Penetrance is a critical parameter for assessing the disease liability of CFTR variants

BackgroundMajor issues of newborn screening (NBS) for CF are the assessment of disease liability of variants and of the penetrance of clinical CF, notably in inconclusive diagnosis. The penetrance of CF is defined as the risk of a particular genotype to lead to a CF phenotype.MethodsWe aimed to get insight into the penetrance of CF for fifteen CFTR variants: 5 frequent CF-causing and 10 classified as of varying clinical consequence (VCC) or associated with a CFTR-related disorder (CFTR-RD) in CFTR2 or CFTR-France databases. The penetrance was approached by: (1) comparison of variant allelic frequencies in CF patients (CFTR2) and in the general population; (2) estimation of the likelihood of a positive NBS test for the 14 compound heterozygous with F508del and the F508del homozygous genotypes, defined as the ratio of detected/expected number of neonates with a given genotype in the 2002–2017 period.ResultsA full penetrance was observed for severe CF-causing variants. Five variants were more frequently found in the general population than in CF patients: TG11T5, TG12T5, TG13T5, L997F and R117H;T7. The likelihood of a positive NBS test was 0.03% for TG11T5, 0.3% for TG12T5, 1.9% for TG13T5, 0.6% for L997F, 11.7% for D1152H, and 17.8% for R117H;T7. Penetrance varied greatly for variants with discrepant classification between CFTR2 and CFTR-France: 5.1% for R117C, 12.3% for T338I, 43.5% for D110H and 52.6% for L206W.ConclusionThese results illustrate the contribution of genetics population data to assess the disease liability of variants for diagnosis and genetic counselling purposes.     

The CFTR polymorphisms poly-T,TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.     

Severity of the S1251N allele in cystic fibrosis is affected by the presence of the F508C variant in cis

BackgroundIn cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation.MethodsGenetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated.ResultsOrganoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation.ConclusionsIn vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF.     

Frequency of the hyperactive W493R ENaC variant in carriers of a CFTR mutation

BackgroundThe basic defect of the autosomal recessive disorder cystic fibrosis (CF) manifests in chloride hyposecretion and sodium hyperabsorption. CF-like disease has been reported in a heterozygous carrier of F508del CFTR and the hyperactive variant p.W493R-SCNN1A of the epithelial sodium channel (ENaC).MethodsThe hypothesis that heterozygosity for p.W493R-SCNN1A and one loss-of-function CFTR mutation causes or predisposes to CF or CF-like disease was tested in 441 parents of a child with CF.Resultsp.W493R-SCNN1A was detected in three female carriers of F508del CFTR who did not show any symptoms of respiratory or intestinal disease that could be interpreted as the manifestation of CF or CFTR-related disorder. Frequency of p.W493R was lower in CF parents than in Caucasian control subjects.ConclusionsA hyperactive ENaC does not necessarily cause CF-like disease in a CF gene carrier, but its low frequency in CF parents suggests that it is a risk factor.     

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.     

Expression of the inflammatory regulator A20 correlates with lung function in patients with cystic fibrosis

BackgroundA20 and TAX1BP1 interact to negatively regulate NF-κB-driven inflammation. A20 expression is altered in F508del/F508del patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also assessed.MethodsPrimary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n = 7, R117H/F508del, n = 6) and controls (age-matched, n = 8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR.ResultsSilencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control NECs and reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not TAX1BP1 expression, was proportional to FEV₁ in all CF patients (r = 0.968, p < 0.001).ConclusionsA20 expression is reduced in CF and is proportional to FEV₁. Pending confirmation in a larger study, A20 may prove a novel predictor of CF inflammation/disease severity.     

The relationship between sweat chloride levels and mortality in cystic fibrosis varies by individual genotype

Rationale

The association between CFTR genotype, sweat chloride and mortality has been inconsistent, but no previous analyses have examined the association stratified by individual genotypes.

Extensive CFTR sequencing through NGS in Brazilian individuals with cystic fibrosis: unravelling regional discrepancies in the country

BackgroundThe Brazilian population has a tri-hybrid composition with a high degree of ethnic admixture. We hypothesized that Brazilian individuals with CF from different Brazilian regions have a specific distribution of CFTR variants.MethodsIndividuals with CF with data available in the Patient Registry and without an established genotype were submitted to CFTR sequencing by Next Generation Sequencing (NGS) methodology, and results were anonymously incorporated to the Registry Database. Genotyping results were expressed as ‘positive’, ‘inconclusive’ or ‘negative’. Logistic regression models were performed to investigate the association between demographic/clinical variables and genotyping results. Mediation analysis was conducted to estimate direct and indirect effects of Brazilian region on a binary positive genotyping response.ResultsIn October 2017, data from 4,654 individuals with CF were available, and 3,104(66.7%) of them had a genotyping result. A total of 236 variants (114 new variants) were identified, with F508del identified in 46% of the alleles tested. Genotyping revealed 2,002(64.5%) individuals positive, 757(24.4%) inconclusive and 345(11.1%) negative. Distribution of genotype categories was markedly different across Brazilian Regions, with greater proportions of negative individuals in the North (45%) and Northeast (26%) regions. Newborn screening (CF-NBS) and age at diagnosis were identified as mediators of the effect of Brazilian region on a positive genotyping result.ConclusionsThis large initiative of CFTR genotyping showed significant regional discrepancies in Brazil, probably related to socio-economic conditions, lack of adequate CF-NBS and poor access to reliable sweat testing. These results may be useful to indicate Regions where CF care demands more attention.     

Cystic fibrosis in black African children in South Africa: a case control study

BackgroundCystic fibrosis (CF) is described more commonly in Caucasian populations in whom p.Phe508del is the most common mutation. There is a paucity of data of CF in black African children. The aim of this study was to describe and compare the presentation and outcomes of black African children with CF to those with p.Phe508del genotype.MethodsA retrospective case-controlled study was conducted from January 2000 – March 2018 of children with CF attending two CF centres in South Africa. Presentation, genotype, nutrition and pulmonary function outcomes of black African children were compared to matched controls with the p.Phe508del mutation.ResultsThirty-four black African children (cases) with median age of diagnosis (5.5 months, IQR 2.0-15.0) were matched to 34 controls. Among cases, 3120+1G->A CFTR mutation was most commonly identified; homozygous n=22 (64.7%) and heterozygous=7(20.5%). Compared to controls, cases at diagnosis were more malnourished and fewer presented with neonatal bowel obstruction [cases n=2 (5.9%) vs. controls n=10 (29.4%); p = 0.03]. Nutrition and pulmonary function (FEV1 in children ≥ 6 years) outcomes and changes over time from ages 3-16 years were similar in both groups; median FEV1 z-score at age 6,10 and 14 years was -0.9 (±1.5), -1.8 (±2.0) and -1.8 (±1.9) respectively for all patients. Deaths were recorded in three cases (8.8%) and one control (2.9%) (p = 0.6).ConclusionBlack African children with CF were more malnourished at diagnosis, and fewer presented with neonatal bowel obstruction. Cases and controls had comparable nutritional, pulmonary function and early mortality outcomes.     

Complete CFTR gene sequencing in 5,058 individuals with cystic fibrosis informs variant-specific treatment

BackgroundCystic fibrosis (CF) is a recessive condition caused by variants in each CF transmembrane conductance regulator (CFTR) allele. Clinically affected individuals without two identified causal variants typically have no further interrogation of CFTR beyond examination of coding regions, but the development of variant-specific CFTR-targeted treatments necessitates complete understanding of CFTR genotype.MethodsWhole genome sequences were analyzed on 5,058 individuals with CF. We focused on the full CFTR gene sequence and identified disease-causing variants in three phases: screening for known and structural variants; discovery of novel loss-of-function variants; and investigation of remaining variants.ResultsAll variants identified in the first two phases and coding region variants found in the third phase were interpreted according to CFTR2 or ACMG criteria (n = 371; 16 [4.3%] previously unreported). Full gene sequencing enabled delineation of 18 structural variants (large insertions or deletions), of which two were novel. Additional CFTR variants of uncertain effect were found in 76 F508del homozygotes and in 21 individuals with other combinations of CF-causing variants. Both causative variants were identified in 98.1% (n = 4,960) of subjects, an increase of 2.3 percentage points from the 95.8% (n = 4,847) who had a registry- or chart-reported disease-causing CFTR genotype. Of the remaining 98 individuals, 78 carried one variant that has been associated with CF (CF-causing [n = 70] or resulting in varying clinical consequences n = 8]).ConclusionsComplete CFTR gene sequencing in 5,058 individuals with CF identified at least one DNA variant in 99.6% of the cohort that is targetable by current molecular or emerging gene-based therapeutic technologies.     

Cystic fibrosis carrier frequency and estimated prevalence of the disease in Morocco

BackgroundThe epidemiology of cystic fibrosis (CF) is poorly known in North African populations, in particular in Morocco and the CF carrier frequency in the general Moroccan population has never been evaluated.MethodsTo estimate the prevalence of CF mutations in Morocco, blood samples from 150 healthy Moroccans were tested for frequent CFTR mutations and the intron 8 polyT variant.ResultsTwo subjects were heterozygous for F508del and eight others for the (T)5 variant.ConclusionThese findings indicate that the Moroccan population is at risk for CF and CFTR-related disorders. CF prevalence could be in the range of that found in European populations. Wider studies are necessary to identify the clinical pattern and accurately determine the prevalence and molecular basis of CF in Morocco.     

A survey of the prevalence,management and outcome of infants with an inconclusive diagnosis following newborn bloodspot screening for cystic fibrosis (CRMS/CFSPID) in six Italian centres

ObjectiveWe evaluated the prevalence, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene profile, clinical data, management and outcome for infants with a CFTR-related metabolic syndrome/CF Screen Positive, Inconclusive Diagnosis (CRMS/CFSPID) designation from six Italian centres.MethodsAll newborn bloodspot screening (NBS) positive infants born from January 2011 to August 2018 with a CF diagnosis or a CRMS/CFSPID designation were enrolled. Data on sweat testing, genetics, clinical course and management were collected.ResultsWe enrolled 257 CF patientsand 336 infants with a CRMS/CFSPID designation (CF: CRMS/CFSPID ratio of 1:1.30).Blood immuno-reactive trypsinogen (IRT) was significantly lower in CRMS/CFSPID infants and the F508del variant accounted for only 20% of alleles. Children with CRMS/CFSPID showed a milder clinical course, pancreatic sufficiency compared to CF infants. Varied practice across centres was identified regarding sweat testing, chest radiograph (8-100%) and salt supplementation (11-90%). Eighteen (5.3%) CRMS/CFSPID infants converted or were reclassified to diagnosis of CF. Four infants (1.3%) developed a clinical feature consistent with a CFTR-related disorder (1.2%). Twenty-seven were re-classified as healthy carriers (8.0%) and 16 as healthy infants (4.8%).ConclusionsWe have identified considerable variability in the evaluation and management of infants with an inconclusive diagnosis following NBS across six Italian centres. CRMS/CFSPID is more regularly seen in this population compared to countries with higher prevalence of F508del.Conversion to a CF diagnosis was recorded in 18 (5.3%) of CRMS/CFSPID infants and in 16 was as a result of increasing sweat chloride concentration.     

Molecular screening of CFTR gene in Egyptian patients with congenital bilateral absence of the vas deferens: a preliminary study

In the current study, we enrolled 14 Egyptian infertile males with isolated congenital bilateral absence of the vas deferens (CBAVD). Screening for the most commonly reported 36 CFTR mutations, and the intron 8 (T)n splice variant was performed by multiplex PCR followed by reversed hybridisation. Samples with the 5T variant were picked for DNA sequencing of intron 8/exon 9 region to identify the number of adjacent TG repeats. The p.Phe508del and the p.Ser1251Asn mutations were detected in heterozygous state in three patients (10.7% of alleles) and in one patient (3.6% of alleles), respectively, while the 5T variant was detected in five patients (28.6% of alleles). Among those five patients, four had TG12 repeats and one had TG13 repeats confirming the pathogenic penetrance of all 5T alleles in Egyptian CBAVD patients. The allelic frequencies of the mutations p.Phe508del, p.Ser1251Asn and the 5T variant in 60 Egyptian cystic fibrosis patients were 24.2%, 3.3% and 2.5% respectively. The mutation p.Ser1251Asn was detected for the first time in isolated CBAVD patient in our study. Due to the high prevalence of p.Phe508del mutation and 5T variant in Egyptian CBAVD patients, we recommend their screening initially, ideally followed by full CFTR gene sequencing in unidentified patients.     

Is there evidence for correct diagnosis in cystic fibrosis registries?

BackgroundCystic fibrosis (CF) spans a wide spectrum. Therefore, benchmarking between registries implies comparing similar cohorts.Objective and methodsExplore patient characteristics in Belgian (B), French (F), German (G) and Dutch (NL) registries (total N = 13,122) and determine whether they fulfill predefined diagnostic criteria.ResultsUsing as case definition sweat chloride > 60 mmol/L or 2 CFTR mutations identified, CF diagnosis was not documented in 2.8, 5.7, 6.5 and 21.6% of subjects in the F, B, NL, and G registries. Restricting CFTR mutation interpretation to 124 CF causing mutations in CFTR2, these numbers rose to 10.5, 10.4, 14.5 and 24.3% respectively. Excluding these subjects impacted on outcomes. The impact differed between countries; the largest changes seen were a decrease in % adults from 51.9 to 47.8% in G, a decrease in % pancreas sufficiency from 17.0 to 13.0 in F, an increase in % homozygous for F508del from 55.3 to 63.7 in NL and a decrease of % with sweat chloride ≤ 60 mmol/L from 8.4 to 1.1 in B.ConclusionCF diagnosis is not documented in 10 to 24% of patients included in CF registries. Excluding these patients for analyses leads to significant changes in outcomes.     

Nasal potential difference in suspected cystic fibrosis patients with 5T polymorphism

Background5T polymorphism is a CFTR mutation with unclear clinical consequences: the phenotype varies from healthy individuals to Cystic Fibrosis (CF). The aim of this study was to evaluate if nasal potential difference (NPD) and sweat testing correlate with symptoms and CF diagnosis in 5T patients.Methods86 patients with 5T who had undergone NPD measurement, were included (6 homozygous (5T/5T), 41 with a PI-CF causing mutation in trans (5T/PI-CF), 11 with a PS-CF causing mutation in trans (5T/PS-CF) and 28 without a known mutation in trans (5T/?). Data including age, phenotype, sweat chloride and follow up were collected.Results33% of the 5T/5T patients had abnormal NPD results, compared to 70% in 5T/PI-CF; 33% in 5T/PS-CF and 29% in 5T/?. The percentage of high or borderline sweat chloride was highest in 5T/PI-CF, and 5T/?, compared to 5T/5T and 5T/PS-CF (91, 96, 80, and 63%, respectively). TGm (number of TG repeats in intron 8) analysis was performed in 21 5T/PI-CF patients. TG11 was associated with lower sweat chloride, lower percentage of abnormal NPD and less progression of symptoms compared to TG12 and TG13.ConclusionThere is much variation in clinical status among 5T patients. All patients in this study with 5T/PS CF, all patients with both normal NPD and sweat test, and most patients with TG11 were stable or improving over time. Therefore, NPD measurement and TGm status aid to assess if a patient is at high risk for developing CF or CFTR-related disease and if specific follow up in a CF center is required.     

Modification of the salivary secretion assay in F508del mice — The murine equivalent of the human sweat test

BackgroundIn 2005 Best and Quinton established the salivary secretion assay in mice for the in vivo characterization of new drugs against cystic fibrosis (CF). However, limited data are available and the predictive value of this in vivo assay for treatment effects in CF patients is not fully understood.MethodsTherefore, we revisited the salivary secretion assay and systematically investigated the salivary secretion rates in different murine backgrounds and sexes, as well as in different CF mouse models. Moreover, we established quantification of salivary chloride content.ResultsWe found a strain- and sex-dependency of salivary secretion rates and were able to confirm the decreased β-adrenergic salivary secretion response in CFTR knockout mice (CFTR^tm1Unc) as well as in the F508del CFTR mice of different origins (CFTR^tm1Kth and CFTR^tm1Eur). In heterozygous Cftr +/? and Cftr +/F508del mice, the isoprenaline-stimulated salivary secretion rate and the Cl^? content were intermediate between values measured in WT and CF mice, indicating that this assay is also able to detect CF carriership. Pilocarpine-induced abnormalities in saliva chloride content in CF mice resembled the changes observed in the human sweat test.ConclusionsDetermination of murine salivary chloride content in combination with salivary secretion rate in CF mice may render the salivary secretion assay as a powerful tool for validation of new CF treatments.     

Ventilatory pattern and energy expenditure are altered in cystic fibrosis mice

BackgroundAltered ventilatory pattern and increased energy expenditure are facets of the complex cystic fibrosis (CF) phenotype. It is not known whether these are inherent attributes of CF, secondary consequences of lung infection or other disease complications.MethodsStudies were performed in congenic C57BL/6J, F508del (Cftr^tm1kth) and CF gut-corrected (F508del) mice. Ventilatory patterns were measured using whole-body plethysmography. Indirect calorimetry was used to determine oxygen consumption, carbon dioxide production and resting energy expenditure.ResultsCF mice (F508del and F508del gut-corrected) have a significantly faster respiratory rate and increased ventilatory pattern variability as compared to non-CF mice. F508del but not CF gut-corrected mice had significantly increased energy expenditure per gram body weight.ConclusionsCF mice exhibit a faster, more variable ventilatory pattern. These changes were present in the absence of detectable infection or illness due to gastrointestinal obstruction. Increased resting energy expenditure does not completely account for these differences.     

Antisense oligonucleotide eluforsen improves CFTR function in F508del cystic fibrosis

BackgroundCystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. In this study we assessed the effect of antisense oligonucleotide eluforsen on CFTR biological activity measured by Nasal Potential Difference (NPD) in patients with the most common mutation, F508del-CFTR.MethodsThis multi-centre, exploratory, open-label study recruited adults with CF homozygous or compound heterozygous for the F508del-CFTR mutation. Subjects received intranasal eluforsen three times weekly for 4 weeks. The primary endpoint was the within-subject change from baseline in total chloride transport (Cl-free+iso), as assessed by NPD. Secondary endpoints included within-subject change from baseline in sodium transport.ResultsIn the homozygous cohort (n = 7; per-protocol population), mean change (90% confidence interval) in Cl-free+iso was ?3.0 mV (?6.6; 0.6) at day 15, ?4.1 mV (?7.8; ?0.4, p = .04) at day 26 (end of treatment) and ? 3.7 mV (?8.0; 0.6) at day 47. This was supported by improved sodium transport as assessed by an increase in average basal potential difference at day 26 of +9.4 mV (1.1; 17.7, p = .04). The compound heterozygous cohort (n = 7) did not show improved chloride or sodium transport NPD values. Eluforsen was well tolerated with a favourable safety profile.ConclusionsIn F508del-CFTR homozygous subjects, repeated intranasal administration of eluforsen improved CFTR activity as measured by NPD, an encouraging indicator of biological activity.