L1077P CFTR pathogenic variant function rescue by Elexacaftor-Tezacaftor-Ivacaftor in cystic fibrosis patient-derived air-liquid interface (ALI) cultures and organoids: in vitro guided personalized therapy of non-F508del patients.

Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

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.

Sperm DNA Fragmentation and Sperm-Borne miRNAs: Molecular Biomarkers of Embryo Development?

The evaluation of morpho-functional sperm characteristics alone is not enough to explain infertility or to predict the outcome of Assisted Reproductive Technologies (ART): more sensitive diagnostic tools are needed in clinical practice. The aim of the present study was to analyze Sperm DNA Fragmentation (SDF) and sperm-borne miR-34c-5p and miR-449b-5p levels in men of couples undergoing ART, in order to investigate any correlations with fertilization rate, embryo quality and development. Male partners (n = 106) were recruited. Semen analysis, SDF evaluation and molecular profiling analysis of miR-34c-5p and miR-449b-5p (in 38 subjects) were performed. Sperm DNA Fragmentation evaluation- a positive correlation between SDF post sperm selection and the percentage of low-quality embryos and a negative correlation with viable embryo were found. SDF > 2.9% increased the risk of obtaining a non-viable embryo by almost 4-fold. Sperm miRNAs profile­we found an association with both miRNAs and sperm concentration, while miR-449b-5p is positively associated with SDF. Moreover, the two miRNAs are positively correlated. Higher levels of miR-34c-5p compared to miR-449b-5p increases by 14-fold the probability of obtaining viable embryos. This study shows that SDF, sperm miR-34c-5p, and miR-449b-5p have a promising role as biomarkers of semen quality and ART outcome.

Characteristic Hallmarks of Aging and the Impact on Carcinogenesis.

Evidence shows that there is a synergistic, bidirectional association between cancer and aging with many shared traits. Age itself is a risk factor for the onset of most cancers, while evidence suggests that cancer and its treatments might accelerate aging by causing genotoxic and cytotoxic insults. Aging has been associated with a series of alterations that can be linked to cancer: i) genomic instability caused by DNA damage or epigenetic alterations coupled with repair errors, which lead to progressive accumulation of mutations; ii) telomere attrition with possible impairment of telomerase, shelterin complex, or the trimeric complex (Cdc13, Stn1 and Ten1 - CST) activities associated with abnormalities in DNA replication and repair; iii) altered proteostasis, especially when leading to an augmented proteasome, chaperon and autophagy-lysosome activity; iv) mitochondrial dysfunction causing oxidative stress; v) cellular senescence; vi) stem cells exhaustion, intercellular altered communication and deregulated nutrient sensing which are associated with microenvironmental modifications which may facilitate the subsequential role of cancer stem cells. Nowadays, anti-growth factor agents and epigenetic therapies seem to assume an increasing role in fighting aging-related diseases, especially cancer. This report aims to discuss the impact of age on cancer growth.

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.

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.

CRMS/CFSPID Subjects Carrying D1152H CFTR Variant: Can the Second Variant Be a Predictor of Disease Development?

BACKGROUND: There are no predictive factors of evolution of cystic fibrosis (CF) screen positive inconclusive diagnosis subjects (CFSPIDs). AIM: to define the role of the second CFTR variant as a predictive factor of disease evolution in CFSPIDs carrying the D1152H variant. METHODS: We retrospectively evaluated clinical characteristics and outcome of CFSPIDs carrying the D1152H variant followed at five Italian CF centers. CFSPIDs were divided in two groups: Group A: compound heterozygous for D1152H and a CF-causing variant; Group B: compound heterozygous for D1152H and a: (i) non CF-causing variant, (ii) variant with varying clinical consequences, or (iii) variant with unknown significance. The variants were classified according to CFTR2 mutation database. RESULTS: We enrolled 43 CFSPIDs with at least one D1152H variant: 28 (65.1%) were classified in the group A, and 15 (34.9%) in the Group B. CFSPIDs of group A had the first IRT significantly higher compared to those of group B (p < 0.05) and had a more severe clinical outcome during the follow-up. At the end of the study period, after a mean follow-up of 40.6 months (range 6-91.6), 4 (9.3%) out of 43 CFSPIDs progressed to CFTR-RD or CF. All these subjects were in the group A. CONCLUSIONS: The genetic profile could help predict the risk of disease evolution in CFSPIDs carrying D1152H, revealing the subjects that need a more frequent follow-up.

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling.

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.

Alcohol dependence and serotonin transporter functional polymorphisms 5-HTTLPR and rs25531 in an Italian population.

AIMS: The role of the serotonin transporter gene (SLC6A4) in alcohol dependence (AD) is still unclear. In this paper, we have evaluated the association of the SLC6A4 gene polymorphisms 5-HTTLPR and rs25531 in AD and assessed the polymorphic patterns both in alcoholics and in healthy people of an Italian population. METHODS: Genotyping of the 5-HTTLPR (L/S) and rs25531 (A/G) polymorphisms of the SLC6A4 gene was performed on 403 alcoholics outpatients and 427 blood donors. RESULTS: Comparing AD and control populations and taking into account statistical correction for multiple testing, we found no statistically significant differences for 5-HTTLPR (L/S) and rs25531 polymorphisms in terms of either genotypes or alleles frequencies. By univariate ANOVA, a statistically significant difference was found in the onset of AD: the mean age of onset resulted to be of 25.4 years in males in respect to 28.1 in females. In particular in males, the early AD onset was different, in a statistically significant manner, depending on the presence of at least one S or Lg allele (24.6 years) in respect to La homozygotes (27.5 years) (P = 0.03). CONCLUSIONS: These findings suggest that genetic factors contribute, together with gender and age, to the onset differences in alcohol-dependent phenotypes.

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.

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.