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

RETRACTED: Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events

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Validazione della Diagnosi Molecolare di Rene Policistico Autosomico Dominante Mediante la Tecnologia Next Generation Sequencing

Abstract non disponibil

Amniotic Mesenchymal Stem Cells: A New Source for Hepatocyte-Like Cells and Induction of CFTR Expression by Coculture with Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33–50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype

Why, when and how to investigate primary ciliary dyskinesia in adult patients with bronchiectasis

Bronchiectasis represents the final pathway of several infectious, genetic, immunologic or allergic disorders. Accurate and prompt identification of the underlying cause is a key recommendation of several international guidelines, in order to tailor treatment appropriately. Primary ciliary dyskinesia (PCD) is a genetic cause of bronchiectasis in which failure of motile cilia leads to poor mucociliary clearance. Due to poor ciliary function in other organs, individuals can suffer from chronic rhinosinusitis, otitis media and infertility. This paper explores the current literature describing why, when and how to investigate PCD in adult patients with bronchiectasis. We describe the main PCD diagnostic tests and compare the two international PCD diagnostic guidelines. The expensive multi-test diagnostic approach requiring a high level of expertise and specialist equipment, make the multifaceted PCD diagnostic pathway complex. Therefore, the risk of late or missed diagnosis is high and has clinical and research implications. Defining the number of patients with bronchiectasis due to PCD is complex. To date, few studies outlining the aetiology of adult patients with bronchiectasis conduct screening tests for PCD, but they do differ in their diagnostic approach. Comparison of these studies reveals an estimated PCD prevalence of 1-13% in adults with bronchiectasis and describe patients as younger than their counterparts with moderate impairment of lung function and higher rates of chronic infection with Pseudomonas aeruginosa. Diagnosing PCD has clinical, socioeconomic and psychological implications, which affect patients' life, including the possibility to have a specific and multidisciplinary team approach in a PCD referral centre, as well as a genetic and fertility counselling and special legal aspects in some countries. To date no specific treatments for PCD have been approved, standardized diagnostic protocols for PCD and recent diagnostic guidelines will be helpful to accurately define a population on which planning RCT studies to evaluate efficacy, safety and accuracy of PCD specific treatments

Workload measurement for molecular genetics laboratory: A survey study

Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory

Identification of the 5T-12TG allele of the cystic fibrosis transmembrane conductance regulator gene in hypertrypsinaemic newborns

Abstract In order to increase knowledge of the pathogenic effect of the 5T-12TG allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we evaluated its presence in 24 hypertrypsinaemic newborns with borderline sweat tests. Among 20 CFTR-identified alterations, the 5T-12TG haplotype was the second most frequent mutation (14.6%) over F508del. Conclusion: Our study suggests the need for searching for this allele in hypertrypsinaemic infants with inconclusive sweat tests

Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events

Article retracted by authors

Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events

Article retracted by authors

RETRACTED: Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events

The article entitled “Ehlers-Danlos syndrome caused by the c.934C>T, p. Arg312Cys mutation in COL1A1 gene: an Italian family without cardiovascular events” has been retracted because the description and characterization of the disease in a family may have been previously published. Upon publication of this article we were notified by an author of a study appearing in 2016 in another journal claiming that characteristics and symptoms of the family described closely matched their study, and that the two studies describe the same family. Whereas constituent family members appearing in both articles were not identical (differing by one member), symptoms and diagnoses of each family proband appeared to be consistent in both studies, leading to the editors’ conclusion that it is likely that the same family was being described in two separate articles.The corresponding author of the article in Dermatology Online Journal was informed of this incident, and responded with the assertion that they were unaware of the study published in 2016, and provided no additional information. They further requested that their article be retracted. In light of the available information and author’s request, the editors of Dermatology Online Journal have retracted this article.The original article was published on July15, 2018 and corrected on September 15, 2018.The original article was published on July15, 2018 and corrected on September 15, 2018