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.

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells.

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.

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.

The Immune Response to SARS-CoV-2 Vaccine in a Cohort of Family Pediatricians from Southern Italy.

In Italy, from January 2021, the Ministry of Health indicated a vaccination plan against COVID for frail patients and physicians based on a three-dose scheme. However, conflicting results have been reported on which biomarkers permit immunization assessment. We used several laboratory approaches (i.e., antibodies serum levels, flow cytometry analysis, and cytokines release by stimulated cells) to investigate the immune response in a cohort of 53 family pediatricians (FPs) at different times after the vaccine. We observed that the BNT162b2-mRNA vaccine induced a significant increase of specific antibodies after the third (booster) dose; however, the antibody titer was not predictive of the risk of developing the infection in the six months following the booster dose. The antigen stimulation of PBMC cells from subjects vaccinated with the third booster jab induced the increase of the activated T cells (i.e., CD4+ CD154+); the frequency of CD4+ CD154+ TNF-α+ cells, as well as the TNF-α secretion, was not modified, while we observed a trend of increase of IFN-γ secretion. Interestingly, the level of CD8+ IFN-γ+ (independently from antibody titer) was significantly increased after the third dose and predicts the risk of developing the infection in the six months following the booster jab. Such results may impact also other virus vaccinations.

Comparative Analysis of a Human Neutralizing mAb Specific for SARS-CoV-2 Spike-RBD with Cilgavimab and Tixagevimab for the Efficacy on the Omicron Variant in Neutralizing and Detection Assays.

The recent pandemic years have prompted the scientific community to increasingly search for and adopt new and more efficient therapeutic and diagnostic approaches to deal with a new infection. In addition to the development of vaccines, which has played a leading role in fighting the pandemic, the development of monoclonal antibodies has also represented a valid approach in the prevention and treatment of many cases of CoronaVirus Disease 2019 (COVID-19). Recently, we reported the development of a human antibody, named D3, showing neutralizing activity against different SARS-CoV-2 variants, wild-type, UK, Delta and Gamma variants. Here, we have further characterized with different methods D3's ability to bind the Omicron-derived recombinant RBD by comparing it with the antibodies Cilgavimab and Tixagevimab, recently approved for prophylactic use of COVID-19. We demonstrate here that D3 binds to a distinct epitope from that recognized by Cilgavimab and shows a different binding kinetic behavior. Furthermore, we report that the ability of D3 to bind the recombinant Omicron RBD domain in vitro results in a good ability to also neutralize Omicron-pseudotyped virus infection in ACE2-expressing cell cultures. We point out here that D3 mAb maintains a good ability to recognize both the wild-type and Omicron Spike proteins, either when used as recombinant purified proteins or when expressed on pseudoviral particles despite the different variants, making it particularly useful both from a therapeutic and diagnostic point of view. On the basis of these results, we propose to exploit this mAb for combinatorial treatments with other neutralizing mAbs to increase their therapeutic efficacy and for diagnostic use to measure the viral load in biological samples in the current and future pandemic waves of coronaviruses.

Corrigendum: Age-Related Differences in the Expression of Most Relevant Mediators of SARS-CoV-2 Infection in Human Respiratory and Gastrointestinal Tract.

[This corrects the article DOI: 10.3389/fped.2021.697390.].

Age-Related Differences in the Expression of Most Relevant Mediators of SARS-CoV-2 Infection in Human Respiratory and Gastrointestinal Tract.

Background: Clinical features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection seem to differ in children compared to that in adults. It has been hypothesized that the lower clinical severity in children could be influenced by differential expression of the main host functional receptor to SARS-CoV-2, the angiotensin-converting enzyme 2 (ACE2), but data are still conflicting. To explore the origin of age-dependent clinical features of coronavirus disease 2019 (COVID-19), we comparatively evaluated the expression in children and adult subjects of the most relevant mediators of the SARS-CoV-2 infection: ACE2, angiotensin-converting enzyme 1 (ACE1), transmembrane serine protease-2 (TMPRSS2), and neuropilin-1 (NRP1), at upper respiratory tract and small intestine level. Methods: The expression of ACE2, ACE1, TMPRSS2, and NRP1 in nasal epithelium and in small intestine epithelium was investigated by quantitative real-time PCR analysis. Results: We found no differences in ACE2, ACE1, and TMPRSS2 expression in the nasal epithelium comparing children and adult subjects. In contrast, nasal epithelium NRP1 expression was lower in children compared to that in adults. Intestinal ACE2 expression was higher in children compared to that in adults, whereas intestinal ACE1 expression was higher in adults. Intestinal TMPRSS2 and NRP1 expression was similar comparing children and adult subjects. Conclusions: The lower severity of SARS-CoV-2 infection observed in children may be due to a different expression of nasal NRP1, that promotes the virus interaction with ACE2. However, the common findings of intestinal symptoms in children could be due to a higher expression of ACE2 at this level. The insights from these data will be useful in determining the treatment policies and preventive measures for COVID-19.

Elexacaftor-Tezacaftor-Ivacaftor Therapy for Cystic Fibrosis Patients with The F508del/Unknown Genotype.

The new CFTR modulator combination, elexacaftor/tezacaftor/ivacaftor (Trikafta) was approved by the FDA in October 2019 for treatment of Cystic Fibrosis in patients 6 years of age or older who have at least one F508del mutation in one allele and a minimal-function or another F508del mutation in the other allele. However, there is a group of patients, in addition to those with rare mutations, in which despite the presence of a F508del in one allele, it was not possible to identify any mutation in the other allele. To date, these patients are excluded from treatment with Trikafta in Italy, where the CF patients carrying F508del/unknown represent about 1.3% (71 patients) of the overall Italian CF patients. In this paper we show that the Trikafta treatment of nasal epithelial cells, derived from F508del/Unknown patients, results in a significant rescue of CFTR activity. Based on our findings, we think that the F508del/Unknown patients considered in this study could obtain clinical benefits from Trikafta treatment, and we strongly suggest their eligibility for this type of treatment. This study, adding further evidence in the literature, once again confirms the validity of functional studies on nasal cells in the cystic fibrosis theratyping and personalized medicine.