for any association with suggested risk factors and effects.ResultsCFRD increased by approximately ten percentage points every decade from ten years of age. Prevalence was higher in females in the younger age groups. CFRD was associated with severe CF genotypes (OR = 3.11, 95%CI: 2.77–3.48), pancreatic insufficiency (OR = 1.46, 95%CI: 1.39–1.53) and female gender (OR = 1.28, 95%CI: 1.21–1.34). Patients with CFRD had higher odds of being chronically infected with Pseudomonas aeruginosa, Burkholderia cepacia complex and Stenotrophomonas maltophilia than patients without CFRD, higher odds of having FEV1% of predicted <40% (OR = 1.82, 95%CI: 1.70–1.94) and higher odds of having BMI SDS ≤−2 than patients without CFRD (OR = 1.24, 95%CI: 1.15–1.34).ConclusionsSevere genotype, pancreatic insufficiency and female gender remain considerable intrinsic risk factors for early acquisition of CFRD. CFRD is associated with infections, lower lung function and poor nutritional status. Early diagnosis and aggressive treatment of CFRD are more important than ever with increasing life span.     

Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation

BackgroundW1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations.MethodsWe compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors.ResultsCas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR.ConclusionAltogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.     

A 10-year large-scale cystic fibrosis carrier screening in the Italian population

BackgroundCystic Fibrosis (CF) is one of the most common autosomal recessive genetic disorders, with the majority of patients born to couples unaware of their carrier status. Carrier screenings might help reducing the incidence of CF.MethodsWe used a semi-automated reverse-dot blot assay identifying the 47 most common CFTR gene mutations followed by DGGE/dHPLC analysis.ResultsResults of a 10-year (1996–2006) CF carrier screening on 57,999 individuals with no prior family history of CF are reported. Of these, 25,104 were couples and 7791 singles, with 77.9% from the Italian Veneto region. CFTR mutations were found in 1879 carriers (frequency 1/31), with ΔF508 being the most common (42.6%). Subjects undergoing medically assisted reproduction (MAR) had significantly (p < 0.0001) higher CF carrier frequency (1/22 vs 1/32) compared to non-MAR subjects.ConclusionsIf coupled to counselling programmes, CF carrier screening tests might help reducing the CF incidence.     

Retrospective analysis of stored dried blood spots from children with cystic fibrosis and matched controls to assess the performance of a proposed newborn screening protocol in Switzerland

BackgroundNewborn screening (NBS) for Cystic Fibrosis (CF) has been introduced in many countries, but there is no ideal protocol suitable for all countries. This retrospective study was conducted to evaluate whether the planned two step CF NBS with immunoreactive trypsinogen (IRT) and 7 CFTR mutations would have detected all clinically diagnosed children with CF in Switzerland.MethodsIRT was measured using AutoDELFIA Neonatal IRT-Kit in stored NBS cards.ResultsBetween 2006 and 2009, 66 children with CF were reported, 4 of which were excluded for various reasons (born in another country, NBS at 6 months, no informed consent). 98% (61/62) had significantly higher IRT compared to matched control group. There was one false negative IRT result in an asymptomatic child with atypical CF (normal pancreatic function and sweat test).ConclusionsAll children but one with atypical CF would have been detected with the planned two step protocol.     

Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip

BackgroundCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which results in impaired airway mucociliary clearance, inflammation, infection, and respiratory insufficiency. The development of new therapeutics for CF are limited by the lack of reliable preclinical models that recapitulate the structural, immunological, and bioelectrical features of human CF lungs.MethodsWe leveraged organ-on-a-chip technology to develop a microfluidic device lined by primary human CF bronchial epithelial cells grown under an air-liquid interface and interfaced with pulmonary microvascular endothelial cells (CF Airway Chip) exposed to fluid flow. The responses of CF and healthy Airway Chips were analyzed in the presence or absence of polymorphonuclear leukocytes (PMNs) and the bacterial pathogen, Pseudomonas aeruginosa.ResultsThe CF Airway Chip faithfully recapitulated many features of the human CF airways, including enhanced mucus accumulation, increased cilia density, and a higher ciliary beating frequency compared to chips lined by healthy bronchial epithelial cells. The CF chips also secreted higher levels of IL-8, which was accompanied by enhanced PMN adhesion to the endothelium and transmigration into the airway compartment. In addition, CF Airway Chips provided a more favorable environment for Pseudomonas aeruginosa growth, which resulted in enhanced secretion of inflammatory cytokines and recruitment of PMNs to the airway.ConclusionsThe human CF Airway Chip may provide a valuable preclinical tool for pathophysiology studies as well as for drug testing and personalized medicine.     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis

BackgroundThe pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized.ResultsThe aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype–phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed.Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype–phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate–severe phenotypic consequences at any given time.ConclusionsThe genotype–phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.     

Disease progression in patients with cystic fibrosis treated with ivacaftor: Data from national US and UK registries

BackgroundIvacaftor is the first in a class of drugs, CFTR modulators, that target the underlying defect in cystic fibrosis (CF). This long-term observational safety study evaluated CF disease progression in patients treated with ivacaftor in a real-world setting for up to 5 years.MethodsData from existing US and UK CF patient registries were used to assess longitudinal patterns in lung function, nutritional status, pulmonary exacerbations and hospitalizations, CF-related diabetes (CFRD), and Pseudomonas aeruginosa in ivacaftor-treated vs untreated comparator cohorts matched by age, sex, and disease severity.ResultsUS analyses included 635 ivacaftor-treated patients and 1874 comparators followed for 5 years from year 1 of market availability (2012–2016). Evaluation of outcome patterns from pretreatment baseline (2011) through year 5 (2016), showed that relative to comparators, ivacaftor-treated patients had better preserved lung function (mean change in percent predicted FEV₁, −0.7 percentage points with ivacaftor vs −8.3 percentage points in comparators) and improved nutritional status (mean body mass index change +2.4 kg/m² with ivacaftor vs +1.6 kg/m² in comparators). US patients treated with ivacaftor had significantly lower frequencies of exacerbations and hospitalizations in each of the 5 years of follow-up relative to pretreatment baseline and comparators. Favorable trends in CFRD and P. aeruginosa prevalence were also observed. Findings from the smaller UK registry were directionally similar to and consistent with US findings.ConclusionsThis observational study represents the largest longitudinal analysis of patients treated with ivacaftor in a real-world setting. The findings support disease modification by CFTR modulation with ivacaftor.     

Cardiovascular complications in cystic fibrosis: A review of the literature

Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to dysfunction of the CFTR protein. CFTR dysfunction leads to disease in the respiratory and gastrointestinal systems. Disorders of the cardiovascular system in individuals with CF are usually attributed to secondary effects from progressive lung disease. However, CFTR has been localized to vascular endothelium and smooth muscle, suggesting that CFTR dysfunction may directly impact cardiovascular function. As treatments for CF improve and life-expectancy increases, the risk of vascular disease may increase in prevalence related to primary and secondary CFTR dysfunction, chronic systemic inflammation, nutritional health and hyperglycemia in individuals with CF related diabetes. Here we review the available literature on CF and the cardiovascular system, examining the secondary effects and evidence for direct CFTR dysfunction in the heart, aorta, pulmonary vessels, and vasculature, as well as future directions and treatment options.