Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation.

In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

Art meets science: The Cosmopolitan Chicken Research Project.

The Cosmopolitan Chicken Project is an artistic undertaking of renowned artist Koen Vanmechelen. In this project, the artist interbreeds domestic chickens from different countries aiming at the creation of a true Cosmopolitan Chicken as a symbol for global diversity. The unifying theme is the chicken and the egg, symbols that link scientific, political, philosophical and ethical issues. The Cosmopolitan Chicken Research Project is the scientific component of this artwork. Based on state of the art genomic techniques, the project studies the effect of the crossing of chickens on the genetic diversity. Also, this research is potentially applicable to the human population. The setup of the CC®P is quite different from traditional breeding experiments: starting from the crossbreed of two purebred chickens (Mechelse Koekoek x Poule de Bresse), every generation is crossed with a few animals from another breed. For 26 of these purebred and crossbred populations, genetic diversity was measured (1) under the assumption that populations were sufficiently large to maintain all informative SNP within a generation and (2) under the circumstances of the CCP breeding experiment. Under the first assumption, a steady increase in genetic diversity was witnessed over the consecutive generations, thus indeed indicating the creation of a "Cosmopolitan Chicken Genome". However, under the conditions of the CCP, which reflects the reality within the human population, diversity is seen to fluctuate within given boundaries instead of steadily increasing. A reflection on this might be that this is because, in humans, an evolutionary optimum in genetic diversity is reached. Key words.

A quality control study of CFTR mutation screening in 40 different European laboratories. The European Concerted Action on Cystic Fibrosis.

A quality control study was performed to determine the accuracy of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutation screening in 40 different genetic screening laboratories throughout Europe. A total of 9 different samples were investigated blindly by the participating laboratories. Only 25/40 laboratories, i.e. 62.5%, were able to type all samples correctly for the mutations for which they routinely screened. Only 2 of the 9 samples were correctly typed in all 40 laboratories. The lowest accuracy rate was 80% for 1 sample. 12.5% of the participating laboratories interpreted the F508C polymorphism as a true CF disease mutation and 23.5% interpreted the delta I507 mutation as a delta F508 mutation. For the delta F508 mutation, a false-negative result of 3.75% was obtained. It is clear that the accuracy of CFTR typing should be improved.

Limited expansion of the (CAG)n repeat of the Huntington gene: a premutation (?).

Huntington's disease (HD) is an autosomal dominant disorder with choreic movements, psychiatric manifestations and cognitive dysfunction. Recently the IT15 gene on chromosome 4p has been identified containing an unstable and expanded trinucleotide repeat in patients with HD. We report on the characteristics of this repeat in 248 individuals from 41 Belgian HD families. The length of the expanded repeat was defined precisely and reproducibly on an ALF sequencer and correlated well with the age of onset (r = -0.72). Paternal transmission of the expanded repeat resulted on average in a significantly longer repeat length (+2.79 repeats) than maternal transmission (-0.29 repeats). (CAG)n repeat of a premutation (?) size was observed in this population with subsequent expansion in the disease range. Presymptomatic or prenatal testing using only linked markers may be problematic in these cases.

Recommendations for quality improvement in genetic testing for cystic fibrosis. European Concerted Action on Cystic Fibrosis.

These recommendations for quality improvement of cystic fibrosis genetic diagnostic testing provide general guidelines for the molecular genetic testing of cystic fibrosis in patients/individuals. General strategies for testing as well as guidelines for laboratory procedures, internal and external quality assurance, and for reporting the results, including the requirements of minimal services in mutation testing, the nomenclature for describing mutations, procedures to control false-positive amplification reactions and to validate tests, and guidelines to implement a quality system in a molecular diagnostic laboratory are reviewed.

Phosphorylation site independent single R-domain mutations affect CFTR channel activity.

We investigated CFTR channel activity of mature R-domain mutants showing single alterations at sites other than the predicted phosphorylation sites. All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. The anion permeability sequence for all three mutants was the same as that of wild type CFTR. Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Single channel conductances were unchanged in all mutants. Our results suggest that additional sites in the R-domain other than phosphorylation sites influence gating of CFTR channels.

Suppressive interactions between mutations located in the two nucleotide binding domains of CFTR.

The S1235R locus in CFTR was studied in combination with alleles found at the M470V and G628R loci. While R628 caused a maturational defect, R1235 did not. The impact of R1235 was found to be influenced by the alleles present at the G628R and M470V loci. At the single channel level, R1235-V (R1235 on a V470 background) was characterized by an open probability significantly higher than V470-wildtype CFTR. M470, which on its own increases CFTR chloride transport activity when compared to V470-wildtype CFTR, suppressed the activity of R1235 in such a way that a protein with an open probability not significantly different from V470-wildtype CFTR was obtained. While R628-V CFTR had similar current densities as V470-wildtype CFTR in Xenopus laevis oocytes, R1235-V resulted in current densities that were more than twofold higher than those of V470-wildtype CFTR. However, the current densities generated by R1235/R628-V (R1235 and R628 on a V470 background) CFTR were significant lower than R1235-V or R628-V CFTR.

Characterization of mutations located in exon 18 of the CFTR gene.

In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I11139V and deltaM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild-type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.

A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1). It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR. Determination of the three-dimensional structure of NBF1 would help to understand this defect. We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1-ATPase protein. This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.

Rapid detection of hypervariable regions by the polymerase chain reaction technique.

The polymerase chain reaction (PCR) technique has provided a substantial improvement for the detection and analysis of known genetic polymorphisms. Here, we describe the application of this method for the detection of variable number of tandem repeat (VNTR) sequences. With the use of unique oligonucleotide primers, flanking the repeat sequence, and the thermostable Taq DNA polymerase, the hypervariable regions 3' of the Ha-ras gene, 3' of the apolipoprotein B gene, and 5' to the joining segments of the heavy-chain immunoglobulin gene could be amplified. Alleles up to 2,000 bp could be visualized directly on ethidium bromide-stained agarose gels. Larger alleles were seen only after traditional Southern blot analysis with an internal probe. The value of this new approach for the detection of VNTRs is illustrated in a case of paternity dispute.

European Epidemiologic Registry of Cystic Fibrosis (ERCF): comparison of major disease manifestations between patients with different classes of mutations.

SUMMARY. By August 1997, 11,749 patients with cystic fibrosis had been enrolled in the European Epidemiologic Registry of Cystic Fibrosis (ERCF). Genotype analysis had been performed on 8,963 (76%) of these patients, and the majority had one or two identifiable mutations. Patients with known mutations were classified according to the type of mutation (Classes I-V), and were grouped according to the class of mutation on both chromosomes. This resulted in six subgroups, including all patients homozygous for Class I (I/I, n = 72), for Class II (II/II, n = 5,020), and for Class III mutations, (III/III, n = 23). Since there were only 23 patients homozygous for Class III mutations, a fourth group was made up of patients who were compound heterozygous for a Class II and III mutation (II/III, n = 265). There were only five patients homozygous for Class IV mutations, and consequently a fifth group was made up of all patients carrying at least one Class IV mutation, regardless of the nature of the mutation on the other chromosome (IV/any, n = 187). None were homozygous for Class V mutations; consequently, a sixth group consisted of patients carrying at least one Class V mutation (V/any, n = 22). Mean age was highest in groups III/III, IV/any, and V/any (15.6, 16, and 17 years, respectively) as opposed to 12.4 years in group II/II and 13.4 in group II/III, but both group III/III and V/any were small, and the confidence interval of the mean was large. The percentage of patients receiving pancreatic enzymes was lower in groups IV/any and V/any than in any of the other groups, i.e., approximately 50% of patients 18 years or older in both groups as opposed to between 90-100% of all other patients regardless of age. The prevalence of diabetes mellitus increased with age from 2.6% in patients < 18 years to 22.1% in patients 18 years or older in the large group II/II, but was only 1.5% in patients 18 years or older in group IV/any. Disregarding the small group III/III, abnormally elevated liver enzymes and/or bilirubin (1.5 x upper normal limit) was much less frequent in group IV/any than in any of the other groups, both overall and in patients aged 18 years or more. The course of lung disease appeared to be less dependent on genotype than pancreatic function, with only minor differences between groups; however, the mean values of both FVC % and FEV(1) % were slightly higher in group IV/any than all other groups in both younger and older patients. The same was found for the prevalence of some major clinical signs of severe lung disease, such as clubbing, hyperinflation, and crepitations. Overall mean weight expressed as an age percentile was markedly higher in group IV/any than in any other group, which may be related to the finding of a much lower prevalence of chronic P. aeruginosa infection in patients 18 years or older belonging to group IV/any (and V/any) than in any other group. In conclusion, the presence of a class IV mutation appears to offer some degree of protection against pancreatic insufficiency, diabetes mellitus, and liver disease. We confirmed that lung disease follows a milder clinical course in patients with a class IV mutation and that the presence of a class IV mutation (and possibly class V) is associated with a delay in the onset of P. aeruginosa infection.

Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.

BACKGROUND: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC). RESULTS: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. CONCLUSIONS: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

Identification of polysaccharides in pharmaceuticals by capillary gas chromatography.

A sensitive method for the identification of polysaccharides in pharmaceuticals is described. Polysaccharides are isolated by gel filtration and subsequently hydrolysed. The monomeric carbohydrates obtained are transformed into oxime-trimethylsilyl derivatives and analysed by capillary gas chromatography. Profiles of 13 different natural or semi-synthetic polysaccharides are discussed. The profiles of the hydrolysis products can be used to identify the polysaccharides mentioned above. Possible interferences by other polymers are given. The method can be used to identify most polysaccharides used as pharmaceutical adjuvants.

An Algerian child homozygous for the M470V polymorphism and for a deletion of two nucleotides in exon 10 of the CFTR gene, shows severe cystic fibrosis symptoms.

When screening for the presence of major cystic fibrosis mutations in Algerian cystic fibrosis families by heteroduplex formation, aberrant heteroduplexes were observed for exon 10 in one family. Here we describe the clinical and molecular findings in a severely affected child of this family, homozygous for the 1609delCA and for the M470V polymorphism.

The relative frequency of CFTR mutation classes in European patients with cystic fibrosis.

More than 1900 different mutations in the CFTR gene have been reported. These are grouped into classes according to their effect on the synthesis and/or function of the CFTR protein. CFTR repair therapies that are mutation or mutation class specific are under development. To progress efficiently in the clinical phase of drug development, knowledge of the relative frequency of CFTR mutation classes in different populations is useful. Therefore, we describe the mutation class spectrum in 25,394 subjects with CF from 23 European countries. In 18/23 countries, 80% or more of the patients had at least one class II mutation, explained by F508del being by far the most frequent mutation. Overall 16.4% of European patients had at least one class I mutation but this varied from 3 countries with more than 30% to 4 countries with less than 10% of subjects. Overall only respectively 3.9, 3.3 and 3.0% of European subjects had at least one mutation of classes III, IV and V with again great variability: 14% of Irish patients had at least one class III mutation, 7% of Portuguese patients had at least one class IV mutation, and in 6 countries more than 5% of patients had at least one class V mutation.

New clinical diagnostic procedures for cystic fibrosis in Europe.

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.

Recommendations for the classification of diseases as CFTR-related disorders.

Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops. A CFTR-RD may be defined as "a clinical entity associated with CFTR dysfunction that does not fulfil diagnostic criteria for CF". The utility of sweat testing, mutation analysis, nasal potential difference, and/or intestinal current measurement for the differential diagnosis of CF and CFTR-RD is discussed. Algorithms which use genetic and functional diagnostic tests to distinguish CF and CFTR-RDs are presented. According to present knowledge, congenital bilateral absence of vas deferens (CBAVD), acute recurrent or chronic pancreatitis and disseminated bronchiectasis, all with CFTR dysfunction, are CFTR-RDs.

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories.

Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.