Some of these rare human entries have additional information associated with them, including disease associations, genotype information and allele origin, as some variations are somatic rather than germ-line events. Genotypes and allele frequencies information for various populations from different studies, including data form the HapMap project, are also available.
As in dbSNP, genotypes and allele frequencies information are available for a large number of variants [ 40 ]. This project aimed to discover novel genes and mechanisms contributing to various disorders by sequencing the protein coding regions of the human genome i. LSDBs are now recognized as the best mode of collecting and curating lists of mutations related to human genetic diseases [ 42 ].
They compile in a single bioinformatics tool disease-causing and non-disease-causing sequence variations identified by genetics laboratories in families with a history of a given Mendelian disease. Here we choose to detail three LSDB dedicated to CFTR that provide complementary information for the interpretation and the characterization of variants identified in diagnostics practice. CFMDB allows the direct submission of new variants by laboratories, by filling out an on- line standardized form with the possibility to detail phenotypic data, genotype i.
The key point of this database is to collect the largest number of CFTR sequence variations identified in patients, relatives and partners. On the other side, because the submission procedure applies only to the initial report of each variant, CFMDB does not provide frequency data, available with the two other databases described below.
Finally, contributors do not always follow HGVS recommendations and a same variant can be reported by several laboratories under different names, possibly leading to misinterpretation or misreporting in diagnosis reports. CFTR2 [ 45 ] is a website designed to provide information about specific CF mutations to patients, researchers and the general public. For each mutation included in the database, it provides information about whether a given mutation causes cystic fibrosis when combined with another CF-causing mutation and clinical and biological information sweat chloride, lung function, pancreatic status and pseudomonas infection rates in patients carrying the mutation.
A specific section for health practitioners and scientists provides more in-depth and research-related information. Mutations that have not been fully analysed are considered of unknown clinical significance. The major advantages of CFTR2 are i the collection of detailed clinical characteristics on large cohorts of individuals [ 46 ] that provide useful information related to a given genotype, and ii results of functional testing that are key arguments for their final interpretation [ 47 ].
However, this database only collects clinical and genetic data of CF patients from national registers that can lead to a bias of phenotypic spectrum assessment of several mutations considered as CF-causing mutations while they were also reported in CFTR-RD patients in trans of other CF-causing mutations. CFTR -France [ 48 ] has been developed since with the aim to collect, store and process any category of variants identified in the CFTR gene, thanks to the collaboration of nine French laboratories with high expertise in the molecular analyses of this gene.
Its specificity is to compile and annotate any category of variations disease-causing, non-disease-causing and variants of unknown clinical significance that have been identified by collaborators in patients affected with CF or CFTR-RD, in foetuses with abnormal ultrasonography e. The database includes the main clinical data of these individuals, genetic information from familial segregation studies and various variant annotations frequency in patients and controls populations, sequence homology, predicted or experimentally assessed functional impact, etc.
Thus, CFTR -France, by collecting all phenotypes, reflects the phenotypic spectrum of a large number of mutations. It also reports mutations in complex alleles with association frequencies related to all individuals recorded in the database , and gives the up-to-date HGVS nomenclature of mutations. Data collected in CFTR -France are provided by level 2 specialised and reference laboratories, so that patients analysed only by level 1 laboratories searching for the most common mutations are not included in the database.
A public access program is in progress for the medical and scientific community and for patients and families. Multiple sequence alignment of orthologous sequences reveal what positions have been conserved through evolution, and these positions are supposed to be important for protein function. Annotation can enhance prediction for variants located in structurally and functionally important domains, but this information is often sparse.
The issue of the efficiency of prediction tools in assessing possible pathogenicity of missense variants in the CFTR gene is of major interest, since they constitute the vast majority of VUS identified in patients. Diagnostics laboratories frequently use those tools and particularly in problematic situations. Unfortunately their performance has not been clearly established and results i.
Predictions of the impact of non-synonymous substitutions in CFTR are mainly based on multiple sequence alignment of orthologous sequences. It is classically recommended to use several prediction tools to obtain concordant predictions that could be considered for variant interpretation. Table 5 summarizes bioinformatics programs classically used by diagnostics laboratories [ 51 - 53 ] and the new software SuSPect [ 54 ]. Bioinformatics tools for the prediction of amino acid changes: websites, characteristics and output format [ 55 - 59 ].
A recent work has emphasised the importance of sequence alignments on the performance of prediction tools [ 60 ]. The authors constructed custom multiple sequence alignments called phenotype-optimized sequence ensembles POSEs that was tested on a training set of CFTR mutations. A previous work already suggested that providing SIFT or PolyPhen-2 with custom alignments increased their performance relative to the default alignments employed by the algorithms [ 61 ].
Splicing mechanisms comprise exon recognition within large pre-mRNA molecules and the precise removal of flanking introns. These core human splice site motifs contain only a part of the information that defines exons, whereas the rest corresponds to less conserved splicing regulatory elements. A schematic of key splicing motifs and regulatory elements. Many bioinformatics tools have been developed to predict which splicing modification is the most probable for a given sequence variation — exon skipping, cryptic splice sites activation, use of de novo splice sites — or if the variant may be considered as neutral regarding its impact on splicing.
Most algorithms were developed based on biostatistical and experimental analyses of information contained in the genomic sequence. They provide a score depending on the strength of the considered splice site. Indeed, the strength of splicing motifs is a key parameter to predict the impact of a sequence variation.
Performance of these tools has been widely studied by comparing the results of predictions with experimental assays for various genes including CFTR [ 39 , 63 - 66 ]. This work provided guidelines for the proper use of these tools and for the interpretation of prediction results.
Main characteristics of several Splicing prediction tools [ 74 - 79 ]. It is important to note that consequences on splicing of exonic synonymous and non-synonymous CFTR variants must be assessed, as suggested by recent of experimental studies [ 39 , 80 ]. The examples of insertion of intronic sequences called pseudo-exons or cryptic exons in mature transcripts of various genes are becoming ever more numerous and their role in human diseases has been largely demonstrated.
We saw in section 2. Bioinformatics tools described above, which assess the impact of variants on splicing, can also be used to evaluate deep intronic mutations. Indeed, prediction tools allowed the selection of possible disease-causing mutations i. The type of cells used for transfection depends on the tissue that is studied and the clinical context. Stable expression is usually obtained by lentivirus transduction and transient transfection by chemical agent Polyfect, interferin. In this case, the endogenous CF molecular and cellular context inflammation should also be considered.
Minigenes are autonomic cyclic entity containing promoter and exons and are produced by clonal amplification in bacteria [ 81 , 82 ]. They contain a genomic segment from the gene of interest here CFTR that includes exon and flanking intronic regions length can range from ten to thousands of nucleotides, an average of bp or only intronic regions in the case of evaluation of potential creation of a pseudo-exon.
These regions of interest are framed by two invariable exons, which are part of the system. Every assay of transfection in cell lines compares the wild-type and mutated through directed mutagenesis constructs [ 84 ].
All CFTR exons are needed to produce a mature and functional protein. Thus, a modification of transcript in the in vitro system suggests that the assessed CFTR change has a deleterious effect on exon splicing. An ever-increasing number of mini-gene studies have been performed to assess the pathogenicity of CFTR variants [ 39 , 60 , 80 , 84 ]. This strategy, despite its limitations, is of high interest in the overall strategy for the characterization of rare sequence variations.
To assess molecular consequences of large rearrangements concerning one or more exons, a truncated CFTR cDNA can be inserted in the expression vector [ 85 - 87 ]. Transient or stable transfection can be performed in eukaryotic cells describe in III. Then, measurement of mRNA expression and evaluation of function and localization of the CFTR protein can be performed for each alternative transcript construct, compared to wild-type. Automated real-time RT-PCR allows the relative straightforward quantification of mRNA transcripts with specific primers and appropriate reference genes for normalization.
Protein assessment consists in the implementation of complementary experiments for protein quantification, evaluation of its maturation or its cellular localization. Main techniques are detailed below. Long-term pulse-chase experiments can provide additional information on the lifetime of CFTR on cellular compartments [ 88 ]. However, most difficulties noted in IF experiments relate to non-specific antibody staining and the effect of sample processing on characteristics of cell development. Moreover, confusion between cell surface where CFTR is active and subsurface where it would not may occur.
Finally, this remains a qualitative or semi-quantitative method. CFTR function and activity, i.
CFTR-specific chloride conductance, can be determined by patch-clamp electrophysiology, halide selective electrode technique, radioisotope efflux assays and by fluorescence-based halide efflux measurement. To date, the easiest approach developed consists in Iodide efflux based on fluorescence measurement. It may be further adjusted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function. Technical limitations include the need to perform this assay in specialized centres using expensive imaging equipment.
All these methods offer the possibility to evaluate the functional consequences of molecular abnormalities on CFTR and finally improve the classification of variants. Nasal or lung airway epitheliums are optimal. These tissues are accessible by minimally invasive interventions and display an endogenous expression of CFTR transcripts and protein. Moreover, nasal and bronchial epitheliums show the same cellular composition ciliated, goblet, columnar and immune cells although ratios differ slightly [ 89 ].
Since quantification and detection of aberrant splicing and quantification or localization of proteins are possible in human tissues, information that they bring is crucial to assess the effect of variants and to propose a functional classification. However, this approach has its limitations and requires other functional assays to perform large-scale genotype—phenotype correlation studies.
In addition, especially for nasal tissue, the low quantity of cells collected out of Moreover, highly variable CFTR expression in heterogeneous cell types, in healthy individuals and in p. Other genetic see below or environmental parameters could also modify CFTR expression levels.
The Sanger DNA sequencing method. This background information should facilitate an understanding of the application of genetic techniques in studies presented in the literature and in the daily practice of clinical medicine. Polymerase chain reaction amplification of DNA followed by restriction enzyme analysis enables diagnosis of diseases such as sickle cell anemia from a single sample of blood. Therefore, it remains imperative that, along with advances in our understanding of the human genome, work continues in the fields of ethics and sociology to help resultant new information be used in the best way possible for the overall good of humanity. Curr Med Res Opin. Because this reaction is exponential, 30 cycles produce more than one million copies of the targeted DNA segment.
Culture of primary cells from CF patients can be performed with brushed nasal or bronchial cells after biopsies. Wild-type endogenous CFTR protein is expressed at the apical membrane of polarized cells. Therefore, in vitro monolayer culture seems no longer adapted. Obtaining polarized cells is promoted by air—liquid interface culture ALI , proposed since the s, by an ex vivo system of collagen-coated porous membrane on which cells are platted after a phase of monolayer amplification or directly after nasal brushing.
Basal adherent cells differentiate in all airways epithelial cell types, which organize into a pseudo-stratified epithelium [ 91 ]. This model offers the opportunity to perform functional assays described above to determine CFTR dysfunction. Molecular defect induced by a specific mutation can be qualitatively determined if the cell donor is homozygous for this mutation.
Technical limitations such as bacterial or fungi contaminations or absence of adherence complicate culture of cells obtained from CF patients.
Moreover, further studies are needed to determine if extrapolation is possible between observations in primary cells directly after brushing and after several weeks in culture media, particularly for quantitative level assessment. ICM was developed as a research tool to assess CFTR function in the s and has been used as a diagnostic test since the early s [ 92 , 93 ]. At least four superficial rectal biopsies per patient, obtained by suction, are needed and mounted on adequate tissue sliders.
This combination of ionic responses discriminates patients with CF from healthy subjects but not CF patients with pancreatic insufficiency PI or sufficiency PS. Moreover, mild mutations could result in a false-negative ICM. ICM is not altered by secondary damage on tissue and thus better reveals the primary CFTR dysfunction compared with nasal potential difference, see below.
Finally, setup and maintenance of dedicated equipment by experienced and trained staff limits its use. In atypical clinical context these in vivo tests can give additional arguments to further explore the CFTR locus cf. Figure 2 that describes the molecular diagnostics step-by-step strategy.
Furthermore, they provide complementary information for the interpretation of CFTR variations. Sweat electrolytes are higher in the most severely affected and are lower in those with mild mutation, who have partial rescue of channel function [ 95 - 97 ]. There are two major advantages for the use of sweat test in the evaluation of the CFTR mutation severity: i stability of the measure throughout life and ii non-invasive way of measurement which maintains skin integrity. Indeed, sweat electrolytes levels reflect the primary defect on the CFTR protein and do not highlight secondary consequences of its absence or dysfunction on affected organs.
This method provides a unique evaluation of the purely secretory function of CFTR in vivo. It has been shown to be more sensitive in individuals carrying mutations that commonly exhibit normal or borderline sweat chloride rates, such as c. The transepithelial NPD measurement estimates the net ion conductance across the nasal airway epithelium and indirectly measure CFTR activity. However, other sources of phenotypic variability modifiers genes or environment and technical limitations i.
CFTR expression and NPD response can also be modified if nasal epithelium is affected by rhinosinusitis, polyposis or exposure to smoking [ ]. However, despite the difficulty of setting up this technique, NPD assessment could be used as a complementary step to support CFTR dysfunction in inconclusive clinical cases and then to confirm the deleterious effect of CFTR variants identified in these patients. On one hand, NGS approaches offer new possibilities by multiplexing samples and provide a wider coverage of the CFTR locus including deep intronic regions.
NGS assay design can also include additional modifiers genes [ 31 ]. On the other hand, molecular diagnoses in emergency contexts challenge the possibility of sample multiplexing, and the increased number of VUCS will require complex functional analyses. However, as tools described above are constantly improving, the knowledge about CFTR variations is rapidly expanding, allowing geneticists and clinicians to provide patients with high quality information and adequate genetic counselling.
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