Downregulation of epithelial sodium channel (ENaC) activity in cystic fibrosis cells by epigenetic targeting.

The pathogenic mechanism of cystic fibrosis (CF) includes the functional interaction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with the epithelial sodium channel (ENaC). The reduction of ENaC activity may constitute a therapeutic option for CF. This hypothesis was evaluated using drugs that target the protease-dependent activation of the ENaC channel and the transcriptional activity of its coding genes. To this aim we used: camostat, a protease inhibitor; S-adenosyl methionine (SAM), showed to induce DNA hypermethylation; curcumin, known to produce chromatin condensation. SAM and camostat are drugs already clinically used in other pathologies, while curcumin is a common dietary compound. The experimental systems used were CF and non-CF immortalized human bronchial epithelial cell lines as well as human bronchial primary epithelial cells. ENaC activity and SCNN1A, SCNN1B and SCNN1G gene expression were analyzed, in addition to SCNN1B promoter methylation. In both immortalized and primary cells, the inhibition of extracellular peptidases and the epigenetic manipulations reduced ENaC activity. Notably, the reduction in primary cells was much more effective. The SCNN1B appeared to be the best target to reduce ENaC activity, in respect to SCNN1A and SCNN1G. Indeed, SAM treatment resulted to be effective in inducing hypermethylation of SCNN1B gene promoter and in lowering its expression. Importantly, CFTR expression was unaffected, or even upregulated, after treatments. These results open the possibility of CF patients' treatment by epigenetic targeting.

Theratyping cystic fibrosis in vitro in ALI culture and organoid models generated from patient-derived nasal epithelial conditionally reprogrammed stem cells.

QUESTION: Cystic fibrosis (CF) is due to pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent improvements have enabled pharmacological therapy aiming at restoring mutated CFTR expression and function. CFTR "modulators" have revolutionised the CF therapeutic landscape, particularly the last approved, Trikafta. This drug combination is indicated by the United States Food and Drug Administration and very recently by the European Medicines Agency for genotypes carrying at least one copy of CFTR with the F508del pathogenic variant. However, several genotypes are not yet eligible for Trikafta treatment. MATERIALS/PATIENTS AND METHODS: We exploited an innovative cellular approach allowing highly efficient in vitro expansion of airway epithelial stem cells (AESCs) through conditional reprogramming from nasal brushing of CF patients. This approach, coupled to the development of AESC-derived personalised disease models, as organoids and air-liquid interface (ALI) cultures, revealed highly suitable for CFTR pharmacological testing. RESULTS AND ANSWER TO THE QUESTION: We fully validated the experimental models and implemented the CFTR functional assays and biochemical CFTR protein characterisation, which allowed the evaluation of the efficacy of clinically available modulators in restoring CFTR maturation and function of each patient-derived "avatar" (theratyping). F508del homozygous genotypes, used as controls, confirmed the higher clinical activity of Trikafta in comparison with older modulators. In addition, Trikafta showed its efficacy on three rare genotypes previously not eligible for treatment with modulators, opening the way to clinical translation. Finally, encouraging results for innovative drug combinations were obtained.

DNA Methylation Patterns Correlate with the Expression of SCNN1A, SCNN1B, and SCNN1G (Epithelial Sodium Channel, ENaC) Genes.

The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype-phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, ß and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5'-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF.

A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis.

Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype-phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype-phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.

Quantitative Evaluation of CFTR Gene Expression: A Comparison between Relative Quantification by Real-Time PCR and Absolute Quantification by Droplet Digital PCR.

In the precision medicine era of cystic fibrosis (CF), therapeutic interventions, by the so-called modulators, target the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The levels of targetable CFTR proteins are a main variable in the success of patient-specific therapy. In turn, the CFTR protein level depends, at least in part, on the level of CFTR mRNA. Many mechanisms can modulate the CFTR mRNA level, for example, transcriptional rate, stability of the mRNA, epigenetics, and pathogenic variants that can affect mRNA production and degradation. Independently from the causes of variable CFTR mRNA levels, their exact quantitative assessment is of great importance in CF. Methods with high analytical sensitivity, precision, and accuracy are mandatory for the quantitative evaluation aimed at the amelioration of the diagnostic, prognostic, and therapeutic aspects. This paper compares, for the first time, two CFTR gene expression quantification methods: a well-established method for the relative quantification of CFTR mRNA using a real-time PCR and an innovative method for its absolute quantification using a droplet digital PCR. No comprehensive methods for absolute CFTR quantification via droplet digital PCR have been published so far. The accurate quantification of CFTR expression at the mRNA level is a critical step for the personalized therapeutic approaches of CF.

A template for mutational data analysis of the CFTR gene.

BACKGROUND: Automated DNA sequencing produces large amounts of data that need to be analyzed by appropriate software. Personalization of software can be a difficult and time-consuming task, especially if a large number of mutations have to be analyzed. METHODS: The Applied BioSystems SeqScape software, based on the KB basecaller algorithm, is a versatile tool that can be used for mutational analysis and for data quality assessment of sequences belonging to any gene of interest. Using this software we analyzed over 1400 sequences of CFTR exons and adjacent intronic zones, representing over 500,000 bases. RESULTS: We present an up to date specific template and a linked set of instructions for automated labeling of all point mutations and polymorphisms of the CFTR gene, whose mutations cause cystic fibrosis (the most common genetic disease among Caucasian individuals). We also describe our refined software settings for mutational analysis, in order to keep to a minimum the need of manual validation. CONCLUSIONS: The use of our template greatly simplifies the mutational analysis of the CFTR gene, reducing human intervention. In our opinion, it might not only be useful to researchers that already perform CFTR mutational analysis by sequencing methods but it should also improve the approach in those laboratories that already use ABI PRISM instrumentation for a limited mutational analysis of the CFTR gene. Similar mutational templates can also be used for other disease causing genes, thus improving molecular genetics protocols.

Quantitative Evaluation of CFTR Pre-mRNA Splicing Dependent on the (TG)mTn Poly-Variant Tract.

Genetic analysis in cystic fibrosis (CF) is a difficult task. Within the many causes of variability and uncertainty, a major determinant is poor knowledge of the functional effect of most DNA variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. In turn, knowledge of the effect of a CFTR variant has dramatic diagnostic, prognostic and, in the era of CF precision medicine, also therapeutic consequences. One of the most challenging CFTR variants is the (TG)mTn haplotype, which has variable functional effect and controversial clinical consequences. The exact quantification of the anomalous splicing of CFTR exon 10 (in the HGVS name; exon 9 in the legacy name) and, consequently, of the residual wild-type functional CFTR mRNA, should be mandatory in clinical assessment of patients with potentially pathological haplotype of this tract. Here, we present a real time-based assay for the quantification of the proportion of exon 10+/exon 10- CFTR mRNA, starting from nasal brushing. Our assay proved rapid, economic and easy to perform. Specific primers used for this assay are either disclosed or commercially available, allowing any laboratory to easily perform it. A simplified analysis of the data is provided, facilitating the interpretation of the results. This method helps to enhance the comprehension of the genotype-phenotype relationship in CF and CFTR-related disorders (CFTR-RD), crucial for the diagnosis, prognosis and personalized therapy of CF.

A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.

PURPOSE: To evaluate the role of complex alleles, with two or more mutations in cis position, of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the definition of the genotype-phenotype relationship in cystic fibrosis (CF), and to evaluate the functional significance of the highly controversial L997F CFTR mutation. METHODS: We evaluated the diagnosis of CF or CFTR-related disorders in 12 unrelated subjects with highly variable phenotypes. According to a first CFTR mutational analysis, subjects appeared to be compound heterozygotes for a classic mutation and the L997F mutation. A further CFTR mutational analysis was conducted by means of a protocol of extended sequencing, particularly suited to the detection of complex alleles. RESULTS: We detected a new [R117L; L997F] CFTR complex allele in the four subjects with the highest sweat test values and CF. The eight subjects without the complex allele showed the most varied biochemical and clinical outcome and were diagnosed as having mild CF, CFTR-related disorders, or even no disease. CONCLUSIONS: The new complex allele partially explains the variable phenotype in CF subjects with the L997F mutation. CFTR complex alleles are likely to have a role in the definition of the genotype-phenotype relationship in CF. Whenever apparently identical CFTR-mutated genotypes are found in subjects with divergent phenotypes, an extensive mutational search is mandatory.

Two novel and correlated CF-causing insertions in the (TG)mTn tract of the CFTR gene.

Two novel and related pathogenic variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene were structurally and functionally characterized. These alterations have not been previously described in literature. Two patients with diagnosis of Cystic Fibrosis (CF) based on the presence of one mutated allele, p.Phe508del, pathological sweat test and clinical symptoms were studied. To complete the genotypes of both patients, an extensive genetic and functional analysis of the CFTR gene was performed. Extensive genetic characterization confirmed the presence of p.Phe508del pathogenic variant and revealed, in both patients, the presence of an insertion of part of intron 10 in intron 9 of the CFTR gene, within the (TG)m repeat, with a variable poly-T stretch. The molecular lesions resulted to be very similar in both patients, with only a difference in the number of T in the poly-T stretch. The functional characterization at RNA level revealed a complete anomalous splicing, without exon 10, from the allele with the insertion of both patients. Consequently, the alleles with the insertions are expected not to contribute to the formation of a functional CFTR protein. Molecular and functional features of these alterations are compatible with the definition of novel CF-causing variants of the CFTR gene. This also allowed the completion of the genetic characterization of both patients.

DAT gene polymorphisms (rs28363170, rs393795) and levodopa-induced dyskinesias in Parkinson's disease.

L-dopa-induced dyskinesias (LID) is a common motor side effect of levodopa therapy of Parkinson's disease (PD). The identified predictors may only partially account for the risk of developing LID and genetic factors may contribute to this variability. The present study is aimed to investigate whether polymorphisms in the dopamine transporter gene (DAT) are associated with the risk of developing LID. Genotyping of the 40-bp VNTR (rs28363170) and rs393795 (A/C) polymorphisms of the DAT gene was performed in a well-characterized cohort of 181 Italian PD patients in treatment with L-DOPA for 3 years or more. The results of our study show that there is no difference in dyskinesias prevalence among carriers of the two DAT gene polymorphisms. However, the combination of the two genotypes 10R/10R (rs28363170) and A carrier (rs393795) of the DAT gene reduces the risk of LID occurrence during long-term therapy with l-DOPA with respect to the PD subjects who did not carry these alleles (OR = 0.31; 95% CI, 0.09-0.88). Also based on a logistic regression analysis, the 10R/10R and the A carrier allele of the rs393795 polymorphisms of the DAT gene, could reduce the susceptibility to develop LID during levodopa therapy adjusted by demographical and clinical variables (OR = 0.19; 95% CI, 0.05-0.69). Additional studies further investigating the rs28363170 and rs393795 polymorphisms with LID in PD are needed to clarify their role in different ethnicities.

Polymorphism of the 3'-UTR of the dopamine transporter gene (DAT) in New World monkeys.

Genetic polymorphism in the 3'-untranslated region (3'-UTR) of the dopamine transporter (DAT) gene has been reported in both human and nonhuman primates, and the variable number of tandem repeats (VNTR) polymorphism has been related to several neurological and psychiatric disorders. As New World primates have been employed as models in biomedical research in these fields, in the present study we assessed genetic variation in the DAT gene in 25 robust capuchin monkeys (Sapajus spp.) and 39 common marmosets (Callithrix jacchus). Using enzymatic amplification followed by sequencing of amplified fragments, a VNTR polymorphism in the 3'-UTR region of the DAT gene was identified in both robust capuchins and common marmosets. The polymorphic tandem repeat of 40-bp basic units is similar to the human VNTR consensus sequence, with size variants composed of 9, 10, and 11 units in marmosets and 8, 9, 13, and 17 basic units in capuchins. We found behavioral evidence that carrying the 10-repeat DAT allele promotes flexible choice and maximization of foraging in marmosets tested in an operant choice paradigm. Moreover, in an intertemporal choice task, capuchins with longer repeat variants show less self-controlled choices than capuchins with at least one short repeat variant. Future research should focus on the relationship between these DAT polymorphisms, dopamine reuptake via the dopamine transporter, and behavioral and cognitive variation across New World monkey individuals.

The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes.

Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR.

The Impact on Genetic Testing of Mutational Patterns of CFTR Gene in Different Clinical Macrocategories of Cystic Fibrosis.

More than 2000 sequence variations of the cystic fibrosis transmembrane conductance regulator gene are known. The marked genetic heterogeneity, poor functional characterization of the vast majority of sequence variations, and an uncertain genotype-phenotype relationship complicate the definition of mutational search strategies. We studied the effect of the marked genetic heterogeneity detected in a case series comprising 610 patients of cystic fibrosis (CF), grouped in different clinical macrocategories, on the operative characteristics of the genetic test designed to fully characterize CF patients. The detection rate in each clinical macrocategory and at each mutational step was found to be influenced by genetic heterogeneity. The definition of a single mutational panel that is suitable for all clinical macrocategories proved impossible. Only for classic CF with pancreas insufficiency did a reduced number of mutations yield a detection rate of diagnostic value. All other clinical macrocategories required an extensive genetic search. The search for specific mutational classes appears to be useful only in specific CF clinical forms. A flowchart defining a mutational search that may be adopted for different CF clinical forms, optimized in respect to those already available, is proposed. The findings also have consequences for carrier screening strategies.