Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75

Use of benzodiazepine and Z‐drugs and mortality in older adults after myocardial infarction

BACKGROUND: The adverse cardiovascular effects of benzodiazepines and Z-drugs (jointly referred as BZDRs) have been of concern. Yet, little is known about the use of BZDRs in relation to mortality risk among older adults with myocardial infarction history (post-MI). METHODS: This study is a secondary analysis of the Alpha Omega Cohort study, comprising post-MI patients aged 40-60 years. Self-reported information on the use of BZDRs, including types and dose, was collected at baseline. Four categories of mortality were examined, namely all-cause mortality, cardiovascular (CVD) mortality, cancer mortality, and non-CVD/non-cancer mortality. Associations between BZDRs use, by types and doses, and mortality were estimated with Cox regression models, adjusted for demographic and classic cardiovascular risk factors. RESULTS: A total of 433 (8.9%) out of 4837 (21.8% females) patients reported BZDRs use at baseline. During a median follow-up of 12.4 years, 2287 deaths were documented, of which 825 (36.1%) were due to CVD. BZDRs use was related to a statistically significantly higher risk of all-cause and CVD mortality; adjusted hazard ratios [95% CI] were (1.31 [1.41, 1.52]) and (1.43 [1.14, 1.81]), respectively. These relationships were dose-dependent-patients using BZDRs on an as-needed basis had similar risks compared to the non-uses, whereas patients with a daily use schedule and increasing doses had higher risks (p-value for trend: <0.001). CONCLUSION: BZDRs use was independently associated with a higher risk of all-cause and cardiovascular mortality in older post-MI patients, and there was evidence for a dose-dependent relationship. CLINICAL TRIAL REGISTRATION: NCT00127452 (www.gov)

A New COL3A1 Mutation in Ehlers-Danlos Syndrome Vascular Type with Different Phenotypes in the Same Family

Vascular Ehlers-Danlos syndrome (vEDS) is a rare and severe connective tissue disorder caused by mutations in the collagen type III alpha I chain (COL3A1) gene. We describe a pathogenetic heterozygous COL3A1 mutation c.3140 G>A, p. Gly1047Asp, identified using next-generation sequencing, in a 40-year-old Italian female. The genetic test performed on her relatives, which present different clinical phenotypes, confirmed that they carry the same mutation in heterozygous state. This finding confirms that mutations causing vEDS have an incomplete penetrance

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o-Cells

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o-line, CFBE) on Transwell\uae filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction-(GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC: CFBE, 1: 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells

The Italian registry of pulmonary non-tuberculous mycobacteria - IRENE:The study protocol

Background: A substantial increase in pulmonary and extra-pulmonary diseases due to non-tuberculous mycobacteria (NTM) has been documented worldwide, especially among subjects suffering from chronic respiratory diseases and immunocompromised patients. Many questions remain regarding the epidemiology of pulmonary disease due to NTM (NTM-PD) mainly because reporting of NTM-PD to health authorities is not mandated in several countries, including Italy. This manuscript describes the protocol of the first Italian registry of adult patients with respiratory infections caused by NTM (IRENE). Methods: IRENE is an observational, multicenter, prospective, cohort study enrolling consecutive adult patients with either a NTM respiratory isolate or those with NTM-PD. A total of 41 centers, including mainly pulmonary and infectious disease departments, joined the registry so far. Adult patients with all of the following are included in the registry: 1) at least one positive culture for any NTM species from any respiratory sample; 2) at least one positive culture for NTM isolated in the year prior the enrolment and/or prescribed NTM treatment in the year prior the enrolment; 3) given consent to inclusion in the study. No exclusion criteria are applied to the study. Patients are managed according to standard operating procedures implemented in each IRENE clinical center. An online case report form has been developed to collect patients' demographics, comorbidities, microbiological, laboratory, functional, radiological, clinical, treatment and outcome data at baseline and on an annual basis. An IRENE biobank has also been developed within the network and linked to the clinical data of the registry. Conclusions: IRENE has been developed to inform the clinical and scientific community on the current management of adult patients with NTM respiratory infections in Italy and acts as a national network to increase the disease's awareness

When and how ruling out cystic fibrosis in adult patients with bronchiectasis

Background: Bronchiectasis is the final result of different processes and most of the guidelines advocate for a careful evaluation of those etiologies which might be treated or might change patients' management, including cystic fibrosis (CF). Main body: CFTR mutations have been reported with higher frequency in bronchiectasis population. Although ruling out CF is considered as a main step for etiological screening in bronchiectasis, CF testing lacks of a standardized approach both from a research and clinical point of view. In this review a list of most widely used tests in CF is provided. Conclusions: Exclusion of CF is imperative for patients with bronchiectasis and CFTR testing should be implemented in usual screening for investigating bronchiectasis etiology. Physicians taking care of bronchiectasis patients should be aware of CFTR testing and its limitations in the adult population. Further studies on CFTR expression in human lung and translational research might elucidate the possible role of CFTR in the pathogenesis of bronchiectasis

When and how ruling out cystic fibrosis in adult patients with bronchiectasis

Background: Bronchiectasis is the final result of different processes and most of the guidelines advocate for a careful evaluation of those etiologies which might be treated or might change patients' management, including cystic fibrosis (CF). Main body: CFTR mutations have been reported with higher frequency in bronchiectasis population. Although ruling out CF is considered as a main step for etiological screening in bronchiectasis, CF testing lacks of a standardized approach both from a research and clinical point of view. In this review a list of most widely used tests in CF is provided. Conclusions: Exclusion of CF is imperative for patients with bronchiectasis and CFTR testing should be implemented in usual screening for investigating bronchiectasis etiology. Physicians taking care of bronchiectasis patients should be aware of CFTR testing and its limitations in the adult population. Further studies on CFTR expression in human lung and translational research might elucidate the possible role of CFTR in the pathogenesis of bronchiectasis