Search

Background: Real-world population studies have shown waning immunity, over time, after receiving the two doses of the BNT162b2 COVID-19 vaccine. Studies reporting the long-term humoral response are important to drive future vaccination strategies like the introduction of the booster dose. Yet, available literature on long follow-up periods is scarce. Covidiagnostix is a multicenter study aiming to assess the antibody response in >1000 healthcare professionals (HCPs) who received the BNT162b2 vaccine., Methods: Serum was tested at time-0 (T0), before the first dose and then at T1, T2, T3 and T4, respectively, 21, 42, 177 and 302 days after T0. Antibodies against the SARS-CoV-2 nucleocapsid-protein were measured to assess SARS-CoV-2 infections, whereas antibodies against the receptor-binding domain of the spike protein were measured to assess vaccine response., Results: The antibody titer observed 10 months post-vaccination showed a decrease of approximately 80% from the peak measured at T2, yet the median titer of the seronegatives HCPs was still higher than seropositives before vaccination. We identified 12 post-vaccination infected HCPs within 6 months after receiving the first dose and another 12 post-vaccination infected HCPs between 6 and 10 months post-vaccination., Conclusion: Vaccination induced a humoral response which is well detectable even 10 months post-vaccination. Yet a high anti-spike serum antibody titer does not guarantee protection from infection. Differences in symptomatology between SARS-CoV-2 infections occurred within the first 6 months post-vaccination and the following 4 months, and differences in COVID-19 prevalence and vaccination coverage observed in these two time intervals were consistent with a decrease in vaccine efficacy 6 months after receiving the first dose., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background and Aims: Vaccines, to limit SARS-CoV-2 infection, were produced and reliable assays are needed for their evaluation. The WHO produced an International-Standard (WHO-IS) to facilitate the standardization/comparison of serological methods. The WHO-IS, produced in limited amount, was never tested for reproducibility. This study aims at developing a reproducible and accessible working standard (WS) to complement the WHO-IS., Materials and Methods: Sera from vaccinated individuals were used to produce the WSs. The WHO-IS, the WSs and single serum samples (n = 48) were tested on 6 quantitative serological devices. Neutralization assays were performed for the 48 samples and compared with their antibody titers., Results: The WS carry an antibody titer 20-fold higher than the WHO-IS. It was reproducible, showed both good linearity and insignificant intra- and inter-laboratory variability. However, the WSs behave differently from the WHO-IS. Analysis of the 48 samples showed that single correlation factors are not sufficient to harmonize results from different assays. Yet, all the devices predict neutralization activity based on the antibody titer., Conclusions: A reproducible and highly concentrated WS, specific for IgG against SARS-CoV-2 Spike-glycoprotein was produced. Such characteristics make it particularly suited for the harmonization of commercially available assays and the consequent evaluation of post-vaccinated individuals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Triadin is a protein expressed in cardiac and skeletal muscle that has an essential role in the structure and functional regulation of calcium release units and excitation-contraction coupling. Mutations in the triadin gene (TRDN) have been described in different forms of human arrhythmia syndromes with early onset and severe arrhythmogenic phenotype, including triadin knockout syndrome., Objective: The purpose of this study was to characterize the pathogenetic mechanism underlying a case of severe pediatric malignant arrhythmia associated with a defect in the TRDN gene., Methods: We used a trio whole exome sequencing approach to identify the genetic defect in a 2-year-old boy who had been resuscitated from sudden cardiac arrest and had frequent episodes of ventricular fibrillation and a family history positive for sudden death. We then performed in vitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype., Results: We identified a novel homozygous missense variant (p.L56P) in the TRDN gene in the proband that was inherited from the heterozygous unaffected parents. Expression of a green fluorescent protein (GFP)-tagged mutant human cardiac triadin isoform (TRISK32-L56P-GFP) in heterologous systems revealed that the mutation alters protein dynamics. Furthermore, when co-expressed with the type 2 ryanodine receptor, caffeine-induced calcium release from TRISK32-L56P-GFP was relatively lower compared to that observed with the wild-type construct., Conclusion: The results of this study allowed us to hypothesize a pathogenic mechanism underlying this rare arrhythmogenic recessive form, suggesting that the mutant protein potentially can trigger arrhythmias by altering calcium homeostasis., (Copyright © 2019 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

A major effort has been focused on the detection of oncogenes' mutations in diverse types of clinical specimens including formalin-fixed and paraffin embedded tissues, presently the gold-standard samples, up to plasma, that constitute a noninvasive alternative source of tumor DNA. The reliable detection of mutations in circulating tumor DNA requires a high analytical sensitivity. Here, we applied three different highly sensitive methodologies (COLD-PCR, a microarray-based approach and the droplet digital PCR, ddPCR) to identify mutations in the plasma of 30 metastatic colorectal cancer patients previously genotyped on tissue biopsy. The methods showed a modest concordance rate with respect to the results obtained on tissue biopsies: 63.3% by ddPCR, 63% by microarray and 55.6% by COLD-PCR. This could be ascribed either to the different timing between tissue and liquid biopsy collection, which could reflect a different stage of disease progression or to the diverse sensitivity of the methodologies applied. Indeed, if we compare the results obtained on plasma samples, the concordance rates were higher especially by comparing ddPCR versus COLD-PCR (92.6%). Thus, we consider both methodologies as useful procedures easily transferable in a clinical setting. Notably, the ddPCR allows a quantitative assessment of the fractional abundance of the mutation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in infancy, affecting preterm children with low birth weight. The disease has a multifactorial aetiology with a significant genetic component; until now published association studies have identified several candidate genes but only few of these data has been replicated. In this pilot study, we approached exome sequencing aimed at identifying non-common variants, which are expected to have a stronger phenotypic effect., Materials and Methods: We performed this study on 26 Italian severely affected BPD preterm unrelated newborns, homogeneously selected from a large prospective cohort. We used an Illumina HiSeq 2000 for sequencing. Data analysis was focussed on genes previously associated to BPD susceptibility and to new candidates in related pathways, highlighted by a prioritization analysis performed using ToppGene Suite., Results: By exome sequencing, we identified 3369 novel variants, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (TLR) family. All of them have been confirmed with Sanger sequencing., Conclusions: Potential candidate genes have been discovered in this preliminary study; the pathogenic role of identified variants will need to be confirmed with functional and segregation studies and possibly with further methods, able to evaluate the collective influence of rare variants. Moreover, additional candidates will be tested and genetic analysis will be extended to all affected children., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A companion diagnostic test provides information that is essential for the safe and effective use of a corresponding therapeutic product as indicated in the drug instructions. The implementation of a companion diagnostic follows the rules of a molecular test for somatic mutations in a routine clinical laboratory environment and needs guidance on practical aspects, including the choice of the proper analytical method and the procedures for internal and external quality controls. Selection of the appropriate assay for detection of genetic alterations depends on several factors: the type of mutation under study, the sample to be assayed and its preparation procedure. In addition, the results of a molecular assay require a complex interpretation process of the analytical data as the patient's genotype, the translation of the identified variant into a predicted phenotype and knowledge on restrictions of the method used. In relation to these aspects herein we report an opinion paper of the Working Group Personalized Laboratory Medicine jointly constituted by the European Federation of Laboratory Medicine (EFLM) and by the European Society of Pharmacogenomics and Theranostics (ESPT) using, as an example, the BRAF genotype analysis in tumor tissue samples for identification of melanoma patients that can benefit treatment with BRAF inhibitors. The manuscript is focused on the following aspects: i) medical rationale, ii) methodologies of analysis, iii) laboratory performance evaluation and iv) the laboratory specific report for the clinicians. The critical evaluation of these aspects would be useful for the implementation of a companion diagnostic in the clinical laboratory., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Pre-mRNA splicing defects may have an important impact on clinical phenotype in several diseases, but often their pathogenic role is difficult to demonstrate. The aim of this study was to validate an in vitro method to assess the effects of putative splicing variants., Materials and Methods: We studied three novel variants in vitro using a novel minigene approach and compared results with in silico and ex vivo strategies from patient samples., Results: For the c.1146C>T variant in the LMNA gene, in vitro and ex vivo studies were concordant with the prediction obtained by in silico tools, confirming the loss of 13 bp at the end of exon 6. In the second case (c.1140+1G>A, SCN5A gene), in vitro experiments identified the insertion of 94 intronic bp in exon 9 as well as exon 9 skipping, but these results were not correctly predicted by ex vivo data and in silico tools. In the third case (c.1608+1C>T, LMNA gene) in vitro and ex vivo studies suggested the recognition of an exonic cryptic site leading to the loss of 29 bp in exon 9, not predicted by in silico analysis., Conclusion: Our results revealed how in silico tools are often unreliable requiring "wet" RNA analysis. Since ex vivo studies are not always feasible, the use of an in vitro construct represents an efficient and useful method for the evaluation of damaging effects of unknown splicing variants, especially in diagnostic laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)