Putting bicarbonate on the spot: pharmacological insights for CFTR correction in the airway epithelium.

Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl-) and bicarbonate (HCO3 -) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3 - secretion by studying transepithelial HCO3 - fluxes. Methods: HCO3 - secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3 - transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO3 - secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3 - transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3 - transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO3 - is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3 - secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.

Theratyping cystic fibrosis patients to guide elexacaftor/tezacaftor/ivacaftor out-of-label prescription.

BACKGROUND: Around 20% of people with cystic fibrosis (pwCF) do not have access to the triple combination elexacaftor/tezacaftor/ivacaftor (ETI) in Europe because they do not carry the F508del allele on the CF transmembrane conductance regulator (CFTR) gene. Considering that pwCF carrying rare variants may benefit from ETI, including variants already validated by the US Food and Drug Administration (FDA), a compassionate use programme was launched in France. PwCF were invited to undergo a nasal brushing to investigate whether the pharmacological rescue of CFTR activity by ETI in human nasal epithelial cell (HNEC) cultures was predictive of the clinical response. METHODS: CFTR activity correction was studied by short-circuit current in HNEC cultures at basal state (dimethyl sulfoxide (DMSO)) and after ETI incubation and expressed as percentage of normal (wild-type (WT)) CFTR activity after sequential addition of forskolin and Inh-172 (ΔI ETI/DMSO%WT). RESULTS: 11 pwCF carried variants eligible for ETI according to the FDA label and 28 carried variants not listed by the FDA. ETI significantly increased CFTR activity of FDA-approved CFTR variants (I601F, G85E, S492F, M1101K, R347P, R74W;V201M;D1270N and H1085R). We point out ETI correction of non-FDA-approved variants, including N1303K, R334W, R1066C, Q552P and terminal splicing variants (4374+1G>A and 4096-3C>G). ΔI ETI/DMSO%WT was significantly correlated to change in percentage predicted forced expiratory volume in 1 s and sweat chloride concentration (p<0.0001 for both). G85E, R74W;V201M;D1270N, Q552P and M1101K were rescued more efficiently by other CFTR modulator combinations than ETI. CONCLUSIONS: Primary nasal epithelial cells hold promise for expanding the prescription of CFTR modulators in pwCF carrying rare mutants. Additional variants should be discussed for ETI indication.

Standards for the care of people with cystic fibrosis (CF): A timely and accurate diagnosis.

There is considerable activity with respect to diagnosis in the field of cystic fibrosis (CF). This relates primarily to developments in newborn bloodspot screening (NBS), more extensive gene analysis and improved characterisation of CFTR-related disorder (CFTR-RD). This is particularly pertinent with respect to accessibility to variant-specific therapy (VST), a transformational intervention for people with CF with eligible CFTR gene variants. This advance reinforces the need for a timely and accurate diagnosis. In the future, there is potential for trials to assess effectiveness of variant-specific therapy for CFTR-RD. The guidance in this paper reaffirms previous standards, clarifies a number of issues, and integrates emerging evidence. Timely and accurate diagnosis has never been more important for people with CF.

Nonsense mutations accelerate lung disease and decrease survival of cystic fibrosis children.

RATIONALE: Limited information is available on the clinical status of people with Cystic Fibrosis (pwCF) carrying 2 nonsense mutations (PTC/PTC). The main objective of this study was to compare disease severity between pwCF PTC/PTC, compound heterozygous for F508del and PTC (F508del/PTC) and homozygous for F508del (F508del+/+). METHODS: Based on the European CF Society Patient Registry clinical data of pwCF living in high and middle income European and neighboring countries, PTC/PTC (n = 657) were compared with F508del+/+ (n = 21,317) and F508del/PTC(n = 4254).CFTR mRNA and protein activity levels were assessed in primary human nasal epithelial (HNE) cells sampled from 22 PTC/PTC pwCF. MAIN RESULTS: As compared to F508del+/+ pwCF; both PTC/PTC and F508del/PTC pwCF exhibited a significantly faster rate of decline in Forced Expiratory Volume in 1 s (FEV1) from 7 years (-1.33 for F508del +/+, -1.59 for F508del/PTC; -1.65 for PTC/PTC, p < 0.001) until respectively 30 years (-1.05 for F508del +/+, -1.23 for PTC/PTC, p = 0.048) and 27 years (-1.12 for F508del +/+, -1.26 for F508del/PTC, p = 0.034). This resulted in lower FEV1 values in adulthood. Mortality of pediatric pwCF with one or two PTC alleles was significantly higher than their F508del homozygous pairs. Infection with Pseudomonas aeruginosa was more frequent in PTC/PTC versus F508del+/+ and F508del/PTC pwCF. CFTR activity in PTC/PTC pwCF's HNE cells ranged between 0% to 3% of the wild-type level. CONCLUSIONS: Nonsense mutations decrease the survival and accelerate the course of respiratory disease in children and adolescents with Cystic Fibrosis.

Non-invasive prenatal diagnosis of single gene disorders with enhanced relative haplotype dosage analysis for diagnostic implementation.

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

The French Compassionate Program of elexacaftor-tezacaftor-ivacaftor in people with cystic fibrosis with advanced lung disease and no F508del CFTR variant.

BACKGROUND: The European Medicines Agency has approved the cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination elexacaftor-tezacaftor-ivacaftor (ETI) for people with cystic fibrosis (pwCF) carrying at least one F508del variant. The United States Food and Drug Administration (FDA) also approved ETI for pwCF carrying one of 177 rare variants. METHODS: An observational study was conducted to evaluate the effectiveness of ETI in pwCF with advanced lung disease that were not eligible to ETI in Europe. All patients with no F508del variant and advanced lung disease (defined as having a percent predicted forced expiratory volume (ppFEV1)<40 and/or being under evaluation for lung transplantation) and enrolled in the French Compassionate Program initiated ETI at recommended doses. Effectiveness was evaluated by a centralized adjudication committee at 4-6 weeks in terms of clinical manifestations, sweat chloride concentration and ppFEV1. RESULTS: Among the first 84 pwCF included in the program, ETI was effective in 45 (54%) and 39 (46%) were considered to be non-responders. Among the responders 22/45 (49%) carried a CFTR variant that is not currently approved by FDA for ETI eligibility. Important clinical benefits, including suspending the indication for lung transplantation, a significant decrease in sweat chloride concentration by a median [IQR] -30 [-14;-43]mmol·l-1 (n=42; p<0.0001) and an improvement in ppFEV1 by+10.0 [6.0; 20.5] (n=44, p<0.0001), were observed in those for whom treatment was effective. CONCLUSION: Clinical benefits were observed in a large subset of pwCF with advanced lung disease and CFTR variants not currently approved for ETI.

SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing.

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/.

WITHDRAWN: Clinical interpretation of PRSS1 gene variants in patients with pancreatitis.

Since the description of the PRSS1 gene encoding the cationic trypsinogen as being involved in dominant hereditary pancreatitis, more than 50 PRSS1 gene variants have been reported. Among those that have been classified as pathogenic, some have a high penetrance and others have a low penetrance. Assessing the clinical relevance of PRSS1 variants is often complicated in the absence of functional evidence and interpretation regarding rare variants is not very easy in clinical practice. The aim of this study was to review the PRSS1 variants and to classify them according to their degree of deleterious effect. This classification was based on the results of in vitro experiments and on population data, in comparing the allelic frequency of each variant in patients with pancreatitis and in unaffected individuals. This review should help geneticists and clinicians in charge of patient...s care and genetic counseling to interpret results of genetic studies.

Non-invasive prenatal diagnosis of single gene disorders by paternal mutation exclusion: 3 years of clinical experience.

OBJECTIVES: Cell-free fetal DNA (cffDNA) analysis is performed routinely for aneuploidy screening, RhD genotyping or sex determination. Although applications to single gene disorders (SGD) are being rapidly developed worldwide, only a few laboratories offer cffDNA testing routinely as a diagnosis service for this indication. In a previous report, we described a standardised protocol for non-invasive exclusion of paternal variant in SGD. Three years later, we now report our clinical experience with the protocol. DESIGN: Descriptive study. SETTING: Multi-centre French. POPULATION: Indications for referral included pregnancies at risk of 25% or 50% of paternally inherited SGD, and pregnancies associated with an increased risk of SGD due to a de novo variant, either from strongly suggestive ultrasound findings or from a possible parental germinal mosaicism in the context of a previously affected child. METHODS: Non-invasive prenatal diagnosis was performed using custom assays for droplet digital PCR. Feasibility, diagnostic performance and turn-around time were evaluated. RESULTS: Mean time for a new assay design and validation was evaluated at 14 days, and mean result reporting time was 6 days. All referred pathogenic variants could be targeted except one located in a complex genomic region. A result was obtained for every 198 referrals except two. CONCLUSION: This service was successfully implemented as a routine laboratory practice. It has been widely adopted by French clinicians and patients for paternal variant exclusion in various disorders. TWEETABLE ABSTRACT: A robust approach to non-invasive prenatal exclusion of paternal pathogenic variant in a diagnosis setting.

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

BACKGROUND: Newborn 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 METHODS: We 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. RESULTS: Extensive 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). CONCLUSION: This 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.

[Genetics of complex and syndromic palmoplantar keratoderma]. / Génétique des kératodermies palmoplantaires complexes et syndromiques.

Palmoplantar keratodermas (PPK) comprise a heterogenous group of acquired and hereditary disorders marked by excessive thickening of the epidermis of palms and soles. Hereditary PPKs can be classified into 3 groups: 1) isolated non-syndromic PPKs; 2) complex non-syndromic PPKs associated with other ectodermal defects; and 3) syndromic PPKs associated with extracutaneous manifestations. All types of inheritance have been observed: autosomal dominant, autosomal recessive, X-linked recessive, and mitochondrial. Some of these disorders are restricted to geographic isolates. This review describes the different genetic causes of hereditary syndromic and complex PPKs for which the genes have been identified. The identification of pathogenic variants has consequences in terms of genetic counseling, appropriate medical care and follow-up, especially for PPKs predisposing to hearing loss, cardiomyopathies, benign tumors or carcinomas. In addition, the development of targeted therapies based on pathophysiology of disorders should allow a more effective treatment of these conditions in the near future.

Clinical interpretation of PRSS1 variants in patients with pancreatitis.

Since the description of the PRSS1 gene encoding the cationic trypsinogen as being involved in dominant hereditary pancreatitis, more than 50 PRSS1 variants have been reported. Among the PRSS1 variants that have been classified as pathogenic, some have a high penetrance and others have a low penetrance. Assessing the clinical relevance of PRSS1 variants is often complicated in the absence of functional evidence and interpretation of rare variants is not very easy in clinical practice. The aim of this study was to review the different variants identified in the PRSS1 gene and to classify them according to their degree of deleterious effect. This classification was based on the results of several in vitro experiments and on population data, in comparing the allelic frequency of each variant in patients with pancreatitis and in unaffected individuals. This review should help geneticists and clinicians in charge of patient's care and genetic counseling to interpret molecular results.

Exon identity influences splicing induced by exonic variants and in silico prediction efficacy.

BACKGROUND: Minigenes and in silico prediction tools are commonly used to assess the impact on splicing of CFTR variants. Exon skipping is often neglected though it could impact the efficacy of targeted therapies. The aim of the study was to identify exon skipping associated with CFTR variants and to evaluate in silico predictions of seven freely available software. METHODS: CFTR basal exon skipping was evaluated on endogenous mRNA extracted from non-CF nasal cells and on two CFTR minigene banks. In silico tools and minigene systems were used to evaluate the impact of CFTR exonic variants on exon skipping. RESULTS: Data showed that out of 65 CFTR variants tested, 26 enhanced exon skipping and that in silico prediction efficacy was of 50%-66%. Some in silico tools presented predictions with a bias towards the occurrence of splicing events while others presented a bias towards the absence of splicing events (non-detection including true negatives and false negatives). Classification of exons depending on their basal exon skipping level increased prediction rates up to 80%. CONCLUSION: This study indicates that taking basal exon skipping into account could orientate the choice of the in silico tools to improve prediction rates. It also highlights the need to validate effects using in vitro assays or mRNA studies in patients. Eventually, it shows that variant-guided therapy should also target exon skipping associated with variants.

The Role of Extended CFTR Gene Sequencing in Newborn Screening for Cystic Fibrosis.

There has been considerable progress in the implementation of newborn screening (NBS) programs for cystic fibrosis (CF), with DNA analysis being part of an increasing number of strategies. Thanks to advances in genomic sequencing technologies, CFTR-extended genetic analysis (EGA) by sequencing its coding regions has become affordable and has already been included as part of a limited number of core NBS programs, to the benefit of admixed populations. Based on results analysis of existing programs, the values and challenges of EGA are reviewed in the perspective of its implementation on a larger scale. Sensitivity would be increased at best by using EGA as a second tier, but this could be at the expense of positive predictive value, which improves, however, if EGA is applied after testing a variant panel. The increased detection of babies with an inconclusive diagnosis has proved to be a major drawback in programs using EGA. The lack of knowledge on pathogenicity and penetrance associated with numerous variants hinders the introduction of EGA as a second tier, but EGA with filtering for all known CF variants with full penetrance could be a solution. The issue of incomplete knowledge is a real challenge in terms of the implemention of NBS extended to many genetic diseases.

Two frequent loss-of-function mutations in Aurora Kinase C gene in Algerian infertile men with macrozoospermia.

Macrozoospermia is associated with severe male infertility. To date, the only gene implicated in this phenotype is the Aurora Kinase C gene. We report in this work the genetic screening of AURKC mutations in 34 patients with macrozoospermia among 3,536 Algerian infertile men. Nineteen patients (56%) were homozygotes for the c.144delC mutation, eight (23.52%) homozygotes for the c.744C>G (p.Y248*) mutation and two (5.88%) compound heterozygotes. No AURKC mutation was identified in five patients (14.7%). Interestingly and although it is generally accepted that nearly all positive mutated AURKC patients have close to 100% large-head spermatozoa, our results showed that 11 patients with AURKC mutations (32.35%) had large-headed spermatozoa lower than 70% (7 with c.144delC and 4 with p.Y248*), and no mutation was found in 2 patients who had >70% of macrocephalic spermatozoa. Twenty ICSI attempts were performed before genetic screening resulting in 39 embryos but no pregnancy was obtained. The sequencing of AURKC exons 3 and 6 is appropriate as a first-line genetic exploration in these patients to avoid unsuccessful ICSI attempts. A percentage of large head spermatozoa beyond 25% and a percentage of multiflagellar spermatozoa beyond 10% are predictive of a positive mutation diagnosis.

Clinical interpretation of SPINK1 and CTRC variants in pancreatitis.

Since the description of the SPINK1 gene encoding the serine protease inhibitor Kazal type 1 and the CTRC gene encoding the Chymotrypsin C as being involved in chronic pancreatitis, more than 56 SPINK1 and 87 CTRC variants have been reported. Assessing the clinical relevance of SPINK1 and CTRC variants is often complicated in the absence of functional evidence and interpretation of rare variants is not very easy in clinical practice. The aim of this study was to review the different variants identified in these two genes and to classify them according to their degree of damaging effect. This classification was based on the results of in vitro experiments, in silico analysis using different prediction tools, and on population data, in comparing the allelic frequency of each variant in patients with pancreatitis and in unaffected control individuals. This review should help geneticists and clinicians in charge of patient's care and genetic counseling to interpret the results of genetic studies.