Search tips
Search criteria

Results 1-25 (945887)

Clipboard (0)

Related Articles

1.  The CFTR polymorphisms poly-T, TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens 
Asian Journal of Andrology  2012;14(5):687-690.
Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.
PMCID: PMC3734982  PMID: 22842702
CFTR; congenital bilateral absence of the vas deferens; IVS8-5T; male infertility; M470V
2.  Large genomic rearrangements in the CFTR gene contribute to CBAVD 
BMC Medical Genetics  2007;8:22.
By performing extensive scanning of whole coding and flanking sequences of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). Conventional PCR-based methods of mutation analysis do not detect gross DNA lesions. In this study, we looked for large rearrangements within the whole CFTR locus in the 32 CBAVD patients with only one or no mutation.
We developed a semi-quantitative fluorescent PCR assay (SQF-PCR), which relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different DNA samples. We confirmed the gross alterations by junction fragment amplification and identified their breakpoints by direct sequencing.
We detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or CFTRdele2], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or CFTRdele 22_24], in two males carrying a typical CBAVD mutation on the other parental CFTR allele. We present the first bioinformatic tool for exon phasing of the CFTR gene, which can help to rename the exons and the nomenclature of small mutations according to international recommendations and to predict the consequence of large rearrangements on the open reading frame.
Identification of large rearrangements further expands the CFTR mutational spectrum in CBAVD and should now be systematically investigated. We have designed a simple test to specifically detect the presence or absence of the two rearrangements identified in this study.
PMCID: PMC1876208  PMID: 17448246
3.  Gene Mutation in MicroRNA Target Sites of CFTR Gene: A Novel Pathogenetic Mechanism in Cystic Fibrosis? 
PLoS ONE  2013;8(3):e60448.
Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3′ untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.
PMCID: PMC3608608  PMID: 23555973
4.  Mutations in the Cystic Fibrosis Transmembrane Regulator Gene and In Vivo Transepithelial Potentials 
Aim: To examine the relationship between cystic fibrosis transmembrane regulator gene mutations (CFTR) and in vivo transepithelial potentials.
Methods: We prospectively evaluated 162 men including 31 healthy subjects, 21 obligate heterozygotes, 60 with congenital bilateral absence of the vas deferens (CBAVD) and 50 with CF by extensive CFTR genotyping, sweat chloride and nasal potential difference testing.
Results: Six (10%) men with CBAVD carried no CFTR mutations, 18 (30%) carried one mutation, including the 5T variant, and 36 (60%) carried mutations on both alleles, for a significantly higher rate carrying one or more mutations than healthy controls (90% versus 19%, p < 0.001). There was an overlapping spectrum of ion channel measurements among the men with CBAVD, ranging from values in the control and obligate heterozygote range at one extreme, to values in the CF range at the other. All pancreatic-sufficient patients with CF and 34 of 36 patients with CBAVD with mutations on both alleles carried at least one mild mutation. However, the distribution of mild mutations in the two groups differed greatly. Genotyping, sweat chloride and nasal potential difference (alone or in combination) excluded CF in all CBAVD men with no mutations. CF was confirmed in 56% and 67% of CBAVD men carrying 1 and 2 CFTR mutations, respectively.
Conclusion: Abnormalities of CFTR transepithelial function correlate with the number and severity of CFTR gene mutations.
PMCID: PMC2648063  PMID: 16840743
CFTR mutations; congenital bilateral absence of the vas deferens; cystic fibrosis; nasal potential difference; sweat chloride
5.  Linkage disequilibrium between the M470V variant and the IVS8 polyT alleles of the CFTR gene in CBAVD. 
Journal of Medical Genetics  1998;35(7):594-596.
Congenital bilateral absence of the vas deferens (CBAVD) is a cause of male sterility mostly resulting from mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The most common defect is the 5T variant at the branch/acceptor site of intron 8, which induces high levels of exon 9 skipping leading to non-functional protein. However, this 5T variant has incomplete penetrance and variable expressivity, suggesting that some other regulatory factors may modulate the splicing of exon 9. To identify such factors, we report here the genetic analysis of a polymorphic locus, M470V, located in exon 10 of the CFTR gene in 60 patients with CBAVD, compared to a normal control population. The statistical analysis showed strong linkage disequilibrium between the 5T allele and the V allele of the M470V polymorphism in the CBAVD population, but not in the normal population. The V allele in a gene carrying 5T could, however, contribute to lowering the level of normal transcripts, as already suggested by in vitro transcriptional studies. These genetic findings, together with previous studies, suggest involvement of the M470V variant in the modulation of the splicing of exon 9 of the CFTR gene.
PMCID: PMC1051371  PMID: 9678705
6.  p.Ser1235Arg should no longer be considered as a cystic fibrosis mutation: results from a large collaborative study 
Among the 1700 mutations reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a missense mutation, p.Ser1235Arg, is a relatively frequent finding. To clarify its clinical significance, we collected data from 104 subjects heterozygous for the mutation p.Ser1235Arg from the French CF network, addressed for various indications including classical CF, atypical phenotypes or carrier screening in subjects with or without a family history. Among them, 26 patients (5 having CF, 10 CBAVD (congenital bilateral absence of the vas deferens) and 11 with CF-like symptoms) and 14 healthy subjects were compound heterozygous for a second CFTR mutation. An exhaustive CFTR gene analysis identified a second mutation in cis of p.Ser1235Arg in all CF patients and in 81.8% CBAVD patients. Moreover, epidemiological data from >2100 individuals found a higher frequency of p.Ser1235Arg in the general population than in CF or CBAVD patients. These data, added to the fact that in silico analysis and functional assays suggest a benign nature of this substitution, give several lines of evidence against an association of p.Ser1235Arg with CF or CBAVD.
PMCID: PMC3039504  PMID: 20717170
cystic fibrosis; diagnosis; genetic counseling; mutation
7.  The CFTR Met 470 Allele Is Associated with Lower Birth Rates in Fertile Men from a Population Isolate 
PLoS Genetics  2010;6(6):e1000974.
Although little is known about the role of the cystic fibrosis transmembrane regulator (CFTR) gene in reproductive physiology, numerous variants in this gene have been implicated in etiology of male infertility due to congenital bilateral absence of the vas deferens (CBAVD). Here, we studied the fertility effects of three CBAVD–associated CFTR polymorphisms, the (TG)m and polyT repeat polymorphisms in intron 8 and Met470Val in exon 10, in healthy men of European descent. Homozygosity for the Met470 allele was associated with lower birth rates, defined as the number of births per year of marriage (P = 0.0029). The Met470Val locus explained 4.36% of the phenotypic variance in birth rate, and men homozygous for the Met470 allele had 0.56 fewer children on average compared to Val470 carrier men. The derived Val470 allele occurs at high frequencies in non-African populations (allele frequency  = 0.51 in HapMap CEU), whereas it is very rare in African population (Fst  = 0.43 between HapMap CEU and YRI). In addition, haplotypes bearing Val470 show a lack of genetic diversity and are thus longer than haplotypes bearing Met470 (measured by an integrated haplotype score [iHS] of −1.93 in HapMap CEU). The fraction of SNPs in the HapMap Phase2 data set with more extreme Fst and iHS measures is 0.003, consistent with a selective sweep outside of Africa. The fertility advantage conferred by Val470 relative to Met470 may provide a selective mechanism for these population genetic observations.
Author Summary
Cystic fibrosis (CF) is the most common lethal recessive disorder in European-derived populations and is characterized by clinical heterogeneity that involves multiple organ systems. Over 1,600 disease-causing mutations have been identified in the cystic fibrosis transmembrane regulator (CFTR) gene, but our understanding of genotype–phenotype correlations is incomplete. Male infertility is a common feature in CF patients; but, curiously, CF–causing mutations are also found in infertile men who do not exhibit any other CF–related complications. In addition, three common polymorphisms in CFTR have been associated with infertility in otherwise healthy men. We studied these three polymorphisms in fertile men and show that one, called Met470Val, is associated with variation in male fertility and shows a signature of positive selection. We suggest that the Val470 allele has risen to high frequencies in European populations due a fertility advantage but that other genetic and, possibly, environmental factors have tempered the magnitude of these effects during human evolution.
PMCID: PMC2880556  PMID: 20532200
8.  Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols 
Asian Journal of Andrology  2010;12(6):819-826.
Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.
PMCID: PMC3739074  PMID: 20657600
congenital bilateral absence of vas deferens; cystic fibrosis transmembrane conductance regulator; male infertility
9.  The CFTR M470V, Intron 8 Poly-T, and 8 TG-Repeats Detection in Chinese Males with Congenital Bilateral Absence of the Vas Deferens 
BioMed Research International  2014;2014:689185.
Purpose. To evaluate the significance of molecular detection of cystic fibrosis transmembrane conductance regulator (CFTR) M470V, intron 8 poly-T, and intron 8 TG-repeats in congenital bilateral absence of the vas deferens (CBAVD). Methods. Eighty-nine male patients with CBAVD and 103 healthy males were included in this study. Polymerase chain reaction was performed to amplify the polymorphic regions using primers from conserved regions. M470V was genotyped using real-time PCR by cycling probe. The exon 9 DNA sequence was determined using an automated sequencer. TG-repeats and poly-T were identified by direct sequencing analysis. Results. The 5T allele distribution was 0.32, 0.66 for 7T, and 0.02 for 9T in CBAVD males, respectively. In contrast, the 5T allele distribution was 0.03, 0.96 for 7T, and 0.01 for 9T in healthy control. Study of the polymorphisms of the upstream of exon 9 revealed a higher frequency of 5T allele in the CBAVD males. All cases with TG13T5 haplotype and TG12T5 homozygous led to CBAVD. The CFTR TG12T5-V470 variant haplotype was associated with CBAVD. Conclusion. The 5T allele of intron 8 of CFTR has clinically significant association with CBAVD. TG13T5 and TG12T5 homozygously led to CBAVD, and TG12T5-V470 may also lead to CBAVD.
PMCID: PMC3914569  PMID: 24551851
10.  CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients 
PLoS ONE  2014;9(2):e89094.
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.
PMCID: PMC3935850  PMID: 24586523
11.  VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1 
Molecular Biology of the Cell  2013;24(19):3016-3024.
Misfolding of cystic fibrosis transmembrane conductance regulator protein (CFTR) causes the fatal lung disease cystic fibrosis. VX-809 was developed to suppress disease-related folding defects in CFTR. VX-809 suppresses folding defects in CFTR by modulating the conformation of membrane-spanning domain 1. VX-808 is thereby able to partially restore function to F508del-CFTR and other disease-related mutants.
Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.
PMCID: PMC3784376  PMID: 23924900
12.  SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men 
PLoS ONE  2014;9(11):e112498.
Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1–2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF) and CFTR-related disorder (CFTR-RD) mutations are found in a high percentage (22.7%) of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.
PMCID: PMC4227699  PMID: 25386751
13.  An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion 
BMC Medical Genetics  2011;12:62.
F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes KRT8 and KRT18 in a candidate gene approach to ask whether variants in KRT8 and/or KRT18 modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.
We have selected contrasting F508del-CFTR homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The KRT8/KRT18 locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal.
KRT8, but not KRT18, showed an association with CF disease severity (Pbest = 0.00131; Pcorr = 0.0185) and CFTR mediated residual chloride secretion (Pbest = 0.0004; Pcorr = 0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire KRT8 gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild KRT8 haplotype 2211 is phylogenetically older than its severe counterpart.
The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.
PMCID: PMC3107781  PMID: 21548936
14.  Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy 
Cystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations.
We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity.
We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC).
Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%).
PMCID: PMC419352  PMID: 15084222
Cystic fibrosis; CFTR mutation screening; DHPLC
15.  Impact of heterozygote CFTR Mutations in COPD patients with Chronic Bronchitis 
Respiratory Research  2014;15(1):18.
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.
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.
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).
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.
PMCID: PMC3925354  PMID: 24517344
16.  Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis 
PLoS Genetics  2014;10(7):e1004376.
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.
Author Summary
Genetic disorders of ion channels can affect the body's ability to function properly in many ways. CFTR, an ion channel regulating movement of chloride and bicarbonate across cell membranes, is important for absorbing and secreting fluids. If the gene responsible for the CFTR channel is mutated severely, the result is cystic fibrosis, a hereditary disorder in which the patient develops thick mucus, especially in the lungs, as well as scarring (fibrosis) in the pancreas. Cystic fibrosis also affects the sweat glands, nasal sinuses, intestines, liver, and male reproductive system. Mutations to the CFTR gene that do not cause cystic fibrosis have been considered benign. However, we discovered 9 CFTR mutations that do not cause cystic fibrosis but do cause inflammation and scarring of the pancreas (chronic pancreatitis). These mutant CFTR channels secrete chloride, which is important in the sweat glands, lungs, and intestines, but not bicarbonate, which is important in the pancreas, sinuses, and male reproductive tract. We found patients with any of these 9 mutations had chronic pancreatitis, and often sinus infections, and male infertility, but not other symptoms of cystic fibrosis. Our computer models and data will help researchers develop better drugs and help physicians treating patients with chronic pancreatitis.
PMCID: PMC4102440  PMID: 25033378
17.  Cystic fibrosis carrier frequencies in populations of African origin 
Journal of Medical Genetics  1999;36(1):41-44.
Cystic fibrosis (CF) is a common autosomal recessive disorder in populations of European descent. However, very little is known about CF in populations of African origin among whom it has been believed to be extremely rare. The aim of this study was to determine if this is the case or whether it is under-reported. A CFTR mutation, 3120+1G→A, which was first reported in three African-American CF patients, has been shown to account for 9-14% of African-American CF chromosomes. It has also been found in 4/6 CF chromosomes in South African blacks and one CF chromosome of Cameroonian origin. In order to determine the carrier frequency of the 3120+1G→A mutation in Africa, 1360 unrelated, healthy subjects were screened. Nine carriers were identified. In addition, two out of five black CF patients with positive sweat tests were found to be heterozygous for the 3120+1G→A mutation and two out of another four black patients with symptoms suggestive of CF, but unconfirmed by sweat tests, were heterozygous for the D1270N mutation. A further three CFTR mutations, A559T, S1255X, and 444delA, which had been found in African-American CF patients, were not identified in the patients or in over 373 healthy subjects tested. The 3120+1G→A mutation has a carrier frequency of 1 in 91 (8/728) in South African blacks with a 95% confidence interval of 1 in 46 to 1 in 197. Since this mutation accounts for between 15% and 65% of CF chromosomes in South African blacks, a corrected CF carrier frequency would be between 1 in 14 and 1 in 59. Hence, the incidence of CF would be predicted to be between 1 in 784 and 1 in 13 924 births in this population. There are several possible reasons why these people are not being detected. Some of these are misdiagnosis as chronic pulmonary infection, malnutrition, tuberculosis, infantile diarrhoea, failure to thrive, or a high infant mortality rate.

Keywords: CF; African blacks; 3120+1G→A mutation; heterozygote frequencies
PMCID: PMC1762947  PMID: 9950364
18.  Complete cystic fibrosis transmembrane conductance regulator gene sequencing in patients with idiopathic chronic pancreatitis and controls 
Gut  2005;54(10):1456-1460.
Background: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene—many of which cause cystic fibrosis—have also been reported in patients with chronic pancreatitis. The authors examine whether mild or severe CFTR mutations, homozygous or compound heterozygous CFTR mutations, or even simple cystic fibrosis carrier status alone increases the risk of developing pancreatitis.
Methods: After exclusion of patients with trypsinogen (PRSS1) mutations, cystic fibrosis, or pulmonary disease, and with known risk factors for pancreatitis 67 patients with idiopathic chronic pancreatitis (ICP) from northwest Germany and 60 geographically and ethnically matched controls were recruited. The entire coding region of the CFTR gene was sequenced in all patients and controls. ICP patients were also analysed for serine protease inhibitor Kazal type 1 (SPINK1) gene mutations.
Results: Abnormal CFTR alleles were found to be twice as frequent in ICP patients as in controls (25/134 v 11/120; p<0.05). Three of four severe CFTR mutations detected in patients were compound heterozygous with another abnormal CFTR allele, whereas among controls three severe CFTR mutations were found in heterozygous cystic fibrosis carriers. In ICP patients 19 uncommon/mild mutations, including combinations of the 5T allele with 12TG repeats, were identified compared with only five in controls (p = 0.012). Heterozygous SPINK1 mutations were detected in eight ICP patients (15% v 1% in controls) but only one also carried an additional mild CFTR mutation.
Conclusions: These data show that not only compound heterozygosity, but also cystic fibrosis carrier status for different types of CFTR mutations, including uncommon/mild mutations, significantly increase the risk of developing pancreatitis. Although 45% of the study’s ICP patients carried predisposing genetic risk factors (for example, mutations in CFTR or SPINK1), the authors found no evidence that the risk conveyed by CFTR mutations depends on co-inherited SPINK1 mutations.
PMCID: PMC1774703  PMID: 15987793
CFTR; gene mutation; idiopathic chronic pancreatitis
19.  CFTR transcription defects in pancreatic sufficient cystic fibrosis patients with only one mutation in the coding region of CFTR 
Journal of medical genetics  2010;48(4):235-241.
Patients with cystic fibrosis (CF) manifest a multisystem disease due to deleterious mutations in each gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). However, the role of dysfunctional CFTR is uncertain in individuals with mild forms of CF (ie, pancreatic sufficiency) and mutation in only one CFTR gene.
Eleven pancreatic sufficient (PS) CF patients with only one CFTR mutation identified after mutation screening (three patients), mutation scanning (four patients) or DNA sequencing (four patients) were studied. Bi-directional sequencing of the coding region of CFTR was performed in patients who had mutation screening or scanning. If a second CFTR mutation was not identified, CFTR mRNA transcripts from nasal epithelial cells were analysed to determine if any PS-CF patients harboured a second CFTR mutation that altered RNA expression.
Sequencing of the coding regions of CFTR identified a second deleterious mutation in five of the seven patients who previously had mutation screening or mutation scanning. Five of the remaining six patients with only one deleterious mutation identified in the coding region of one CFTR gene had a pathologic reduction in the amount of RNA transcribed from their other CFTR gene (8.4–16% of wild type).
These results show that sequencing of the coding region of CFTR followed by analysis of CFTR transcription could be a useful diagnostic approach to confirm that patients with mild forms of CF harbour deleterious alterations in both CFTR genes.
PMCID: PMC3065505  PMID: 21097845
20.  The CF-modifying gene EHF promotes p.Phe508del-CFTR residual function by altering protein glycosylation and trafficking in epithelial cells 
The three-base-pair deletion c.1521_1523delCTT (p.Phe508del, F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most frequent disease-causing lesion in cystic fibrosis (CF). The CFTR gene encodes a chloride and bicarbonate channel at the apical membrane of epithelial cells. Altered ion transport of CFTR-expressing epithelia can be used to differentiate manifestations of the so-called CF basic defect. Recently, an 11p13 region has been described as a CF modifier by the North American CF Genetic Modifier Study Consortium. Selecting the epithelial-specific transcription factor EHF (ets homologous factor) as the likely candidate gene on 11p13, we have genotyped two intragenic microsatellites in EHF to replicate the 11p13 finding in the patient cohort of the European CF Twin and Sibling Study. We could observe an association of rare EHF haplotypes among homozygotes for c.1521_1523delCTT in CFTR, which exhibit a CF-untypical manifestation of the CF basic defect such as CFTR-mediated residual chloride secretion and low response to amiloride. We have reviewed transcriptome data obtained from intestinal epithelial samples of homozygotes for c.1521_1523delCTT in CFTR, which were stratified for their EHF genetic background. Transcripts that were upregulated among homozygotes for c.1521_1523delCTT in CFTR, who carry two rare EHF alleles, were enriched for genes that alter protein glycosylation and trafficking, both mechanisms being pivotal for the effective targeting of fully functional p.Phe508del-CFTR to the apical membrane of epithelial cells. We conclude that EHF modifies the CF phenotype by altering capabilities of the epithelial cell to correctly process the folding and trafficking of mutant p.Phe508del-CFTR.
PMCID: PMC3992571  PMID: 24105369
cystic fibrosis modifier gene; c.1521_1523delCTT (p.Phe508del; F508del) in CFTR; association study; endophenotype; transcription factor; transcriptome
21.  Deletion of CFTR Translation Start Site Reveals Functional Isoforms of the Protein in CF Patients 
Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening autosomal recessive disease affecting Caucasians. We identified a CFTR mutation (c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese CF patients. This study aims at functionally characterizing the effect of this novel mutation.
RNA and protein techniques were applied to both native tissues from CF patients and recombinant cells expressing CFTR constructs to determine whether c.120del23 allows CFTR protein production through usage of alternative internal codons, and to characterize the putative truncated CFTR form(s).
Our data show that two shorter forms of CFTR protein are produced when the initiation translation codon is deleted indicating usage of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation.
The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation.
PMCID: PMC2793277  PMID: 19910674
CFTR; Cystic Fibrosis; Mutation; Truncated proteins; Alternative translation; N-terminus
22.  Deletion of CFTR Translation Start Site Reveals Functional Isoforms of the Protein in CF Patients 
Cellular Physiology and Biochemistry  2009;24(5-6):335-346.
Background/Aims: Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening autosomal recessive disease affecting Caucasians. We identified a CFTR mutation (c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese CF patients. This study aims at functionally characterizing the effect of this novel mutation. Methods: RNA and protein techniques were applied to both native tissues from CF patients and recombinant cells expressing CFTR constructs to determine whether c.120del23 allows CFTR protein production through usage of alternative internal codons, and to characterize the putative truncated CFTR form(s). Results: Our data show that two shorter forms of CFTR protein are produced when the initiation translation codon is deleted indicating usage of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation. Conclusions: The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation.
PMCID: PMC2793277  PMID: 19910674
CFTR; Cystic Fibrosis; Mutation; Truncated proteins; Alternative translation; N-terminus
23.  Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect 
Human Molecular Genetics  2011;21(3):623-634.
We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.
PMCID: PMC3259015  PMID: 22038833
24.  Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors 
PLoS ONE  2012;7(12):e52070.
Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.
PMCID: PMC3528711  PMID: 23284872
25.  Proteomic Identification of Calumenin as a G551D - CFTR Associated Protein 
PLoS ONE  2012;7(6):e40173.
Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, over 1910 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases) encodes for a CFTR chloride channel with normal expression on the cell surface. Nevertheless, it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Co-immunoprecipitated proteins with G551D-CFTR were resolved by 2D-gel electrophoresis (2-DE). Mass Spectrometry revealed that calumenin was present in the protein complex linked to G551D-CFTR. Despite its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR expressing cells, it was more abundant in the G551D-CFTR complex detected by immunoprecipitation. The calumenin-CFTR interaction was also shown by Surface Plasmon Resonance and further confirmed by computational analysis of the predicted calumenin’s partners. Because in our cellular model calumenin was found in the endoplasmic reticulum (ER) by immunofluorescence experiments, we suggest that calumenin is likely involved in the mutated CFTR’s maturation. In conclusion, we showed for the first time that calumenin binds to CFTR and that it is increased in the G551D-CFTR complex. We suggest that it may be involved in the physiopathology of G551D-CFTR and that G551D-CFTR may follow a specific maturation and trafficking pathway. We also hypothesize that UPR may be triggered independently of the retention of G551D-CFTR in the ER because Grp78/Bip expression is increased in the cells. Finally, we propose here that Calumenin is a new CFTR chaperone.
PMCID: PMC3387016  PMID: 22768251

Results 1-25 (945887)