Objective Cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene, continues to present diagnostic challenges. Newborn screening and an evolving ...understanding of CF genetics have prompted a reconsideration of the diagnosis criteria. Study design To improve diagnosis and achieve standardized definitions worldwide, the CF Foundation convened a committee of 32 experts in CF diagnosis from 9 countries to develop clear and actionable consensus guidelines on the diagnosis of CF and to clarify diagnostic criteria and terminology for other disorders associated with CFTR mutations. An a priori threshold of ≥80% affirmative votes was required for acceptance of each recommendation statement. Results After reviewing relevant literature, the committee convened to review evidence and cases. Following the conference, consensus statements were developed by an executive subcommittee. The entire consensus committee voted and approved 27 of 28 statements, 7 of which needed revisions and a second round of voting. Conclusions It is recommended that diagnoses associated with CFTR mutations in all individuals, from newborn to adult, be established by evaluation of CFTR function with a sweat chloride test. The latest mutation classifications annotated in the Clinical and Functional Translation of CFTR project ( http://www.cftr2.org/index.php ) should be used to aid in diagnosis. Newborns with a high immunoreactive trypsinogen level and inconclusive CFTR functional and genetic testing may be designated CFTR - related metabolic syndrome or CF screen positive, inconclusive diagnosis; these terms are now merged and equivalent, and CFTR - related metabolic syndrome/CF screen positive, inconclusive diagnosis may be used. International Statistical Classification of Diseases and Related Health Problems, 10th Revision codes for use in diagnoses associated with CFTR mutations are included.
CFTR modulators have revolutionized the treatment of individuals with cystic fibrosis (CF) by improving the function of existing protein. Unfortunately, almost half of the disease-causing variants in ...CFTR are predicted to introduce premature termination codons (PTC) thereby causing absence of full-length CFTR protein. We hypothesized that a subset of nonsense and frameshift variants in CFTR allow expression of truncated protein that might respond to FDA-approved CFTR modulators. To address this concept, we selected 26 PTC-generating variants from four regions of CFTR and determined their consequences on CFTR mRNA, protein and function using intron-containing minigenes expressed in 3 cell lines (HEK293, MDCK and CFBE41o-) and patient-derived conditionally reprogrammed primary nasal epithelial cells. The PTC-generating variants fell into five groups based on RNA and protein effects. Group A (reduced mRNA, immature (core glycosylated) protein, function <1% (n = 5)) and Group B (normal mRNA, immature protein, function <1% (n = 10)) variants were unresponsive to modulator treatment. However, Group C (normal mRNA, mature (fully glycosylated) protein, function >1% (n = 5)), Group D (reduced mRNA, mature protein, function >1% (n = 5)) and Group E (aberrant RNA splicing, mature protein, function > 1% (n = 1)) variants responded to modulators. Increasing mRNA level by inhibition of NMD led to a significant amplification of modulator effect upon a Group D variant while response of a Group A variant was unaltered. Our work shows that PTC-generating variants should not be generalized as genetic 'nulls' as some may allow generation of protein that can be targeted to achieve clinical benefit.
Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis ...transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of ł0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.
Cystic fibrosis transmembrane conductance regulator (CFTR) modulators correct the basic defect caused by CFTR mutations. Improvements in health outcomes have been achieved with the combination of a ...CFTR corrector and potentiator in people with cystic fibrosis homozygous for the F508del mutation. The addition of elexacaftor (VX-445), a next-generation CFTR corrector, to tezacaftor plus ivacaftor further improved F508del-CFTR function and clinical outcomes in a phase 2 study in people with cystic fibrosis homozygous for the F508del mutation.
This phase 3, multicentre, randomised, double-blind, active-controlled trial of elexacaftor in combination with tezacaftor plus ivacaftor was done at 44 sites in four countries. Eligible participants were those with cystic fibrosis homozygous for the F508del mutation, aged 12 years or older with stable disease, and with a percentage predicted forced expiratory volume in 1 s (ppFEV1) of 40–90%, inclusive. After a 4-week tezacaftor plus ivacaftor run-in period, participants were randomly assigned (1:1) to 4 weeks of elexacaftor 200 mg orally once daily plus tezacaftor 100 mg orally once daily plus ivacaftor 150 mg orally every 12 h versus tezacaftor 100 mg orally once daily plus ivacaftor 150 mg orally every 12 h alone. The primary outcome was the absolute change from baseline (measured at the end of the tezacaftor plus ivacaftor run-in) in ppFEV1 at week 4. Key secondary outcomes were absolute change in sweat chloride and Cystic Fibrosis Questionnaire-Revised respiratory domain (CFQ-R RD) score. This study is registered with ClinicalTrials.gov, NCT03525548.
Between Aug 3 and Dec 28, 2018, 113 participants were enrolled. Following the run-in, 107 participants were randomly assigned (55 in the elexacaftor plus tezacaftor plus ivacaftor group and 52 in the tezacaftor plus ivacaftor group) and completed the 4-week treatment period. The elexacaftor plus tezacaftor plus ivacaftor group had improvements in the primary outcome of ppFEV1 (least squares mean LSM treatment difference of 10·0 percentage points 95% CI 7·4 to 12·6, p<0·0001) and the key secondary outcomes of sweat chloride concentration (LSM treatment difference −45·1 mmol/L 95% CI −50·1 to −40·1, p<0·0001), and CFQ-R RD score (LSM treatment difference 17·4 points 95% CI 11·8 to 23·0, p<0·0001) compared with the tezacaftor plus ivacaftor group. The triple combination regimen was well tolerated, with no discontinuations. Most adverse events were mild or moderate; serious adverse events occurred in two (4%) participants receiving elexacaftor plus tezacaftor plus ivacaftor and in one (2%) receiving tezacaftor plus ivacaftor.
Elexacaftor plus tezacaftor plus ivacaftor provided clinically robust benefit compared with tezacaftor plus ivacaftor alone, with a favourable safety profile, and shows the potential to lead to transformative improvements in the lives of people with cystic fibrosis who are homozygous for the F508del mutation.
Vertex Pharmaceuticals.
Sweat chloride testing for diagnosis of cystic fibrosis (CF) involves sweat induction, collection and handling, and measurement in an analytical lab. We have developed a wearable sensor with an ...integrated salt bridge for real-time measurement of sweat chloride concentration. Here, in a proof-of-concept study, we compare the performance of the sensor to current clinical practice in CF patients and healthy subjects.
Sweat was induced on both forearms of 10 individuals with CF and 10 healthy subjects using pilocarpine iontophoresis. A Macroduct sweat collection device was attached to one arm and sweat was collected for 30 min and then sent for laboratory analysis. A sensor was attached to the other arm and the chloride ion concentration monitored in real time for 30 min using a Bluetooth transceiver and smart phone app.
Stable sweat chloride measurements were obtained within 15 min following sweat induction using the wearable sensor. We define the detection time as the time at which the standard deviation of the real-time chloride ion concentration remained below 2 mEq/L for 5 min. The sweat volume for sensor measurements at the detection time was 13.1 ± 11.4 μL (SD), in many cases lower than the minimum sweat volume of 15 μL for conventional testing. The mean difference between sweat chloride concentrations measured by the sensor and the conventional laboratory practice was 6.2 ± 9.5 mEq/L (SD), close to the arm-to-arm variation of about 3 mEq/L. The Pearson correlation coefficient between the two measurements was 0.97 highlighting the excellent agreement between the two methods.
A wearable sensor can be used to make real-time measurements of sweat chloride within 15 min following sweat induction, requiring a small sweat volume, and with excellent agreement to standard methods.
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Objectives To characterize the clinical phenotypes and genotypic spectrum of cystic fibrosis (CF) in Chinese children. Study design We recruited and characterized the phenotypes of 21 Chinese ...children with CF. All 27 exons and their flanking sequences of the CF transmembrane conductance regulator gene were amplified and sequenced to define the genotypes. Results Bronchiectasis (95.2%) and sinusitis (76.2%) were the most common clinical presentations among our patients. By contrast, pancreatic insufficiency was rare (14.3%). The predominant organism found in the airways was Pseudomonas aeruginosa (66.7%). There were obvious reductions of forced expiratory volume in the first second (mean ± SD: 71.8% ± 17.2% predicted) and forced expiratory flows at 75% of exhaled vital capacity (33.7% ± 20.4% predicted) in children with CF. Overall, we identified 22 different mutations, including 12 missense, 5 nonsense, 2 frameshift, 1 in-frame insertion, 1 splice site, and 1 3′untranslated region mutation. Of these, 7 were novel observations (W216X780G→A, 1092insA, Q359X, D567Y, 2623-126T→C, 3439delA and 4575+110C→G), and the most common types were L88X and I556V. One de novo mutation (1092insA) was also revealed. Except for N1303K and R334W, none of them were present in the common Caucasian CF transmembrane conductance regulator mutation-screening panels. Conclusions There was a 5.7-year delay between the first clinical presentation and the eventual CF diagnosis, suggesting that CF may be underdiagnosed in China. The clinical phenotypes and genotypic spectrum are different from that observed in Caucasians.
Expanding the use of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators and correctors for the treatment of cystic fibrosis (CF) requires precise and accurate biomarkers. Sweat ...chloride concentration provides an in vivo assessment of CFTR function, but it is unknown the degree to which CFTR mutations account for sweat chloride variation.
To estimate potential sources of variation for sweat chloride measurements, including demographic factors, testing variability, recording biases, and CFTR genotype itself.
A total of 2,639 sweat chloride measurements were obtained in 1,761 twins/siblings from the CF Twin-Sibling Study, French CF Modifier Gene Study, and Canadian Consortium for Genetic Studies. Variance component estimation was performed by nested mixed modeling.
Across the tested CF population as a whole, CFTR gene mutations were found to be the primary determinant of sweat chloride variability (56.1% of variation) with contributions from variation over time (e.g., factors related to testing on different days; 13.8%), environmental factors (e.g., climate, family diet; 13.5%), other residual factors (e.g., test variability; 9.9%), and unique individual factors (e.g., modifier genes, unique exposures; 6.8%) (likelihood ratio test, P < 0.001). Twin analysis suggested that modifier genes did not play a significant role because the heritability estimate was negligible (H
= 0; 95% confidence interval, 0.0-0.35). For an individual with CF, variation in sweat chloride was primarily caused by variation over time (58.1%) with the remainder attributable to residual/random factors (41.9%).
Variation in the CFTR gene is the predominant cause of sweat chloride variation; most of the non-CFTR variation is caused by testing variability and unique environmental factors. If test precision and accuracy can be improved, sweat chloride measurement could be a valuable biomarker for assessing response to therapies directed at mutant CFTR.
Of the 2007 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, 202 have been assigned disease liability. California's racially diverse population, along with CFTR sequencing as ...part of newborn screening model, provides the opportunity to examine the phenotypes of children with uncategorized mutations to help inform disease liability and penetrance.
We conducted a retrospective cohort study based on children screened from 2007 to 2011 and followed for two to six years. Newborns that screened positive were divided into three genotype groups: those with two CF-causing mutations (CF-C); those with one mutation of varying clinic consequence (VCC); and those with one mutation of unknown disease liability (Unknown). Sweat chloride tests, pancreatic sufficiency status, and Pseudomonas aeruginosa colonization were compared.
Children with two CF-causing mutations had a classical CF phenotype, while 5% of VCC (4/78) and 11% of Unknown (27/244) met diagnostic criteria of CF. Children carrying Unknown mutations 2215insG with D836Y, and T1036N had early and classical CF phenotype, while others carrying 1525-42G>A, L320V, L967S, R170H, and 296+28A>G had a benign clinical presentation, suggesting that these are non-CF causing.
While most infants with VCC and Unknown CFTR mutations do not meet diagnostic criteria for CF, a small proportion do. These findings highlight the range of genotypes and phenotypes in the first few years of life following CF newborn screening when CFTR sequencing is performed.
Objective As a Mendelian disease, genetics plays an integral role in the diagnosis of cystic fibrosis (CF). The identification of 2 disease-causing mutations in the CF transmembrane conductance ...regulator (CFTR) in an individual with a phenotype provides evidence that the disease is CF. However, not all variations in CFTR always result in CF. Therefore, for CFTR genotype to provide the same level of evidence of CFTR dysfunction as shown by direct tests such as sweat chloride or nasal potential difference, the mutations identified must be known to always result in CF. The use of CFTR genetics in CF diagnosis, therefore, relies heavily on mutation interpretation. Study design Progress that has been made on mutation interpretation and annotation was reviewed at the recent CF Foundation Diagnosis Consensus Conference. A modified Delphi method was used to identify consensus statements on the use of genetic analysis in CF diagnosis. Results The largest recent advance in CF genetics has come through the Clinical and Functional Translation of CFTR (CFTR2) project. This undertaking seeks to characterize CFTR mutations from patients with CF around the world. The project also established guidelines for the clinical, functional, and population/penetrance criteria that can be used to interpret mutations not yet included in CFTR2's review. Conclusions The use of CFTR genetics to aid in diagnosis of CF requires that the mutations identified have a known disease liability. The demonstration of 2 in trans mutations known to always result in CF is satisfactory evidence of CFTR dysfunction. However, if the identified mutations are known to be associated with variable outcomes, or have unknown consequence, that genotype may not result in a CF phenotype. In these cases, other tests of CFTR function may help.
Objective Although the majority of cases of cystic fibrosis (CF) are now diagnosed through newborn screening, there is still a need to standardize the diagnostic criteria for those diagnosed outside ...of the neonatal period. This is because newborn screening started relatively recently, it is not performed everywhere, and even for individuals who were screened, there is the possibility of a false negative. To limit irreversible organ pathology, a timely diagnosis of CF and institution of CF therapies can greatly benefit these patients. Study design Experts on CF diagnosis were convened at the 2015 CF Foundation Diagnosis Consensus Conference. The participants reviewed and discussed published works and instructive cases of CF diagnosis in individuals presenting with signs, symptoms, or a family history of CF. Through a modified Delphi methodology, several consensus statements were agreed upon. These consensus statements were updates of prior CF diagnosis conferences and recommendations. Results CF diagnosis in individuals outside of newborn screening relies on the clinical evidence and on evidence of CF transmembrane conductance regulator (CFTR) dysfunction. Clinical evidence can include typical organ pathologies seen in CF such as bronchiectasis or pancreatic insufficiency but often represent a broad range of severity including mild cases. CFTR dysfunction can be demonstrated using sweat chloride testing, CFTR molecular genetic analysis, or CFTR physiologic tests. On the basis of the large number of patients with bona fide CF currently followed in registries with sweat chloride levels between 30 and 40 mmol/L, the threshold considered “intermediate” was lowered from 40 mmol/L in the prior diagnostic guidelines to 30 mmol/L. The CF diagnosis was also discussed in the context of CFTR-related disorders in which CFTR dysfunction may be present, but the individual does not meet criteria for CF. Conclusions CF diagnosis remains a rare but important condition that can be diagnosed when characteristic clinical features are seen in an individual with demonstrated CFTR dysfunction.
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