Somatische Gentherapie der Mukoviszidose Aktueller Forschungsstand

Background. Numerous gene mutations associated with hereditary disorders have been identified. In cystic fibrosis the hereditary defect is attributed to mutations in one single gene, the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Conventional therapies of CF have dramatically increased the life expectancy of afflicted individuals. However, the ultimate incurability of this disease calls for novel and better therapeutic strategies. As cystic fibrosis is believed to be caused by mutations in one single gene, it has appeared to be the ideal candidate for one of the most tempting approaches in clinical therapy, namely gene therapy. Laboratory protocols for the introduction of genes into various tissues have been developed and applied over the last years. The ease of gene transfer under laboratory conditions gave rise to the hope that rapid advances in gene transfer protocols under clinical settings could be achieved as well. Clinical trials. 25 clinical trials of gene therapy for cystic fibrosis have been initiated using viral and non-viral vectors for gene transfer. The outcome of the CF gene therapy studies as well as of those for other diseases have clearly demonstrated that gene transfer and gene therapy in humans is a much more complex and challenging task than originally thought. Still, the encouraging results achieved in animal models and the rapid progress in vector technology justify the hope that the novel genetic therapies will be applied successfully to the benefit of patients suffering from cystic fibrosis.     

儿童囊性纤维化并发变应性支气管肺曲霉菌病1例报告并文献复习

目的 分析囊性纤维化(CF)并发变应性支气管肺曲霉菌病(ABPA)的临床特征.方法 回顾分析1例确诊为CF并发ABPA患儿的临床资料,分析近10年PubMed、中国知网、万方数据库中报道CF并发ABPA的资料.结果 11岁患儿,男,曾诊断哮喘,因持续咳嗽、喘息入院,初步确诊ABPA.基因检测显示患儿CFTR基因变异,汗...     

Genotype of the cystic fibrosis population of the Hunter Region of New South Wales

Objective : To determine the genotype of patients attending the cystic fibrosis clinic at John Hunter Hospital, Newcastle, Australia.
Methodology : Seventy-five of the 76 patients attending the clinic over a 6 month period had blood collected for genetic analysis of 17 of the cystic fibrosis (CF) gene mutations.
Results : Sixty-one per cent of the patients were homozygous for the ΔF508 mutation and all except one child had at least one ΔF508 mutation.
Discussion : Nearly 80% of the CF genes were the ΔF508 mutation. This prevalence suggests that the obligatory false negative rate of a newborn screening programme for CF based on a combination of immunoreactive trypsin and the ΔF508 gene may be as low as 4-5%.     

Genetic testing for cystic fibrosis.

Cystic fibrosis occurs in 1 in 2000 to 4000 whites, and about 1 in 25 people are heterozygous carriers. The CF gene is cloned, and a single common mutation is found on 70% to 75% of CF chromosomes in most populations. Numerous different mutations are found on the remaining CF chromosomes. DNA analysis for carrier testing should be offered to relatives of CF patients and to reproductive partners of proven carriers. Population based carrier screening could detect about 72% of at-risk couples, and pilot studies are in progress to assess the feasibility of such screening. With regard to these factors, the possibility of gene therapy, and the questions raised, CF serves as a useful model for many genetic disorders.     

The cystic fibrosis defect approached from different angles — new perspectives on the gene,the chloride channel,diagnosis and therapy

The search for the basic defect in cystic fibrosis (CF) has reached a decisive stage since the recent identification of the responsible gene. Electrophysiological and biochemical research had defined the CF defect as a dysregulation of epithelial chloride channels. The putative protein product of the now identified gene shares properties with other known transport proteins, but it is not necessarily itself a chloride channel protein. Elucidation of the primary cellular defect will certainly have important aetiological and hopefully therapeutic implications. The identification of the major gene mutation already has significant consequences for genetic counselling and prenatal diagnosis. Heterozygote detection at the population level awaits identification of the probably heterogenous mutations on about 30% of the CF chromosomes. At present, about 50% of CF patients are homozygous for the recently identified major CF mutation.     

Mukoviszidose

Characteristics

Cystic fibrosis (CF) is an autosomal recessive hereditary systemic disease with a chronic progressive course affecting all exocrine glands. It is caused by a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR gene) which results in precipitation and retention of mucous secretions in the ducts of exocrine glands and a disposition to respiratory infections.

Diagnosis

The diagnosis of CF is based on a pathologically increased chloride concentration or two mandatory disease-triggering CFTR gene mutations. If the genetic tests and/or chloride concentrations in intermediate ranges are uninformative but CF is clinically suspected, extended CFTR functional diagnostics should be carried out. The course of gastrointestinal and pulmonary manifestations must be monitored by regular standardized control investigations.

Therapy

The standard therapy of gastrointestinal symptoms includes substitution of gastric acid-resistant pancreatic enzymes, a high calorie fat-rich diet and substitution of fat soluble vitamins. The essential pillars of the current therapy of pulmonary symptoms are liberal and early antimicrobial chemotherapy, inhalation, anti-inflammatory treatment, sport and physiotherapy. In cases of advanced pulmonary disease processes, long-term oxygen therapy, non-invasive mask ventilation and ultimately lung transplantation are further options.

Conclusions

The pulmonary manifestations are critical factors which currently determine the course and prognosis of cystic fibrosis in nearly 90 % of patients. The earliest possible diagnosis and nursing in a CF center have advantageous effects.     

Physiological Measurements Confirming the Diagnosis of Cystic Fibrosis: the Sweat Test and Measurements of Transepithelial Potential Difference

Post-natal screening allied with genetic mutation testing has altered our perception of cystic fibrosis (CF) as a clinical entity. Increasingly, infants identified through screening programmes have few or no symptoms or present with atypical forms of the disease. We review how the sweat test has evolved to be the gold standard for confirming the diagnosis of CF and examine its limitations. Other physiological measurements, including nasal potential difference and intestinal current measurement, which might aid in establishing the diagnosis, particularly in patients exhibiting a mild phenotype, are also considered.     

Buccal cell DNA mutation analysis for diagnosis of cystic fibrosis in newborns and infants inaccessible to sweat chloride measurement

OBJECTIVES: To assess the application of DNA-based cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis as a primary cystic fibrosis (CF) diagnostic test in preterm and term newborns and infants for whom the quantitative pilocarpine iontophoresis test (QPIT) cannot be used. DESIGN: Retrospective survey. SETTING: DNA Diagnostic Laboratory, Children's Hospital, Boston, Massachusetts. Buccal cell DNA samples were received from inpatients, outpatients, and three neonatal intensive care units. OUTCOME MEASURE: Detection of at least 1 of 12 CFTR mutations. PATIENTS: Between November 1, 1992, and April 30, 1994, 28 newborns and infants under 12 months of age at risk for CF had CFTR DNA mutation analysis performed because a sweat chloride (SC) value could not be obtained. QPIT was either not performed (infant weight <2 kg, QPIT not available at site of hospitalization, or infant not accessible to QPIT laboratory) or was inconclusive (sweat volume <75 mg or indeterminate SC [>/=40, <60 mEq/L]). The postnatal age at time of testing ranged from 1 day to 11 months, and gestational age at birth from 25 to 40 weeks. RESULTS: Six (21%) of 28 infants with unobtainable or indeterminate QPIT had 1 or 2 CFTR mutations detected. Immediate CF diagnosis by direct detection of 2 CFTR mutations was made in 5 of these 6 patients. Definitive CF diagnosis in the infant with 1 CFTR mutation was delayed until an elevation in SC could be documented. The patients with no CFTR mutations detected had a low likelihood of CF. CONCLUSIONS: For infants in whom CF is suspected but QPIT cannot be obtained, buccal cell DNA-based CFTR mutation analysis can be used as a rapid, noninvasive primary diagnostic test. This simple mode of DNA collection may aid in the diagnosis of other inherited disorders in newborns.     

Therapeutic strategies to correct malfunction of CFTR

Cystic fibrosis (CF) is a systemic autosomal recessive inherited disorder that results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although the gene was cloned 11 years ago, there still is no definitive treatment to correct the functional deficit. Current treatment strategies focus on pancreatic enzyme replacement and control of pulmonary inflammation and infection. This review examines novel strategies still in preclinical development or phase 1 clinical trials. Gene therapy is an evolving area of study that offers the potential for a cure for cystic fibrosis. CF lung disease is a significant barrier to effective gene delivery and transfer, but new vectors show promise in overcoming these limitations. There are also new pharmacological therapies aimed at correcting defects in CFTR processing and function. These are tailored to the specific class of mutation but may offer therapeutic benefit to many patients. They include phenylbutyrate, flavonoids, aminoglycosides and xanthines.     

Cystic fibrosis birth rates in Canada: a decreasing trend since the onset of genetic testing

OBJECTIVE: To estimate cystic fibrosis (CF) birth rates in Canada from 1971 to 2000 and to assess the population impact of genetic testing in families with a history of CF, after identification of the CF transmembrane conductance regulator gene in 1989. STUDY DESIGN: Age-at-diagnosis data were obtained from the Canadian Cystic Fibrosis Foundation Patient Data Registry and Canadian births for the corresponding years from Canadian Vital Statistics. Estimates of the CF birth rate in each year were based on a nonparametric model that allows the birth rate to vary across the years and adjusts for censoring of currently undiagnosed patients. RESULTS: The overall CF birth rate from 1971-1987 was 1/2714 with no increasing or decreasing trend. Beginning in 1988, 1 year before identification of the CF transmembrane conductance regulator gene, estimated CF birth rates followed a linear decline to an estimated rate of 1/3608 in 2000. CF birth rates may have stabilized in the last few years, but further decline may occur with implementation of carrier screening in the general population. CONCLUSIONS: These results demonstrate the temporal association of genetic testing and declining CF birth rates in Canada. They may assist in decisions relating to the allocation of resources for prenatal and neonatal CF screening programs.     

Mutation analysis in the diagnosis of cystic fibrosis

Since the characterization of the gene encoding the cystic fibrosis transmembrane conductance regulator protein and identification of its main mutation, F508, causing cystic fibrosis (CF), more than 150 mutations in this gene have been reported, most of them only in a few or even single CF patients. Attempts to use mutation analysis in genetic counselling or for the diagnosis of CF depends on prevalence data of certain mutations in the respective population, and considerable ethnic differences have been reported. In this study we determined the prevalence of the mutations F508, G551D, R553X, and G542X and of genotypes defined by these mutations in 239 CF patients (444 independent CF chromosomes) seen in our clinic. The analysis for those four mutations alone now permits identification of approximately 75% of all mutations in our CF patients. The complete genotype can be resolved in approximately 63% of patients. This represents the diagnostic sensitivity which can be achieved by mutation analysis in patients without a family history of CF. We conclude that in situations where conventional diagnostic tests are not feasible or difficult to interpret, mutation analysis using a limited set of mutations can contribute significantly to an early and specific diagnosis of CF.     

The need for vigilance: the case of a false-negative newborn screen for cystic fibrosis

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disorder in the white population. CF is caused by abnormalities in the gene that codes for the cystic fibrosis transmembrane conductance regulator protein (CFTR) and may result in severe chronic lung disease, poor growth, and malnutrition. Physicians often do not consider CF in the differential diagnosis of an infant with failure to thrive in the presence of a negative newborn screening (NBS) result. In Minnesota, newborn infants are screened for CF by immunoreactive trypsinogen (IRT) testing followed by DNA analysis if the IRT screen result is abnormal. All positive NBS results are followed by confirmatory sweat-testing by pilocarpine iontophoresis. We present here the case of a 1-month-old white boy with failure to thrive, chronic diarrhea, and severe malnutrition. Minnesota state CF NBS results were negative at birth (IRT: 43 ng/mL [96% cutoff value: 52 ng/mL]). Clinical symptoms resulted in sweat-testing by Gibson-Cooke pilocarpine iontophoresis at 1 month of age, and the result was positive (102 mmol Cl(-)/L [normal: ≤30 mmol Cl(-)/L]). CFTR mutation analysis confirmed a homozygous f508del genotype, and stool pancreatic elastase testing revealed severe exocrine pancreatic insufficiency. This case represents the first known false-negative result in Minnesota since the initiation of NBS for CF in 2006, which illustrates the importance of considering CF in the evaluation of an infant with failure to thrive and symptoms of malabsorption, regardless of NBS results.     

Molecular biological analysis of cystic fibrosis--a model example for the strategy of "reverse genetics"]

The elucidation of the basic defect causing cystic fibrosis (CF) is a paradigm for the application of "reverse genetics" to the analysis of human genetic disease. Following this strategy, linkage analysis localized the responsible gene for CF on chromosome 7. Chromosome mediated gene transfer and chromosome walking and jumping led to the isolation of the CFTR-gene and its cDNA. A major 3 bp deletion mutation (DeltaF508) and more than 100 other mutations of this gene have been identified as molecular basis of cystic fibrosis. The CFTR-amino acid sequence, obtained by conversion of the cDNA-sequence, indicates that CFTR belongs to a group of integral membrane transport proteins (ABC-proteins). The normal cAMP-stimulated chloride-transport, lacking in CF-cells is restored by transfer and expression of CFTR-cDNA-recombinants in these cells. CFTR is most likely itself a chloride channel. The molecular identification of this gene has already led to substantial advances in diagnosis and prevention of this disease. New therapeutic approaches by pharmacological means or gene therapy are expected from the further molecular and functional analysis of the CFTR-gene.     

Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis

A study was performed on the delayed diagnosis of cystic fibrosis (CF) in infants who had false-negative results in a neonatal screening programme. The genetic and clinical features of false-negative infants in this screening programme were assessed together with the efficiency of the screening procedure in the Lombardia region. In total, 774 687 newborns were screened using a two-step immunoreactive trypsinogen (IRT) (in the years 1990-1992), IRT/IRT + delF508 (1993-1998) or IRT/IRT + polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) protocol (1998-1999). Out of 196 CF children born in the 10 y period 15 were false negative on screening (7.6%) and molecular analysis showed a high variability in the genotypes. The cystic fibrosis transmembrane regulator (CFTR) gene mutations identified were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 + 10kbC →T, 2789 + 5G →A, 5T-12TG and the novel mutation D110E. In three patients no mutation was identified after denaturing gradient gel electrophoresis of the majority of CFTR gene exons.

Conclusion: The clinical phenotypes of CF children diagnosed by their symptoms at different ages were very mild. None of them presented with a severe lung disease. The majority of them did not seem to have been damaged by the delayed diagnosis. The combination of IRT assay plus genotype analysis (1998-1999) appears to be a more reliable method of detecting CF than IRT measurement alone or combined with only the delF508 mutation.     

Combining immunoreactive trypsinogen and pancreatitis-associated protein assays, a method of newborn screening for cystic fibrosis that avoids DNA analysis

OBJECTIVES: To evaluate the performance of a strategy in which, after immunoreactive trypsinogen (IRT) determination, genetic analysis is replaced by a biological test, the pancreatitis-associated protein (PAP) enzyme-linked immunosorbent assay (ELISA). STUDY DESIGN: The French newborn screening program includes cystic fibrosis (CF) screening by the IRT/CFTR mutation strategy. PAP was assayed on screening cards, in parallel with IRT, in all newborns from 5 French regions (n = 204,749). Analysis of PAP values in CF and non-CF newborns with elevated IRT allowed direct comparison between the current strategy and the proposed IRT/PAP strategy. RESULTS: A protocol in which newborns with IRT >50 ng/mL and PAP >1.8 ng/mL and those with IRT >100 ng/mL and PAP >1.0 ng/mL are directly recalled for sweat testing would have the same performance as the IRT/CFTR mutation strategy. CONCLUSIONS: The IRT/PAP strategy is an alternative for CF newborn screening, which avoids the drawbacks of genetic analysis and is cheaper and easier to implement than the current IRT/CFTR mutation strategy.     

A family with atypical cystic fibrosis: brother and sister with heterozygosity for both G542X and R117H.

Clinical manifestations of cystic fibrosis (CF) are variable. Genetic and environmental factors that determine whether an individual will develop associated complications are still under investigation. The present study reports the genetic analysis of a family with different clinical forms of CF and addresses the difficulty of CF diagnosis in an individual with mutant alleles G542X and R117H because of the variable phenotype associated with R117H mutation. Both children in this family were heterozygous for G542X/R117H with the same thymine sequence (7T/9T) in intron 8 of CF transmembrane conductance regulator. The girl was diagnosed with CF, whereas the boy was diagnosed with azoospermia as the sole clinical manifestation. The possible implication of the hemochromatosis gene as a CF modifier locus was analyzed because the 2 children had the same genotype. No genetic differences were detected between brother and sister that explained the different clinical manifestations of CF.     

Markedly elevated neonatal immunoreactive trypsinogen levels in the absence of cystic fibrosis gene mutations is not an indication for further testing.

AIMS: To investigate the immunoreactive trypsinogen (IRT) values above the usual 99th centile laboratory cut-off and determine the value of offering further testing to those infants with a markedly elevated IRT but no cystic fibrosis transmembrane regulator (CFTR) gene mutation identified by the screening programme. METHODS: All babies born in Victoria, Australia, between 1991 and 2003, were screened by IRT followed by CF gene mutation analysis. RESULTS: Of the 806,520 babies born, 9268 with the highest IRT levels had CFTR mutation analysis. There were 123 DeltaF508 homozygotes and 703 heterozygotes (86 with CF, 617 carriers). A total of 8442 babies had no CFTR gene mutation, of whom 18 (0.21%) had CF. The total number of CF babies with IRT greater than the laboratory cut-off was 227 (2.4%). The IRT results of the CF patients were distributed normally, with the majority above the laboratory cut-off of newborn IRT results. There was no evidence of an excess of babies with CF in the very highest levels of IRT above the 99th centile. CONCLUSIONS: Only a small proportion of babies with a neonatal IRT >99th centile have CF. Additional CF testing for infants with an elevated IRT but no CFTR gene mutation has an extremely low yield, no matter how high the IRT result.     

Role of <Emphasis Type="Italic">CFTR</Emphasis> mutation analysis in the diagnostic algorithm for cystic fibrosis

Background:The cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation identification is being used with increased frequency to aid in the diagnosis of cystic fibrosis (CF) in those suspected with CF.Aim of this study was to identify diagnostic outcomes when CFTR mutational analysis was used in CF diagnosis.CFTR mutational analysis results were also compared with sweat chloride results.Methods:This study was done on all patients at our institution who had CFTR mutation analysis over a sevenyear period since August 2006.Results:A total of 315 patients underwent CFTR mutational analysis.Fifty-one (16.2％) patients had two mutations identified.Among them 32 had positive sweat chloride levels (≥60 mmol/L),while seven had borderline sweat chloride levels (40-59 mmol/L).An additional 70 patients (22.3％) had only one mutation identified.Among them eight had positive sweat chloride levels,and 17 had borderline sweat chloride levels.Fifty-five patients (17.5％) without CFTR mutations had either borderline (n=45) or positive (n=10) sweat chloride results.Three patients with a CF phenotype had negative CFTR analysis but elevated sweat chloride levels.In eighty-three patients (26.4％) CFTR mutational analysis was done without corresponding sweat chloride testing.Conclusions:Although CFTR mutation analysis has improved the diagnostic capability for CF,its use either as the first step or the only test to diagnose CFTR dysfunction should be discouraged and CF diagnostic guidelines need to be followed.     

Rare CFTR mutation 1525-1G>A in a Pakistani patient

Cystic fibrosis (CF) is rare in non-Caucasian populations, and in such populations little is known about the spectrum of mutations and polymorphisms in the cystic fibrosis transmembrane conductance (CFTR) gene. We report the detection of a very rare CFTR mutation 1525-1G>A in intron 9 in a 5-year-old Pakistani child with typical clinical features of CF. It remains to be seen whether mutation 1525-1G>A is characteristic of Pakistani ethnicity with CF or associated with severe phenotypic features.     

Genetic counselling after carrier detection by newborn screening when one parent carries DeltaF508 and the other R117H

Newborn screening (NBS) for cystic fibrosis (CF) has been carried out in Victoria, Australia since 1989. The primary screen is immunoreactive trypsinogen (IRT) followed by DeltaF508 mutation analysis. As part of this process, carrier babies are detected and their parents are routinely offered carrier testing as part of their follow up. The DeltaF508 parent is identified and the other parent has an extended mutation analysis performed in case they are also a carrier. One of the mutations in the extended analysis is R117H which is associated with a broad phenotypic range, from CF with suppurative lung disease, to no clinical disease. We present four healthy DeltaF508 carrier babies identified by our NBS service with both parents identified as carriers, one DeltaF508 and the other R117H. Owing to the variable phenotype associated with R117H we have developed an approach to this difficult genetic counselling situation. Centres offering or considering NBS for CF will need an approach to this problem.