RESUMEN
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.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Exones , Empalme del ARN/genética , Mutación Missense , MutaciónRESUMEN
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.
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Neuropatías Amiloides Familiares , Cardiomiopatías , Insuficiencia Cardíaca , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Gammopatía Monoclonal de Relevancia Indeterminada , Neuropatías Amiloides Familiares/complicaciones , Neuropatías Amiloides Familiares/diagnóstico , Neuropatías Amiloides Familiares/terapia , Cardiomiopatías/diagnóstico , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnósticoRESUMEN
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.
Asunto(s)
Enfermedades Pulmonares/etiología , Edad de Inicio , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/epidemiología , Enfermedades Pulmonares/terapia , Enfermedades Pulmonares Intersticiales/complicaciones , Enfermedades Pulmonares Intersticiales/diagnóstico , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/terapia , Síndrome de Dificultad Respiratoria del Recién Nacido/diagnóstico , Síndrome de Dificultad Respiratoria del Recién Nacido/etiología , Síndrome de Dificultad Respiratoria del Recién Nacido/terapiaRESUMEN
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.
Asunto(s)
Enfermedades del Recién Nacido/genética , Proteínas Asociadas a Surfactante Pulmonar/genética , Humanos , Recién Nacido , MutaciónRESUMEN
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.
Asunto(s)
Butirilcolinesterasa/sangre , Fármacos Neuromusculares no Despolarizantes/farmacología , Pancuronio/farmacología , Adulto , Anciano , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/genética , Mutación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Pruebas de PrecipitinaRESUMEN
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.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Polimorfismo Genético , Alelos , Sustitución de Aminoácidos , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Genotipo , Células HeLa , Humanos , Mamíferos , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , TransfecciónRESUMEN
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.
Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Fibrosis Quística/fisiopatología , Páncreas/fisiopatología , Sustitución de Aminoácidos , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Fenotipo , Relación Estructura-ActividadRESUMEN
The properties of a protein-kinase-A(PKA)-activated Cl(-)-conductive pathway(s) in alkaline phosphatase-enriched microsomes from the rat inner medulla (IMV) were investigated. Transcripts of cystic fibrosis transmembrane regulator (CFTR) were detected by reverse transcription/polymerase analysis of total RNA from the inner medulla, while immunoblot analysis using anti-CFTR antibodies detected a 170-kDa protein in the IMV. The PKA Cl(-)-conductive pathway(s) was studied by measuring the rate of valinomycin-induced microsomal swelling by light scattering. PKA increased the rate of valinomycin-induced swelling of vesicles consistent with the presence of Cl(-)-conductive pathway(s). The pharmacological properties and anion selectivity of the PKA-activated Cl(-)-conductive pathway(s) were similar to those of the CFTR Cl(-) channel. Our results show that a CFTR Cl(-) channel and possibly another cAMP-activated pathway(s) may participate in Cl(-) secretion in the rat inner medulla.
Asunto(s)
Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Médula Renal/fisiología , Microsomas/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Aniones , Secuencia de Bases , Biomarcadores , Canales de Cloruro/biosíntesis , Canales de Cloruro/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Cartilla de ADN , Membranas Intracelulares/fisiología , Cinética , Masculino , Microsomas/efectos de los fármacos , Datos de Secuencia Molecular , Permeabilidad , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Valinomicina/farmacologíaRESUMEN
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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Mapeo Cromosómico , Fibrosis Quística/diagnóstico , Fibrosis Quística/prevención & control , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Femenino , Asesoramiento Genético , Genotipo , Heterocigoto , Humanos , Mutación , Fenotipo , Embarazo , Diagnóstico PrenatalRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Adrenoleucodistrofia/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Mutación , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Exones , Humanos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Fibrosis Quística/genética , Variación Genética , Proteínas de la Membrana/genética , Mutación Puntual , Polimorfismo Genético , Eliminación de Secuencia , Secuencia de Bases , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Exones , Femenino , Humanos , Intrones , Masculino , Linaje , Mapeo RestrictivoRESUMEN
We report the set-up of a denaturant gradient gel electrophoresis (DGGE) assay to screen for mutations in the whole coding sequence of the p53 gene. These DGGE experimental conditions were applied to the analysis of the p53 gene in acute leukemias. Forty adults with acute myelogenous leukemia (AML) and 21 with acute lymphoid leukemia (ALL) were investigated. Eleven of the AML patients were investigated at the time of the initial diagnosis and at relapse. In contrast with most reports based on amplified fragments analyzed by single-strand conformation electrophoresis and focusing on exons 5 to 8, we analyzed the whole coding sequence of the gene. Two of the 40 AML patients displayed a point mutation in exon 7; it was either an A to G substitution that converted Tyr-234 to Cys, or a G to A change that converted Arg-248 to Gln. The screening procedure led to the discovery of several intronic and exonic polymorphisms. These results confirm the low incidence of p53 mutations in acute leukemias and suggest a limited role of the p53 protein in leukemogenesis. The computerized modeling and electrophoresis parameters presented here provide a powerful tool for the exhaustive characterization of p53 mutants in all kinds of malignancies.
Asunto(s)
Genes p53 , Leucemia/genética , Mutación Puntual , Enfermedad Aguda , Secuencia de Bases , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Exones , Humanos , Leucemia Mieloide Aguda/genética , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Programas InformáticosRESUMEN
We propose a newborn cystic fibrosis (CF) screening test based on the analysis of dried blood spot DNA by a strategy involving simple or multiplex denaturing gradient gel electrophoresis (DGGE) of PCR products of CFTR gene fragments, in conjunction with the immunoreactive-trypsin (IRT) assay. From May 1988 to May 1992 we have performed a neonatal screening programme of 42,000 newborns in Brittany. We identified 450 infants with an elevated IRT level. From this cohort, to evaluate the feasibility of measuring IRT in conjunction with mutation analysis in Guthrie cards, a pilot study was initially conducted on 200 individuals with normal IRT and 150 with raised IRT levels. Furthermore, a retrospective study was performed on 189 IRT positive cards, involving mutation scanning of exons 10 and 11 of the CFTR gene, which contains a number of frequent mutations including the deletion delta F508. We show that this approach has several implications for neonatal CF screening especially in decreasing the recall rate and detecting CF carriers.
Asunto(s)
Fibrosis Quística/diagnóstico , Análisis Mutacional de ADN , Sangre Fetal/química , Tamizaje Neonatal/métodos , Reacción en Cadena de la Polimerasa , Algoritmos , Cloruros/análisis , Fibrosis Quística/epidemiología , Fibrosis Quística/genética , Electroforesis en Gel de Poliacrilamida , Exones , Reacciones Falso Positivas , Genes , Humanos , Recién Nacido , Desnaturalización de Ácido Nucleico , Proyectos Piloto , Estudios Retrospectivos , Sensibilidad y Especificidad , Sudor/química , Tripsina/sangreRESUMEN
Primary tracheal epithelial cells obtained from two fetuses with cystic fibrosis (CF) were successfully transfected with a plasmid vector recombined with the large T oncogene of SV40. The resulting tracheal cells were propagated in culture for up to 25 passages and retained the mutations of the CF genes carried by the two fetuses, one heterozygous for the S549N and N1303K substitutions (CFT-1 cells), and the other homozygous for the most common deletion delta F508 (CFT-2 cells). The transfected cells: (a) expressed the SV40 large T oncogene, as determined by immunofluorescence and Northern blot analysis; (b) retained typical epithelial morphology, as assessed by the presence of microvilli, desmosomes, gap junctions, and cytokeratin expression; (c) were fully responsive to the cAMP-stimulating agents isoproterenol, forskolin and vasoactive intestinal peptide for cAMP production and PKA activation; (d) do not produce any tumour in the athymic nude mice; (e) were diploid and tetraploid with a normal chromosomal complement at early passages, and (f) exhibited the abnormal regulation of chloride conductance characteristic of CF. These results indicate that CFT-1 and CFT-2 cells constitute a suitable model for: (a) comparison of the maturation and function of the CFTR protein mutated in the two nucleotide-binding domains; (2) analysis of the biochemical defect in CF epithelial airway cells, (c) development of new therapeutic agents, and correction of the CF defect by gene replacement therapy in vitro.
Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/patología , Proteínas de la Membrana/genética , Oncogenes , Animales , Transformación Celular Neoplásica , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Epitelio/patología , Femenino , Feto/patología , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Mutación , Virus 40 de los Simios/genética , Tráquea/patología , TransfecciónRESUMEN
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.
Asunto(s)
Fibrosis Quística/genética , Proteínas de la Membrana/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Humanos , MutaciónRESUMEN
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.
Asunto(s)
Fibrosis Quística/genética , Análisis Mutacional de ADN/métodos , Alelos , Fibrosis Quística/diagnóstico , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Análisis Mutacional de ADN/estadística & datos numéricos , Electroforesis en Gel de Poliacrilamida/métodos , Estudios de Evaluación como Asunto , Exones , Amplificación de Genes , Tamización de Portadores Genéticos/métodos , Humanos , Proteínas de la Membrana/genética , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
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.