Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides

The CFTR splicing mutation 3849 + 10 kb C --> T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 + 10 kb C --> T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2'-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose- and sequence-dependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders.     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38–82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.     

Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.     

Complete detection of mutations in cystic fibrosis patients of Native American origin

An increased incidence of cystic fibrosis (CF) has been reported in some populations of Native Americans of the Southwest such as the Pueblo, which is a genetic isolate. As the most common mutation found in Caucasians (F508) was absent and only one chromosome carried the G542X mutation, we decided to analyze the entire coding sequence of the CFTR gene in eight Pueblo CF patients. We have identified four different mutations: G542X, R1162X, 3849+10kbCT, and D648V that account for these 16 haplotypes. The R1162X was found on 11 chromosomes. Using intragenic microsatellites, we have compared the haplotypes of those chromosomes to those of Italian origin where the R1162X mutation was initially reported. These haplotypes turned out to be identical, suggesting a common origin and an admixture with Italian or Spanish settlers, followed by typical founder effect. In contrast the 3849+10kbCT mutation, which was found on three chromosomes, is associated with different haplotypes than those on chromosomes carrying the same mutation in Caucasians. A novel mutation, D648V, observed on one chromosome has not been found outside the Pueblo population.     

Impact of heterozygote CFTR Mutations in COPD patients with Chronic Bronchitis

Background

Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population.

Methods

Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV₁/FVC < 0.70 and FEV₁ < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network’s Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations.

Results

Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS).

Conclusions

The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.     

CFTR Activation Raises Extracellular pH of NIH/3T3 Mouse Fibroblasts and C127 Epithelial Cells

Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes of wet epithelia. Since CFTR is permeable to HCO⁻ ₃ and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor™ Microphysiometer was used to study forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15–35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10–20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for CFTR in controlling pHo and complement recent evidence that HCO⁻ ₃ dependent epithelial secretion may be reduced in amount and altered in composition in CF. Received: 20 June 2000/Revised: 13 November 2000     

Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients

Background

Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF.

Methods

Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: ''Residual'': carrying at least one partial function CFTR mutation (class IV or V) and ''Minimal'' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories.

Results

Subjects with ''Minimal'' CFTR function had a higher hazard than patients with ''Residual'' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile).

Conclusions

Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.     

Effect of genotype on selected clinical features of Polish cystic fibrosis adults

Cystic fibrosis (CF), the most common autosomal recessive disorder of Caucasians, is caused by the mutations in the gene encoding CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein. Until now, approximately 1000 mutations of the CFTR gene have been described. The genotype-phenotype relationships in CF are still not completely understood. This study was undertaken in an attempt to characterise the distribution of CFTR mutations and their effect on selected clinical parameters in a group of Polish CF adults. A total number of 38 adult CF patients (mean age 21.6 +/- 6.8); 18 females & 20 males were enrolled in the study. The CFTR gene identification was conducted with the use of PCR & InnoLipa-CF set. The assessed clinical parameters included: age at diagnosis, age, lung function test, X-ray scored in Brasfield score, weight & height. We found that: (1) the genotypes of the studied population were unevenly distributed (65.8%- genotype deltaF508/M), (2) a high percentage of 3849+10kbC-->T was noted, (3) patients homozygous for the deltaF508 mutation were diagnosed significantly earlier and had a lower body mass index, (4) no differences were observed in the patients' length of life or the progression of lung disease. Conclusions: 1. In comparison to other populations, Polish adult CF patients display a relatively higher frequency of mild mutations. 2. Late diagnosis of CF in the studied group may be partially caused by a high percentage of CFTR mutations connected with the mild course of the disease that are difficult to identify. 3. Cystic fibrosis should be more commonly taken into consideration in the differential diagnosis in adult patients with milder symptoms.     

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon within this exon.     

Cystic fibrosis Δf508 mutation screening in Brazilian women with altered fertility

Cystic Fibrosis (CF) is an autosomal recessive disease, caused by mutations in the Cystic Fibrosis Transmembrane Regulator gene (CFTR). The most frequent mutation in CF is ΔF508. The disease is clinically characterized by elevated concentrations of sweat chlorides and abnormally thick mucus. It affects organs such as lung, pancreas, gastrointestinal and reproductive tract. Women with CF commonly present delayed puberty and amenorrhea due to malnutrition. Our objective was to screen the presence of ΔF508 mutation in 24 women with altered fertility. Nine of these women presented reduced fertility without a known cause, four showed polycystic ovaries and two had early menopause. One woman with early menopause was a carrier of the ΔF508 mutation. Our study demonstrates that it is possible that the frequency of CF mutations among patients with altered fertility may be higher than expected. Previous data showed that fibrocystic women can show reduced fertility, maternal mortality associated with pregnancy and increased incidence of spontaneous abortion. We therefore recommend that women with reduced fertility undertake genetic tests for a better evaluation of pregnancy risks and clinical monitoring.     

Electrophysiological evaluation of Cystic Fibrosis Conductance Transmembrane Regulator (CFTR) expression in human monocytes

Background

Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells.

Methods

Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays.

Results

We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTR_inh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis.

Conclusions

We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF.

General significance

Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.     

Analysis of the Spectra of Mutations and Polymorphic Loci of Cystic Fibrosis Transmembrane Conductance Regulator in the Population of Bashkortostan

The spectra of mutations and polymorphic loci of the gene of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in 60 cystic fibrosis (CF) families from Bashkortostan. Mutations delF508, 394delTT, CFTRdele2,3(21 kb), R334W, and S1196X (33.3, 3.3, 1.7, 0.8, and 0.8%, respectively) were identified. The frequencies of tandem tetranucleotide repeat (TTR) alleles were determined for locus IVS6a-GATT of intron 6 of the CFTR gene and two extragenic loci flanking the CFTR gene, D7S23and MET(probes CS.7 and MetH) in mutant and normal chromosomes. Allelic and haplotypic associations of these loci with the mutations found were estimated. An absolute linkage between the 6TTR allele of locus IVS6a-GATT and the delF508 mutation was ascertained. A considerable linkage disequilibrium between thedelF508mutation and the C2 allele of locus D7S23and between this mutation and the A1 allele of locus MET was found. Most of the other mutant chromosomes carried marker alleles 7TTR, C1, and A2. It was demonstrated that 67% of CF chromosomes carrying delF508 had haplotype 6–2–1 for loci IVS6a-GATT/D7S23/MET, respectively. The frequency distribution of haplotypes in CF chromosomes without delF508had a high variance and did not differ significantly from the distribution in normal chromosomes (² = 9.415; p > 0.05)).     

The Mutation Spectrum of the CFTR Gene in Cystic Fibrosis Patients from Bashkortostan

Mutations of CFTR were studied in patients with cystic fibrosis (CF) from Bashkortostan. In total, 15 mutations were observed and 51% of all mutant alleles identified. The most diagnostically significant mutations were delF508 (33.8%), 394delTT (3.52%), CFTRdele2,3(21kb) (1.41%), R334W (1.41%), 3849 + 10kbC T (1.41%), and N1303K (1.41%). Mutations G542X, 2184insA, S1196X, and W1282X were each found in less than 1% patients. Five new mutations and two neutral substitutions were revealed. These were I488M (exon 10), 1811 + 12A C (intron 11), T663S (exon 13), I1226R (exon 19), 4005 + 9A C (intron 20), 2097A C (A655A, exon 13), and 3996G C (V1288V, exon 20). Bashkortostan was shown to differ in the CFTR mutation spectrum from other regions of Russia. The results will allow direct DNA diagnostics of CF in far more families. Molecular screening of probands" relatives will contribute to identification and medical genetic counseling of heterozygous carriers, which is essential for CF prevention.     

Activation of the JNK pathway by nanosecond pulsed electric fields

12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o⁻ cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o⁻ cells compared to normal bronchial epithelial cells 16HBE14o⁻. Surprisingly, messenger RNA level of IFRD1 in CFBE41o⁻ cells was found elevated. Treating CFBE41o⁻ cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.     

The mutation spectrum of the CFTR gene in mucoviscidosis patients from Bashkortostan

Mutations of CFTR were studied in patients with cystic fibrosis (CF) from Bashkortostan. In total, 15 mutations were observed and 51% of all mutant alleles identified. The most diagnostically significant mutations were delF508 (33.8%), 394delTT (3.52%), CFTRdele2.3(21 kb) (1.41%), R334W (1.41%), 3849+ 10 kbC-->T (1.41%), and N1303K (1.41%). Mutations G542X, 2184insA, S1196X, and W1282X were each found in less than 1% patients. Five new mutations and two neutral substitutions were revealed. These were I488M (exon 10), 1811 + 12A-->C (intron 11), T663S (exon 13), I1226R (exon 19), 4005 + 9A-->C (intron 20), 2097A-->C (A655A, exon 13), and 3996G-->C (V1288V, exon 20). Bashkortostan was shown to differ in CFTR mutation spectrum from other regions of Russia. The results will allow direct DNA diagnostics of CF in far more families. Molecular screening of probands' relatives will contribute to identification and medical genetic counseling of heterozygous carriers, which is essential for CF prevention.     

Neutrophil-Mediated Phagocytic Host Defense Defect in Myeloid Cftr-Inactivated Mice

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.     

Les génotypes responsables de mucoviscidose ou d’absence bilatérale des canaux déférents ABCD

Over the last decade, the genetic basis for CBAVD has been identified by its association with CFTR gene mutations, and CBAVD is now generally considered to be a mild or incomplete form of CF. In this review, the author summarizes the main results of compilation of CFTR gene analysis conducted in French laboratories for 3,923 patients with CF and 800 males with CABVD. The degree of clinical expression can be affected by several variables, including the molecular mechanisms by which the various CFTR mutations impair or disrupt the function of the CFTR chloride channel. Phenotypic expression of CFTR mutational genotypes varies from severe, progressive pulmonary disease with pancreatic insufficiency (CF-PI), to mild pulmonary disease with pancreatic sufficiency (PS) or singleorgan forms of “CFTR-opathies”. In CF, a total of 310 different CFTR mutations accounting for 94% of 7,846 CF alleles have generated almost 500 different genotypes, comprising 2 severe mutations in 88% of cases (CF-PI), one severe mutation in trans to a mild mutation in 11% (CF-PS), and 2 mild mutations in 1% of identified genotypes. In CBAVD, 137 mutations scattered over the whole gene were identified in 60% of 1,600 CBAVD alleles during the study. Among the 150 characterized mutational CFTR genotypes, compound heterozygosity was the rule, and the most frequent CBAVD combinations were ΔF508/5T (35%), ΔF508/other mutation (30%, including ΔF508/R117H-7T: 5,6%), and 5T/other mutation (17%). No combination of two severe mutations was found in CBAVD (0%); by contrast with the CF population, 88% of genotypes identified in CBAVD comprised a severe mutation in trans to a mild mutation, and 12% consisted of 2 mild mutations. A total of 22 genotypes were shared by both CF and CBAVD. The role of the 5T allele as a splicing variant with variable, incomplete disease penetrance in CBAVD is reviewed. Other haplotype backgrounds, such as the TG12 sequence and the M470V polymorphism, may influence CFTR splicing and/or function. This study confirms the high molecular heterogeneity of CFTR mutations in CBAVD and emphasizes the importance of extensive CFTR analysis in these patients. Longterm follow-up studies of CBAVD patients are necessary in order to predict the phenotypic consequences of numerous CFTR mutational genotypes.     

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.