Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

BACKGROUND: In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. METHODS AND RESULTS: As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. CONCLUSIONS: This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.     

Cystic fibrosis: typing 48 German families with linked DNA probes

Summary Two hundred and thirty five subjects from 48 German cystic fibrosis (CF) families were typed for restriction fragment length polymorphisms (RFLPs) detected by the probes pmet H, pmet D, and pJ 3.11, known to be tightly linked to the CF gene. Gene and haplotype frequencies suggest a linkage disequilibrium with the CF locus. The analysis of the predictive value of this typing in individual CF families indicates that the combined use of these probes provides a powerful diagnostic system both for carrier detection and prenatal diagnosis. In 33 out of 48 families carriers and non-carriers could be identified, and in 26 of these 33 families prenatal diagnosis could discriminate between affected and unaffected offspring.     

Extensive Sequencing of the CFTR gene: lessons learned from the first 157 patient samples

Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.     

Cystic fibrosis carrier population screening in the primary care setting.

To determine the receptivity of prenatal care providers and their patients to carrier testing for cystic fibrosis (CF), we offered free carrier screening, followed by genetic counseling of carriers, to all prenatal care providers in Rochester, NY, for all their female patients of reproductive age, pregnant or not. Of 124 prenatal care providers, only 37 elected to participate, but many of these offered screening only to pregnant women. The acceptance rate among pregnant women was approximately 57%. The most common reasons for accepting screening were to obtain reassurance (50.7%) and to avoid having a child with CF (27.8 %). The most common reasons for declining screening were not intending to terminate a pregnancy for CF (32.4%) and believing that the chance of having a CF child was very low (32.2%). Compared with decliners, acceptors were more likely to have no children, regarded having a child with CF as more serious, believed themselves more susceptible to having such a child, knew more about CF, would be more likely to terminate a pregnancy if the fetus were shown to have CF, and more strongly supported offering CF screening to women of reproductive age. Of 4,879 women on whom results were obtained, 124 were found to be carriers. Of these 124 carriers, the partners of 106 were tested. Of the five at-risk couples, four requested prenatal diagnosis and one requested neonatal diagnosis. No woman found to be a carrier whose partner tested negative requested prenatal diagnosis. Except for the imperfect knowledge of those testing negative, none of the adverse outcomes predicted for CF carrier testing in the general population were observed in this study.     

Gestational surrogacy and the role of routine embryo screening: Current challenges and future directions for preimplantation genetic testing

Preimplantation genetic screening (PGS) is a component of IVF entailing selection of an embryo for transfer on the basis of chromosomal normalcy. If PGS were integrated with single embryo transfer (SET) in a surrogacy setting, this approach could improve pregnancy rates, minimize miscarriage risk, and limit multiple gestations. Even without PGS, pregnancy rates for IVF surrogacy cases are generally satisfactory, especially when treatment utilizes embryos derived from young oocytes and transferred to a healthy surrogate. However, there could be a more general role for PGS in surrogacy, since background aneuploidy in embryos remains a major factor driving implantation failure and miscarriage for all infertility patients. At present, the proportion of IVF cases involving GS is limited, while the number of IVF patients requesting PGS appears to be increasing. In this report, the relevance of PGS for surrogacy in the rapidly changing field of assisted fertility medicine is discussed. Birth Defects Research (Part C) 108:98–102, 2016. © 2015 Wiley Periodicals, Inc.     

Spatial and temporal distribution of cystic fibrosis and of its mutations in Brittany,France: a retrospective study from 1960

Cystic fibrosis (CF) is the most common severe inherited disorder that affects children in Caucasian populations. The aim of this study was to define the spatial and temporal distribution of CF and its mutations in Brittany (western France) where the frequency of the disease is high. We retrospectively registered all CF patients born in Brittany since 1960 by cross-checking various data sources (e.g. medical care centres, genetics laboratories, hospital archives). Councils were contacted so that the place of residence of patients at birth could be determined. Moreover, the spectrum of CF transmembrane conductance regulator (CFTR) mutations and their spatial distribution across Brittany were determined. A total of 520 patients was registered in this study. The incidence of CF was assessed according to administrative (department, district) and diocesan divisions of Brittany and its evolution analysed over four decades. The incidence of CF was 1/2630, with a west/east gradient that was confirmed over time (Finistère: 1/2071 vs Ille-et-Vilaine: 1/3286). At present, the incidence of CF is decreasing, mainly as a result of prenatal diagnosis. An excellent mutation detection rate of 99.7% was obtained. Western Brittany presented a specific spectrum of mutations: 1078delT (9.4% of mutated alleles in the diocese of Cornouaille), G551D (7.7% in the diocese of Léon), 4005+1G-->A (2.9% in Cornouaille) and W846X (1.5% in western Brittany). On the other hand, the eastern region showed a spectrum more similar to the overall picture in France as a whole. This study enabled a precise measurement of the incidence of CF in Brittany to be obtained. The high frequency of the CFTR mutated alleles may result from founder effects and genetic drifts. Moreover, the study brings together the regional specificities of the CFTR gene and highlights disparities that exist in this part of France, both in incidence and in mutation distribution. These are attributable to different degrees of isolation and of population movements between the eastern and western parts of the region. Given that this is the first time that such a detailed study of the CFTR gene has been performed on a large population, this heightened knowledge of the epidemiology of CF in Brittany should provide a basis for the improvement of diagnostic strategies and refinement of genetic counselling.     

Attitudes toward the prenatal diagnosis of cystic fibrosis: factors in decision making among affected families.

The objective of this study was to explore psychosocial factors underlying decisions about use of prenatal diagnosis for cystic fibrosis (CF), among parents of affected children. Anonymous survey questionnaires, supplemented by voluntary interviews, were used at 12 CF centers in six New England states, for a consecutive sample of families of minor children visiting CF centers during a 4-mo period. In all, 227 (71%) of 318 families responded. We hypothesized that attitudes toward utilization would be affected by (a) intentions to have children, (b) knowledge, (c) perception of risk, (d) the health of the child with CF, (e) expectations about the child's future, (f) attitudes toward abortion, (g) insurance, (h) genetic counseling, and (i) sociodemographic factors (including attendance at religious services). Of the 227 couples who responded, 69% were surgically sterile, over 45 years of age, widowed, or divorced, and 31% were at risk. Of 70 at-risk couples, 44% intended to have more children; of these, 77% had had or were considering CF prenatal diagnosis. Most families knew CF could be diagnosed prenatally; 20% would terminate for CF. Among intended prenatal diagnosis users, 44% would carry a fetus with CF to term, 28% would abort, and 28% were undecided. Stepwise logistic regression showed three variables significantly related to intentions to use prenatal diagnosis: (1) respondent's willingness to abort for CF (P less than .02, odds ratio 3.36), (2) respondent's siblings' approval of abortion for CF (P less than .03, odds ratio 2.99), and (3) respondent listed no accomplishments for the child with CF (P less than .09, odds ratio 3.01). The majority of affected families reject selective abortion for CF; many will curtail childbearing rather than use prenatal diagnosis.     

Prenatal screening for cystic fibrosis carriers: an economic evaluation.

The cloning of the CFTR gene has made it technically possible to avert the unwanted birth of a child with cystic fibrosis (CF). Several large trials offering prenatal CF carrier screening suggest that such screening is practical and that identified carriers generally use the information obtained. Therefore, a critical question is whether the cost of such screening is justified. Decision analysis was performed that used information about choices that pregnant women were observed to make at each stage in the Rochester prenatal carrier-screening trial. The cost of screening per CF birth voluntarily averted was estimated to be $1,320,000-$1,400,000. However, the lifetime medical cost of the care of a CF child in today's dollars was estimated to be slightly>$1,000,000. Therefore, despite both the high cost of carrier testing and the relative infrequency of CF conceptions in the general population, the averted medical-care cost resulting from choices freely made are estimated to offset approximately 74%-78% of the costs of a screening program. At present, if it is assumed that a pregnancy terminated because of CF is replaced, the marginal cost for prenatal CF carrier screening is estimated to be $8,290 per quality-adjusted life-year. This value compares favorably with that of many accepted medical services. The cost of prenatal CF carrier screening could fall to equal the averted costs of CF patient care if the cost of carrier testing were to fall to $100.     

CFTR haplotypes in northern Iranian population

Background

Cystic fibrosis (CF) is a multiorganic autosomal recessive disorder, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). CF is highly heterogeneous in Iranian population and molecular diagnosis based on direct identification of mutations is not completely efficient. The use of polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations.

Methods

60 CF patients and 53 fertile normal subjects originating from North of Iran were analyzed for F508del mutation and c.1210-12T(5_9), c.1408A>G and c.744-33GATT(6_8) polymorphisms.

Results

c.1210-12T[7] is the most prevalent allele in normal individuals and CF non-F508del patients with 87.7%and 86.7% frequencies respectively. c.1408A>G survey showed that frequency of allele G and A is nearly equal in both non-F508del CF patients and normal individuals. c.744-33GATT(6_8) study showed that 7 repeat is the most prevalent allele in normal individuals and non-F508del CF patients with 80.2% and 82.1% frequencies respectively. The [c.1408A; c.1210-12T[9]; c.744-33GATT[6]] haplotype was only associated with mutant alleles including F508del.

Conclusions

The allelic distribution and heterozygosity results suggest that c.1408A>G, c.1210-12T(5_9) and c.744-33GATT(6_8) can contribute to carrier detection and prenatal diagnosis of CF in Iranian families with previous history of the disease.     

Characterization of more than 85% of cystic fibrosis alleles in the Greek population,including five novel mutations

To completely characterize the spectrum of mutations in the cystic fibrosis transmembrane conductance regulator gene in Greek cystic fibrosis (CF) patients, we screened 500 CF chromosomes by denaturing gradient gel electrophoresis followed by direct sequencing. We identified 48 mutations, accounting for 85.6% of CF chromosomes. They included eight novel mutations, three of which we have described before and five (E822X, Y247X, 2752–26A→G, 3152delT, and 2751+T→A), which are described in this report. The detection of such a high proportion of Greek CF mutations is important for improving prenatal and genetic diagnosis of CF in Greece. Received: 10 May 1996     

Genetics of early miscarriage

A miscarriage is the most frequent complication of a pregnancy. Poor chromosome preparations, culture failure, or maternal cell contamination may hamper conventional karyotyping. Techniques such as chromosomal comparative genomic hybridization (chromosomal‐CGH), array-comparative genomic hybridization (array-CGH), fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and quantitative fluorescent polymerase chain reaction (QF-PCR) enable us to trace submicroscopic abnormalities. We found the prevalence of chromosome abnormalities in women facing a single sporadic miscarriage to be 45% (95% CI: 38–52; 13 studies, 7012 samples). The prevalence of chromosome abnormalities in women experiencing a subsequent miscarriage after preceding recurrent miscarriage proved to be comparable: 39% (95% CI: 29–50; 6 studies 1359 samples). More chromosome abnormalities are detected by conventional karyotyping compared to FISH or MLPA only (chromosome region specific techniques), and the same amount of abnormalities compared to QF-PCR (chromosome region specific techniques) and chromosomal‐CGH and array-CGH (whole genome techniques) only. Molecular techniques could play a role as an additional technique when culture failure or maternal contamination occurs: recent studies show that by using array-CGH, an additional 5% of submicroscopic chromosome variants can be detected. Because of the small sample size as well as the unknown clinical relevance of these molecular aberrations, more and larger studies should be performed of submicroscopic chromosome abnormalities among sporadic miscarriage samples. For recurrent miscarriage samples molecular technique studies are relatively new. It has often been suggested that miscarriages are due to chromosomal abnormalities in more than 50%, but the present review has determined that chromosomal and submicroscopic genetic abnormalities on average are prevalent in maximally half of the miscarriage samples. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here.This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.     

Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.     

Molecular analysis of the CYP21 gene and prenatal diagnosis in families with 21-hydroxylase deficiency in northeastern Iran

OBJECTIVES: A rapid and convenient approach for the detection of the most common CYP21 gene mutations in patients with congenital adrenal hyperplasia (CAH) with classical forms of 21-hydroxylase deficiency was used. In addition, a new semiquantitative strategy for the detection of del8-bp was designed. These procedures were used for prenatal diagnosis and genotype-phenotype correlation in northeastern Iran. Design: Molecular analysis of the CYP21 gene for the detection of the 9 most common mutations (CYP21gene deletion, P30L, i2g, del-8bp, I172N, E6 cluster, V281L, Q318X and R356W) was performed on 30 CAH patients and for prenatal diagnosis in 2 cases. METHODS: Restriction fragment length polymorphism, amplification-created restriction sites, allele-specific polymerase chain reaction (PCR) and semiquantitative PCR were performed. RESULTS: We characterized 90% of the CAH chromosomes. The most frequent mutations in the CYP21 gene were del-CYP21 (25%), I172N (22%) and i2g (15%). Unlike in other ethnic groups, there was no R356W mutation, however, a higher rate of del-8bp (10%) was found in our population. Wealso found 6 complex alleles in our patients. For 2 families prenatal CYP21 gene analysis resulted in the diagnosis of healthy fetuses and termination of dexamethasone treatment in the 15th week of gestation. Genotype-phenotype correlation was observed. The rate of homozygosity (50%) was greater than the predicted values due to the higher rate of parental consanguinity in our population. CONCLUSIONS: These molecular procedures proved to be sensitive and rapid for the detection of the most common mutations of the CYP21 gene and prenatal diagnosis. Increased 17-hydroxyprogesterone, found in neonatal CAH screening, can be confirmed by these mutation analyses.     

Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs)

Autosomal recessive childhood spinal muscular atrophy (SMAs) is the second most common neuromuscular disorder and a common cause of infant disability and mortality. SMA patients are classified into three clinical types based on age of onset, and severity of symptoms. About 94% of patients have homozygous deletion of exon 7 in survival motor neuron (SMN1) gene. The neuronal apoptosis inhibitory protein (NAIP) gene was found to be more frequently deleted in the severest form of the disease. This study aimed to comment on the implementation of genetic counseling and prenatal diagnosis of SMAs for 85 fetuses from 75 Egyptian couples at risk of having an affected child. The homozygous deletion of exon 7 in SMN1 gene and the deletion of exon 5 of the NAIP gene were detected using PCR-REFLP and multiplex PCR methods respectively. Eighteen fetuses showed homozygous deletion of exon 7 in SMN1 gene and deletion of exon 5 in NAIP gene. In conclusion prenatal diagnosis is an important tool for accurate diagnosis and genetic counseling that help decision making in high risk families.     

The antibacterial properties of docosahexaenoic omega-3 fatty acid against the cystic fibrosis multiresistant pathogen Burkholderia cenocepacia

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens that cause multiresistant pulmonary infections in patients with cystic fibrosis (CF). In this study, we evaluated the in vitro antimicrobial efficacy of eight unsaturated fatty acids against Burkholderia cenocepacia K56-2, a CF epidemic strain. Docosahexaenoic acid (DHA) was the most active compound. Its action can be either bacteriostatic or bactericidal, depending upon the concentration used. The effect of DHA was also evaluated on two others B.?cenocepacia clinical isolates and compared with one representative member of all the 17 Bcc species. To test whether DHA could have a therapeutic potential, we assessed its efficacy using a Galleria mellonella caterpillar model of B.?cenocepacia infection. We observed that the treatment of infected larvae with a single dose of DHA (50 mM) caused an increase in the survival rate as well as a reduced bacterial load. Moreover, DHA administration markedly increases the expression profile of the gene encoding the antimicrobial peptide gallerimycin. Our results demonstrate that DHA has in vitro and in vivo antibacterial activity against Bcc microorganisms. These findings provide evidence that DHA may be a useful nutraceutical for the treatment of CF patients with lung infections caused by antibiotic multiresistant Bcc microorganisms.     

Azole Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.     

Low frequency of the deltaAF508 mutation of the CFTR gene in a highly admixed population in Bahia, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease in the European (Caucasian) population, with an incidence of 1:2000 to 1:8000. The deltaF508 mutation (66%) is predominant among more than 1300 different mutations of the CFTR gene. The population of the state of Bahia, in northeastern Brazil, is highly admixed (mainly African and Portuguese descendants), and so far, no study has been carried out to assess the molecular basis of CF in this population. We determined the deltaF508 mutation frequency in 503 individuals from the general population of Salvador, the capital of the state of Bahia, and in 144 CF patients from several cities in Bahia. In the general population samples we found 4 individuals heterozygous for the deltaF508 mutation (allele frequency of 0.4%). This frequency was lower than that found in the state of Rio de Janeiro, in southeastern Brazil, and similar to that reported for the state of Paraná, in the far south. In the CF patients we found 9 heterozygous individuals and 8 homozygous individuals (allele frequency of 8.68%) for the deltaF508 mutation. This frequency is considerably lower than the average frequency of CF in the world population and in the Brazilian CF population of European ancestry (47%). These data could be explained by the intense admixture among the population in Bahia, and they suggest a heterogeneous molecular basis for CF in this area of Brazil.