The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

[Cardiac amyloidosis: State of art in 2022]. / Les amyloses cardiaques : état des lieux en 2022.

The 3 main types of cardiac amyloidosis are linked to two protein precursors: AL amyloidosis secondary to free light chain deposits in the context of monoclonal gammopathy (mainly of undetermined significance or myeloma) and transthyretin amyloidosis (ATTR), comprising wild-type transthyretin amyloidosis (ATTRwt for wild type) and hereditary transthyretin amyloidosis (ATTRv for variant). These diseases are underdiagnosed and highly prevalent in common cardiac phenotypes in recent studies (heart failure with preserved ejection fraction, severe aortic stenosis, hypertrophic cardiomyopathy). Myocardial amyloid infiltration affects all cardiac structures and clinically promotes predominantly heart failure, conductive disorders and cardioembolic events. The search for extracardiac signs makes it possible to arouse diagnostic suspicion. Electrocardiogram, echocardiography and cardiac MRI can suspect cardiac amyloidosis. The diagnostic confirmation follows a simple algorithm including a systematic search for monoclonal gammapathy and a disphosphonate scintigraphy. Histological proof is necessary in case of AL or ATTR amyloidosis with concomitant monoclonal gammopathy in order to initiate specific treatment. Due to the late disease onset in ATTRv, genetic testing must be routine in all cases of ATTR. These diseases are no longer perceived as incurable since recent therapeutic innovations. A better knowledge of the disease is more than ever necessary.

Dose-response effect of serum butyrylcholinesterase activity after clinical doses of pancuronium.

INTRODUCTION: Low-dose pancuronium is known to affect serum cholinesterase activity (BChE); however, the dose-response effect of clinical doses of pancuronium on BChE has not been investigated. METHODS: Thirteen ASA I-II patients scheduled for elective surgery requiring muscle relaxation were enrolled in this study. All patients had normal BChE before surgery. Incremental doses of pancuronium (10, 20, 50, and 100 microg/kg) were injected in accordance with surgical needs every 45 min. BChE was measured 3 min after injection by an automatic colorimetric method. RESULTS: BChE decreased significantly in all except one patient in comparison to the baseline (P < 0.05). However all values remained within normal clinical range. A dose of 100 microg/kg yielded significant decrease in comparison to 10 microg/kg but not to other dosages. Linear regression was not significant for the dose-response relationship (P = 0.05). CONCLUSION: After clinical incremental doses of pancuronium, BChE remained within clinical range.

Identification of mutations in the putative ATP-binding domain of the adrenoleukodystrophy gene.

The recently identified adrenoleukodystrophy (ALD) gene is predicted to encode a peroxisomal protein of 745 amino acids that includes one domain for ATP-binding, termed nucleotide-binding fold (NBF). To determine whether mutations occur in the putative NBF of ALD protein, we analyzed by denaturing gradient gel electrophoresis (DGGE) exon 6 and 8 that encode most part of this domain in 50 ALD patients. Four amino acid substitutions, three frameshift mutations leading to premature termination signal, and a splicing mutation were identified. These amino acid substitutions occurred at residues highly conserved in other ATP-binding cassette (ABC) proteins. In addition, a nonsense mutation was detected in exon 4.

Structure-function analysis of a double-mutant cystic fibrosis transmembrane conductance regulator protein occurring in disorders related to cystic fibrosis.

A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N cystic fibrosis transmembrane conductance regulator, expressed in HeLa cells, to evaluate the contribution of each mutation in the phenotype. We found that R74W cystic fibrosis transmembrane conductance regulator appears to be a polymorphism, while D1270N cystic fibrosis transmembrane conductance regulator could be responsible for the congenital bilateral absence of the vas deferens phenotype. The combination of the two produced a more severe effect on the chloride conductance pathway as well as on the phenotype.

[The cystic fibrosis gene, its product CFTR protein and its mutations]. / Le gène de la mucoviscidose, son produit la protéine CFTR et ses mutations.

Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians. Its etiology is complex, but it is chiefly a disease of electrolyte transport characterized by defects in fluid secretion by several epithelia. In this review are analyzed the data obtained since the cloning of the CF gene and the characterization of its product, the CF transmembrane conductance regulator (CFTR) protein, which has been shown to act like a cAMP-regulated chloride channel. This protein is a member of a family of ATP-binding proteins that are membrane-spanning, are found in a number of prokaryotic and eucaryotic cells, and have two ATP-binding domains. Unique to this family of proteins, the CFTR possesses an additional highly charged domain (the R domain). The majority of CF chromosomes (70%) have a single Phenylalanine codon deletion at position 508 of the protein (delta F508). A large number of other rare mutations (more than 230) have also been identified. This rapid accumulation of data is essential to genetic diagnosis and will aid in understanding the structure and function of the protein.

[Cystic fibrosis: the CFTR gene, its mutations, the genetic counseling]. / La mucoviscidose: le gène CFTR, ses mutations, le conseil génétique.

Cystic fibrosis is the most frequent autosomic recessively inherited disease in the European population. The gene implicated in this disease was cloned in 1989 but the consequences of the biochemical defect in the cell have not been fully elucidated. To date, 500 mutations of this 230 kilobase gene have been identified. These molecular anomalies each have an effect on the encoded protein (CFTR) an ion channel which appears to play a role in regulator functions. Results of the gene cloning and research into the different mutations have led to the development of effective strategies for molecular diagnosis facilitating genetic counselling for families at risk and the identification of atypical forms of the disease.

[Lung diseases in children associated with inherited disorders of surfactant metabolism]. / Pathologies respiratoires de l'enfant associées à des anomalies héréditaires du métabolisme du surfactant.

Pulmonary surfactant is a unique mixture of lipids and specific proteins that reduces surface tension at the air-liquid interface, preventing collapse of the lung at the end of expiration. Recessive loss-of-function mutations of pulmonary surfactant protein B (SP-B) was initially described in infants who develop respiratory failure at birth. More recently, mutations in other constitutive surfactant proteins like surfactant protein C or implied in its metabolism like ATP-binding cassette, sub-family A, member 3 (ABCA3) or NK2 homeobox (NKX2-1) were identified in newborn with respiratory distress but also in children with diffuse infiltrative pneumonia. Intra-alveolar accumulation of protein related to surfactant dysfunction leads to cough, hypoxemia and radiological abnormalities including ground-glass opacities and lung cysts. The clinical and radiological features associated with these genetic disorders, along with their treatment and outcome, are reviewed.

[Genetic disorders of surfactant]. / Pathologies génétiques du surfactant.

Lung diseases associated with surfactant metabolism disorders represent a significant but heterogeneous group of rare disorders. Intra-alveolar accumulation of protein related to surfactant dysfunction leads to cough, hypoxemia and radiological diffuse infiltration. Inherited deficiency of pulmonary surfactant protein B (SP-B) was initially described in term newborns who develop severe respiratory failure at birth. More recently, mutations in surfactant protein C (SP-C) or in proteins required for surfactant synthesis such as ATP-binding cassette, sub-family A, member 3 (ABCA3) or NK2 homeobox 1 (NKX2-1) were identified in newborns with respiratory distress but also in children with diffuse infiltrative pneumonia. The aim of this review is to describe the clinical presentation of these diseases but also the diagnostic tools and the treatments options available.

A 3' splice site consensus sequence mutation in the cystic fibrosis gene.

In the cystic fibrosis (CF) gene, recently cloned, a three base pair deletion (delta F508) has been identified in a majority of CF patients. This deletion has been found in 80% of CF chromosomes in families from north west Brittany. In order to identify new mutations we have selected 43 chromosomes negative for the three base pair deletion from these families and directly sequenced exon 11 after DNA amplification by the polymerase chain reaction. We have detected a base change (G----A) at the 3' end of the consensus sequence of intron ten (namely 1717-1). This mutation destroys a splice site in the cystic fibrosis gene which probably produces a mutant allele. This single nucleotide mutation has been reported on two other CF chromosomes.

A rapid, efficient, and sensitive assay for simultaneous detection of multiple cystic fibrosis mutations.

We describe the use of DGGE multiplex systems for rapid analysis of 15 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which about half of the known CF molecular defects are clustered. We have previously determined the spectrum of mutations affecting the CFTR gene in the French population using a strategy based on denaturing gradient gel electrophoresis (DGGE) of amplified gene segments. Analysis of CF patients' DNA with five DGGE multiplex systems permitted us to characterize nearly 35% of non-delta F508 CF alleles and increased the CF allele detection rate to almost 82% in this population. This simple and rapid multiplex analysis strategy, which allows a significant proportion of the most frequent CF mutations in Caucasians to be detected, will be helpful in the implementation of genetic screening programs.

A comprehensive scanning method for rapid detection of beta-globin gene mutations and polymorphisms.

We describe a scanning procedure for the detection of beta-globin gene mutations and the prenatal diagnosis of beta-thalassemias. The method is based on the combined use of PCR and denaturing gradient gel electrophoresis (DGGE) of six amplified fragments encompassing the whole beta-globin coding region and splice junctions, as well as the promoter and 3' untranslated regions. The whole beta-globin gene can be rapidly scanned for the presence of deleterious mutations. The proposed diagnostic strategy provides a major improvement over current approaches to beta-globin gene analysis in both research and clinical laboratories, especially those which analyse DNA samples from individuals belonging to various ethnic or population groups. The use of this procedure has enabled us to detect six novel sequence changes in the beta-globin gene, including two deleterious mutations and four polymorphisms.

Identification of eight mutations and three sequence variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

To determine cystic fibrosis (CF) defects in a sample of 224 non-delta F508 CF chromosomes, we used denaturing gradient gel multiplex analysis of CF transmembrane conductance regulator gene segments, a strategy based on blind exhaustive analysis rather than a search for known mutations. This process allowed us to detect 11 novel variations comprising two nonsense mutations (Q890X and W1204X), a splice defect (405 + 4 A-->G), a frameshift (3293delA), four presumed missense mutations (S912L, H949Y, L1065P, Q1071P), and three sequence polymorphisms (R31C or 223 C/T, 3471 T/C, and T1220I or 3791 C/T). We describe these variations, together with the associated phenotype when defects on both CF chromosomes were identified.

Three point mutations in the CFTR gene in French cystic fibrosis patients: identification by denaturing gradient gel electrophoresis.

The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (delta F508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-delta F508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.

Cystic fibrosis phenotype associated with pancreatic insufficiency does not always reflect the cAMP-dependent chloride conductive pathway defect. Analysis of C225R-CFTR and R1066C-CFTR.

We have previously screened the cystic fibrosis transmembrane conductance regulator (CFTR) gene and identified new disease-causing mutations. C225R and R1066C are both associated with pancreatic insufficiency, but the former mutation is associated with mild and unusual lung disease, whereas the latter is associated with severe lung disease. In the present study, we expressed these mutants heterologously in HeLa cells, and we analyzed protein synthesis by immunoprecipitation and chloride channel function by using a halide-sensitive fluorescent dye, 6-methoxy-N-ethylquinolinium. Immunoprecipitation and functional studies showed that cells transfected with C225R-CFTR exhibit cAMP-dependent chloride fluxes; C225R-CFTR protein is poorly expressed but fully glycosylated and can be compared with R117H-CFTR. R1066C-CFTR protein is not correctly processed and, unlike DeltaF508-CFTR, this defect cannot be corrected by reduced temperature or overexpression in butyrate-treated cells; defective processing may occur at a different step in the biosynthetic pathway. These results point to two different mechanisms underlying the same pancreatic status and suggest that it is unwise to use pancreatic sufficiency and insufficiency to define mild and severe cystic fibrosis (CF) disease, respectively. Finally, the experimental model described here may be helpful to predict the pulmonary status of CF patients bearing mutations located in putative membrane-spanning domains of the CFTR protein.

Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function.

The number of complex cystic fibrosis transmembrane conductance regulator (CFTR) genotypes identified as having double-mutant alleles with two mutations inherited in cis has been growing. We investigated the structure-function relationships of a severe cystic fibrosis (CF)-associated double mutant (R347H-D979A) to evaluate the contribution of each mild mutation to the phenotype. CFTR mutants expressed in HeLa cells were analyzed for protein biosynthesis and Cl(-) channel activity. Our data show that R347H is associated with mild defective Cl(-) channel activity and that the D979A defect leads to misprocessing. The mutant R347H-D979A combines both defects for a dramatic decrease in Cl(-) current. To decipher the molecular mechanism of this phenotype, single and double mutants with different charge combinations at residues 347 and 979 were constructed as charged residues were involved in this complex genotype. These studies revealed that residue 979, located in the third cytoplasmic loop, is critical for CFTR processing and Cl(-) channel activity highlighting the role of charged residues. These results have also important implications for CF, as they show that two mutations in cis can act in concert to alter dramatically CFTR function contributing to the wide phenotypic variability of CF disease.

Exhaustive screening of exon 10 CFTR gene mutations and polymorphisms by denaturing gradient gel electrophoresis: applications to genetic counselling in cystic fibrosis.

Several mutations in the cystic fibrosis (CF) gene have been reported. Ten, including the most prevalent mutation in Caucasians, delta F508, are located in exon 10 of the gene, in addition to five sequence polymorphisms. We have previously presented a strategy based on denaturing gradient gel analysis for the rapid detection of any nucleotide variation in all the exons and their immediate flanking regions. We now report the application of this method to the simultaneous detection of all mutations and polymorphisms located in exon 10, together with the use of exon 10 polymorphisms, especially the most frequent one (M470V), as intragenic markers for prenatal diagnosis of cystic fibrosis in families with at least one affected child and unknown disease-causing mutations.

Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.

The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.