A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease.

Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.

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.

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination.

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.

Validation study of a new chemiluminescent singleplex IgE assay in a set of Italian allergic rhinitis patients.

BACKGROUND: The measurement of specific IgE to allergenic extracts and molecules in patients with allergic rhinitis (AR) is crucial for a precise diagnosis and further immunotherapy. Companies providing in vitro diagnostic methods in allergology continuously strive for the optimization and modernization of such methods. A new generation of automated allergy tests based on chemiluminescence detection and paramagnetic microparticles is now available, with possible advantages in sample volume, cost-effectiveness and avoidance of sample-related interference. OBJECTIVES: To test whether sIgE antibody levels obtained with a new singleplex chemiluminescent method have a good agreement with the corresponding results obtained with a "gold standard" test. METHODS: We tested sera from 368 AR patients. Specific IgE sera levels (kU/L) to a comprehensive panel of 15 allergen extracts and 6 molecules were tested with ImmunoCAP® (Thermo Fisher Scientific Inc, Phadia AB, Uppsala, Sweden) and NOVEOS™ (HYCOR® Biomedical, Garden Grove, CA, USA). We evaluated the qualitative and quantitative performance of the new NOVEOS system in matching the outcome of ImmunoCAP to each of the examined allergens. RESULTS: In relation to ImmunoCAP, the overall diagnostic sensitivity and specificity of sIgE tests with NOVEOS were 90.8% (95% CI = 88.6-92.7) and 96.2% (95% CI = 93.9-97.8), respectively. These values were higher when only molecules were considered (sensitivity = 98.7% [95% CI = 96.4%-99.7%]; specificity = 94.2% [95% CI = 88.4%-97.6%]) and lower when only extracts were considered (sensitivity = 87.6% [95% CI = 84.7%-90.2%]; specificity = 97% [95% CI = 94.4%-98.6%]). Spearman's correlation between the data set of both methods for a ≥ 0.1 kU/L cut-off was 0.84 (p < .001). CONCLUSIONS: The new singleplex NOVEOS system presented good results for qualitative and quantitative comparisons when testing specific serum IgE antibodies against a range of 21 allergens. This novel immunoassay system using only 4 µl of sample per test appears to be robust and reliable and can, therefore, be used as an aid in allergy diagnosis.

@IT2020: An innovative algorithm for allergen immunotherapy prescription in seasonal allergic rhinitis.

BACKGROUND: Allergen immunotherapy (AIT) is the only disease-modifying treatment in patients with seasonal allergic rhinoconjunctivitis (SAR). Its efficacy depends on the precise identification of the triggering allergen. However, diagnostics based on retrospective clinical history and sensitization to whole extracts (SWE) often leads to equivocal results. OBJECTIVES: To assess the usability and impact of a recently established algorithm for a clinical decision support system (@IT2020-CDSS) for SAR and its diagnostic steps [anamnesis, SWE (skin prick test or serum IgE), component resolved diagnosis, CRD, and real-time digital symptom recording, eDiary] on doctor's AIT prescription decisions. METHODS: After educational training on the @IT2020-CDSS algorithm, 46 doctors (18 allergy specialists, AS, and 28 general practitioners, GP) expressed their hypothetical AIT prescription for 10 clinical index cases. Decisions were recorded repeatedly based on different steps of the algorithm. The usability and perceived impact of the algorithm were evaluated. RESULTS: The combined use of CRD and an eDiary increased the hypothetical AIT prescriptions, both among AS and GP (p < .01). AIT prescription for pollen and Alternaria allergy based on anamnesis and SWE was heterogeneous but converged towards a consensus by integrating CRD and eDiary information. Doctors considered the algorithm useful and recognized its potential in enhancing traditional diagnostics. CONCLUSIONS AND CLINICAL IMPLICATIONS: The implementation of CRD and eDiary in the @IT2020-CDSS algorithm improved consensus on AIT prescription for SAR among AS and GP. The potential usefulness of a CDSS for aetiological diagnosis of SAR and AIT prescription in real-world clinical practice deserves further investigation.

IgE antibody repertoire in nasal secretions of children and adults with seasonal allergic rhinitis: A molecular analysis.

BACKGROUND: There is growing interest both in testing IgE in nasal secretions (NS) and in molecular diagnosis of seasonal allergic rhinitis (SAR). Yet, the reliability of nasal IgE detection with the newest molecular assays has never been assessed in a large cohort of pollen allergic patients. OBJECTIVE: To investigate with microarray technology and compare the repertoires of specific IgE (sIgE) antibodies in NS and sera of a large population of children and adults with SAR. METHODS: Nasal secretions were collected with an absorbent device (Merocel 2000® , Medtronic) and a minimal dilution procedure from 90 children and 71 adults with SAR. Total IgE (tIgE) (ImmunoCAP, Thermo Fisher Scientific (TFS)) and sIgE antibodies against 112 allergen molecules (ISAC-112, TFS) were measured in NS and serum. RESULTS: Nasal sIgE was detectable in 68.3% of the patients. The detected nasal sIgE antibodies recognized airborne (88%), vegetable (10%), and animal food or other (<1%) allergen molecules. The prevalence and average levels of sIgE in NS and serum were highly interrelated at population level. A positive nasal sIgE antibody to a given molecule predicted the detection of the same antibody in the patient's serum with a specificity of 99.7% and a sensitivity of 40%. CONCLUSIONS: The concentration of sIgE is much lower in nasal secretions than in the serum. sIgE assays with very high analytical sensitivity and sampling methods with minimal dilution will be therefore needed to validate nasal secretions as alternative to serum in testing the sIgE repertoire.

TG2 regulates the heat-shock response by the post-translational modification of HSF1.

Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response.

Adherence to Prescribed E-Diary Recording by Patients With Seasonal Allergic Rhinitis: Observational Study.

BACKGROUND: Complete diagnosis and therapy of seasonal allergic rhinoconjunctivitis require evidence that exposure to the sensitizing pollen triggers allergic symptoms. Electronic clinical diaries, by recording disease severity scores and pollen exposure, can demonstrate this association. However, patients who spontaneously download an e-diary app show very low adherence to their recording. OBJECTIVE: The objective of our study was to assess adherence of patients with seasonal allergic rhinitis to symptom recording via e-diary explicitly prescribed by an allergist within a blended care approach. METHODS: The @IT-2020 project is investigating the diagnostic synergy of mobile health and molecular allergology in patients with seasonal allergic rhinitis. In the pilot phase of the study, we recruited Italian children (Rome, Italy) and adults (Pordenone, Italy) with seasonal allergic rhinitis and instructed them to record their symptoms, medication intake, and general conditions daily through a mobile app (Allergy.Monitor) during the relevant pollen season. RESULTS: Overall, we recruited 101 Italian children (Rome) and 93 adults (Pordenone) with seasonal allergic rhinitis. Adherence to device use slowly declined during monitoring in 3 phases: phase A: first week, ≥1267/1358, 90%; phase B: second to sixth week, 4992/5884, 80% to 90%; and phase C: seventh week onward, 2063/2606, 70% to 80%. At the individual level, the adherence assessed in the second and third weeks of recording predicted with enough confidence (Rome: Spearman ρ=0.75; P<.001; Pordenone: ρ=0.81; P<.001) the overall patient adherence to recording and was inversely related to postponed reporting (ρ=-0.55; P<.001; in both centers). Recording adherence was significantly higher during the peak grass pollen season in Rome, but not in Pordenone. CONCLUSIONS: Adherence to daily recording in an e-diary, prescribed and motivated by an allergist in a blended care setting, was very high. This observation supports the use of e-diaries in addition to face-to-face visits for diagnosis and treatment of seasonal allergic rhinitis and deserves further investigation in real-life contexts.

A new molecular multiplex IgE assay for the diagnosis of pollen allergy in Mediterranean countries: A validation study.

BACKGROUND: The identification of the primary sensitizing pollen is difficult in Southern European patients with Seasonal Allergic Rhinitis (SAR) if sensitized to various pollen sources with overlapping seasonality. A more precise diagnosis is obtained by IgE assays to allergen molecules, currently available as singleplex or microarrays. OBJECTIVES: To test the analytical performance of a multi-parameter immunoblot molecular "Pollen Test" specifically designed to test IgE antibodies to pollen extracts and molecules clinically relevant in Southern Europe. METHODS: Sera were obtained from 101 children and 98 adults with SAR and tested with a customized multiplex immunoblot assay (EUROLINE Southern European Pollen Profile [ESEP]; EUROIMMUN AG, Luebeck, Germany) containing a comprehensive panel of allergen extracts and molecules. ESEP's outcomes were then compared in selected sera (ESEP positive to negative = 2:1) with those of singleplex IgE assays (ImmunoCAP; ThermoFisher Scientific, Uppsala, Sweden). For each of the examined reagents, qualitative (sensitivity, specificity, accuracy), semi-quantitative (classes) and quantitative (Spearman's rank correlation, Bland-Altmann plots) comparisons were performed. RESULTS: Compared to ImmunoCAP, cumulative ESEP's sensitivity and specificity were 87% (95% CI 84%-90%) and 88% (83%-93%) for extracts and 99% (98%-100%) and 87% (83%-91%) for molecules. Cohen's kappa coefficients (κC ) ranged for extracts from 0.18 (Pellitory) to 0.50 (Cypress) and for molecules from 0.21 (Ole e 1) to 0.68 (Phl p 7). The quantitative outcomes of the two diagnostic tests were highly correlated, with Spearman's rank correlation coefficients always exceeding 0.80. Bland-Altmann plots showed a tendency of ESEP to overestimate serum specific IgE levels, when compared to ImmunoCAP. CONCLUSIONS AND CLINICAL RELEVANCE: Sensitivity and specificity of ESEP in testing serum IgE antibodies against pollen allergen extracts and molecules, in Italian patients with SAR, both exceeded 85%. The advantages and limitations of a multiplex customized immunoblot assay, in the routine clinical use of molecular diagnostics in Southern European pollen allergic patients, deserve to be tested.

Early molecular biomarkers predicting the evolution of allergic rhinitis and its comorbidities: A longitudinal multicenter study of a patient cohort.

BACKGROUND: Pollen-related seasonal allergic rhinoconjunctivitis (SAR) is a very frequent pediatric disease in Westernized countries. Risk factors and disease phenotypes have been thoroughly examined in several cross-sectional studies. By contrast, only a few studies have examined disease evolution in patient cohorts. We investigated predictive biomarkers of disease evolution in a large cohort of children with SAR. METHODS: During 2015-2017 (follow-up), we re-examined 401 patients from those enrolled in 2009-2011 (baseline) by the "Panallergens in Pediatrics" study, a large multicenter survey of Italian children with SAR. Information on clinical history (standard questionnaire, AllergyCARD®; TPS, Italy) and skin prick tests for inhalant and foods extracts (ALK-Abelló, Hørsholm, Denmark) was acquired as at baseline visit. Evolution in clinical and sensitization data of patients was analyzed over time, as well as their association with the main baseline characteristics and atopy risk factors. RESULTS: The average age of participants was 10.4 ± 3.4 years at baseline and 16.2 ± 3.6 years at follow-up. SAR persisted in 93.3% of patients at follow-up and became more frequently associated with asthma (from 36.7% at baseline to 48.6% at follow-up) and oral allergy syndrome (OAS, from 23.4% to 37.7%). Compared to baseline, the prevalence of skin sensitization to some pollens (Phleum pratense, Corylus avellana, Platanus acerifolia, Artemisia vulgaris) and vegetables (hazelnut, wheat, and apple) significantly decreased at follow-up. Earlier onset of SAR and polysensitization at baseline were associated with incident asthma at follow-up. The presence at baseline of serum IgE to the following allergen molecules was identified as biomarkers of clinical evolution: (a) Phl p 1, for persistence of SAR; (b) Phl p 5, for persistence of both rhinitis and asthma; (c) Pru p 3, for new onset of asthma; (d) Bet v 1, for persistence of OAS. CONCLUSIONS: Seasonal allergic rhinoconjunctivitis is clinically heterogeneous in its evolution from childhood to adolescence. The detection of serum IgE to specific molecules (Phl p 1, Phl p 5, Bet v 1, Pru p 3) may be useful as biomarkers to predict SAR persistence and future onset of comorbidities, such as asthma and/or OAS.

Mutation-specific therapies and drug repositioning in cystic fibrosis.

Cystic fibrosis (CF) is an inherited, prematurely lethal rare disease affecting more than 85,000 people worldwide. CF is caused by more than 2000 loss-of-function mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). This review summarizes recent advances in the etiological therapies of CF that aim at repairing the functional defect of CFTR by means of CFTR modulators. We will discuss the state of art of the mutation-specific treatments that are designed to target different steps of the CFTR biogenesis perturbed by mutations in CFTR gene. Moreover, we will discuss how drug repositioning, namely the use of drugs already approved for the treatment of other human diseases, may be repurposed in CF patients to circumvent CFTR dysfunction. Finally, we highlight how the combined use of two or more compounds acting on different disease mechanisms is required to achieve clinical benefit in CF population.

Inhaled medications in cystic fibrosis beyond antibiotics.

Structural lung disease begins very early in children with cystic fibrosis (CF), often in the first three months of life. Inhaled medications represent an attractive therapeutic approach in CF that are routinely used as early intervention strategies. Two aerosolized solutions, hypertonic saline and dornase alfa, have significant potential benefits by improving mucociliary clearance, with minimal associated side-effects. In particular, they favor rehydration of airway surface liquid and cleavage of extracellular DNA in the airways, respectively, consequently reducing rate of pulmonary disease exacerbations. Indirect anti-inflammatory effects have been documented for both drugs, addressing each of the three interrelated elements in the vicious cycle of lung disease in CF: airway obstruction, inflammation and infection. This short review aimed to summarize the main papers that support potential clinical impact of inhaled solutions on pulmonary disease in CF.

Personalization of therapies in rare diseases: a translational approach for the treatment of cystic fibrosis.

High variability in the response rates to treatments can make the interpretation of data from clinical trials very difficult, particularly in rare genetic diseases in which the enrolment of thousands of patients is problematic. Personalized medicine largely depends on the establishment of appropriate early detectors of drug efficacy that may guide the administration (or discontinuation) of specific treatments. Such biomarkers should be capable of predicting the therapeutic response of individual patients and of monitoring early benefits of candidate drugs before late clinical benefits become evident. The identification of these biomarkers implies a rigorous stepwise process of translation from preclinical evaluation in cultured cells, suitable animal models or patient-derived freshly isolated cells to clinical application. In this review, we will discuss how a process of research translation can lead to the implementation of functional and mechanistic disease-relevant biomarkers. Moreover, we will address how preclinical data can be translated into the clinic in a personalized medical approach that can provide the right drug to the right patient within the right timeframe.

Reduced caveolin-1 promotes hyperinflammation due to abnormal heme oxygenase-1 localization in lipopolysaccharide-challenged macrophages with dysfunctional cystic fibrosis transmembrane conductance regulator.

We have previously reported that TLR4 signaling is increased in LPS-stimulated cystic fibrosis (CF) macrophages (MΦs), contributing to the robust production of proinflammatory cytokines. The heme oxygenase-1 (HO-1)/CO pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. In this study, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules enhances CAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations re-established HO-1 and CAV-1 cell surface localization in CF MΦs. Consistent with restoration of HO-1/CAV-1-negative regulation of TLR4 signaling, genetic or pharmacological (CO-releasing molecule 2) induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counterregulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease.

Multiple antimicrobial and immune-modulating activities of cysteamine in infectious diseases.

Infectious diseases are a major threat to global health and cause millions of deaths every year, particularly in developing countries. The emergence of multidrug resistance challenges current antimicrobial treatments, inducing uncertainty in therapeutic protocols. New compounds are therefore necessary. A drug repurposing approach could play a critical role in developing new treatments used either alone or in combination with standard therapy regimens. Herein, we focused on cysteamine, an aminothiol endogenously synthesized by human cells during the degradation of coenzyme-A, which is a drug approved for the treatment of nephropathic cystinosis. Cysteamine influences many biological processes due to the presence of the highly reactive thiol group. This review provides an overview of cysteamine-mediated effects on different viruses, bacteria and parasites, with a particular focus on infections caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacterium tuberculosis, non-tuberculous mycobacteria (NTM), and Pseudomonas aeruginosa. Evidences for a potential use of cysteamine as a direct antimicrobial agent and/or a host-directed therapy, either alone or in combination with other antimicrobial drugs, are described.

The immune and microbial homeostasis determines the Candida-mast cells cross-talk in celiac disease.

Celiac disease (CD) is an autoimmune enteropathy resulting from an interaction between diet, genome, and immunity. Although many patients respond to a gluten-free diet, in a substantive number of individuals, the intestinal injury persists. Thus, other factors might amplify the ongoing inflammation. Candida albicans is a commensal fungus that is well adapted to the intestinal life. However, specific conditions increase Candida pathogenicity. The hypothesis that Candida may be a trigger in CD has been proposed after the observation of similarity between a fungal wall component and two CD-related gliadin T-cell epitopes. However, despite being implicated in intestinal disorders, Candida may also protect against immune pathologies highlighting a more intriguing role in the gut. Herein, we postulated that a state of chronic inflammation associated with microbial dysbiosis and leaky gut are favorable conditions that promote C. albicans pathogenicity eventually contributing to CD pathology via a mast cells (MC)-IL-9 axis. However, the restoration of immune and microbial homeostasis promotes a beneficial C. albicans-MC cross-talk favoring the attenuation of CD pathology to alleviate CD pathology and symptoms.

Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation.

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.