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Objectives The rRT-PCR test, the current gold standard for the detection of coronavirus disease (COVID-19), presents with known shortcomings, such as long turnaround time, potential shortage of reagents, false-negative rates around 15–20%, and expensive equipment. The hematochemical values of routine blood exams could represent a faster and less expensive alternative. Methods Three different training data set of hematochemical values from 1,624 patients (52% COVID-19 positive), admitted at San Raphael Hospital (OSR) from February to May 2020, were used for developing machine learning (ML) models: the complete OSR dataset (72 features: complete blood count (CBC), biochemical, coagulation, hemogasanalysis and CO-Oxymetry values, age, sex and specific symptoms at triage) and two sub-datasets (COVID-specific and CBC dataset, 32 and 21 features respectively). 58 cases (50% COVID-19 positive) from another hospital, and 54 negative patients collected in 2018 at OSR, were used for internal-external and external validation. Results We developed five ML models: for the complete OSR dataset, the area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.83 to 0.90; for the COVID-specific dataset from 0.83 to 0.87; and for the CBC dataset from 0.74 to 0.86. The validations also achieved good results: respectively, AUC from 0.75 to 0.78; and specificity from 0.92 to 0.96. Conclusions ML can be applied to blood tests as both an adjunct and alternative method to rRT-PCR for the fast and cost-effective identification of COVID-19-positive patients. This is especially useful in developing countries, or in countries facing an increase in contagions.

Brugada syndrome (BrS) is an inherited cardiac arrhythmic disorder that can lead to sudden death, with a prevalence of 1:5000 in Caucasian population and affecting mainly male patients in their third to fourth decade of life. BrS is inherited as an autosomal dominant trait; however, to date genetic bases have been only partially understood. Indeed most mutations are located in the SCN5A gene, encoding the alpha-subunit of the Na(+) cardiac channel, but >70% BrS patients still remain genetically undiagnosed. Although 21 other genes have been associated with BrS susceptibility, their pathogenic role is still unclear. A recent next-generation sequencing study investigated the contribution of 45 arrhythmia susceptibility genes in BrS pathogenesis, observing a significant enrichment only for SCN5A. In our study, we evaluated the distribution of putative functional variants in a wider panel of 158 genes previously associated with arrhythmic and cardiac defects in a cohort of 91 SCN5A-negative BrS patients. In addition, to identify genes significantly enriched in BrS, we performed a mutation burden test by using as control dataset European individuals selected from the 1000Genomes project. We confirmed BrS genetic heterogeneity and identified new potential BrS candidates such as DSG2 and MYH7, suggesting a possible genetic overlap between different cardiac disorders.

Background Bronchopulmonary Dysplasia (BPD) is a multifactorial disease with a significant genetic component. Twin studies indicate that heritability of BPD is estimated at 53 to 79% [1]. Association studies have identifiedseveral potential candidate genes encoding components of innate immune and antioxidant defenses, mechanisms of vascular and lung remodeling, matrix remodeling proteins, surfactantproteins [2,3]. We planned a prospective multicentre study aimedto identify rare genetic variants contributing to the BPD phenotypeby exome sequencing using next-generation sequencing (NGS) technology.

Next generation sequencing is currently a cornerstone of genetic testing in routine diagnostics, allowing for the detection of sequence variants with so far unprecedented large scale, mainly in genetically heterogenous diseases, such as neurological disorders. It is a fast-moving field, where new wet enrichment protocols and bioinformatics tools are constantly being developed to overcome initial limitations. Despite the as yet undiscussed advantages, however, there are still some challenges in data analysis and the interpretation of variants. In this review, we address the current state of next generation sequencing diagnostic testing for inherited human disorders, particularly giving an overview of the available high-throughput sequencing approaches; including targeted, whole-exome and whole-genome sequencing; and discussing the main critical aspects of the bioinformatic process, from raw data analysis to molecular diagnosis.

Aims Brugada syndrome (BrS) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia/fibrillation (VT/VF) in young, otherwise healthy individuals. Despite SCN5A being the most commonly known mutated gene to date, the genotype–phenotype relationship is poorly understood and remains uncertain. This study aimed to elucidate the genotype–phenotype correlation in BrS. Methods and results Brugada syndrome probands deemed at high risk of future arrhythmic events underwent genetic testing and phenotype characterization by the means of epicardial arrhythmogenic substrate (AS) mapping, and were divided into two groups according to the presence or absence of SCN5A mutation. Two-hundred probands (160 males, 80%; mean age 42.6 ± 12.2 years) were included in this study. Patients harbouring SCN5A mutations exhibited a spontaneous type 1 pattern and experienced aborted cardiac arrest or spontaneous VT/VF more frequently than the other subjects. SCN5A-positive patients exhibited a larger epicardial AS area, more prolonged electrograms and more frequently observed non-invasive late potentials. The presence of an SCN5A mutation explained >26% of the variation in the epicardial AS area and was the strongest predictor of a large epicardial area. Conclusion In BrS, the genetic background is the main determinant for the extent of the electrophysiological abnormalities. SCN5A mutation carriers exhibit more pronounced epicardial electrical abnormalities and a more aggressive clinical presentation. These results contribute to the understanding of the genetic determinants of the BrS phenotypic expression and provide possible explanations for the varying degrees of disease expression.

Background: Studies reporting the long-term humoral response after receiving the BNT162b2 COVID-19 vaccine are important to drive future vaccination strategies. Yet, available literature is scarce. Covidiagnostix is a multicenter study designed to assess the antibody response in &gt, 1000 healthcare professionals (HCPs) who received the BNT162b2 vaccine. Methods: Serum was tested at time-0 (T0), before the first dose, T1, T2, and T3, respectively, 21, 42, and 180 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 the vaccine response. Neutralization activity against the D614G, B.1.1.7, and B.1.351 variants were also analyzed. Results: Six months post-vaccination HCPs showed an antibody titer decrease of approximately 70%, yet, the titer was still one order of magnitude higher than that of seropositive individuals before vaccination. We identified 12 post-vaccination infected HCPs. None showed severe symptoms. Interestingly, most of them showed titers at T2 above the neutralization thresholds obtained from the neutralization activity experiments. Conclusion: Vaccination induces a humoral response which is well detectable even six months post-vaccination. Vaccination prevents severe COVID-19 cases, yet post-vaccination infection is possible even in the presence of a high anti-S serum antibody titer.

Aim: The need for pharmacogenomic education is becoming more and more urgent. Our aim was to evaluate the progress in pharmacogenomics education since then, and to put forward further recommendations. Methods: A survey was sent to 248 schools of medicine, pharmacy, nursing and health professions around the world. Results: The majority of the study programs (87%) include pharmacogenomics education, which is generally taught as part of the pharmacology curriculum. On average, educators and teachers have selected appropriate and highly relevant pharmacogenomics biomarkers to include in their teaching programs. Conclusions: Based on the results, we can conclude that the state of pharmacogenomics education at the surveyed universities has improved substantially since 2005. ispartof: PHARMACOGENOMICS vol:20 issue:9 pages:643-657 ispartof: location:England status: published

Aims We aimed at addressing the role of late gadolinium enhancement (LGE) in arrhythmic risk stratification of LMNA-associated cardiomyopathy (CMP). Methods and results We present data from a multicentre national cohort of patients with LMNA mutations. Of 164 screened cases, we finally enrolled patients with baseline cardiac magnetic resonance (CMR) including LGE sequences [n = 41, age 35 ± 17 years, 51% males, mean left ventricular ejection fraction (LVEF) by echocardiogram 56%]. The primary endpoint of the study was follow-up (FU) occurrence of malignant ventricular arrhythmias [MVA, including sustained ventricular tachycardia (VT), ventricular fibrillation, and appropriate implantable cardioverter-defibrillator (ICD) therapy]. At baseline CMR, 25 subjects (61%) had LGE, with non-ischaemic pattern in all of the cases. Overall, 23 patients (56%) underwent ICD implant. By 10 ± 3 years FU, eight patients (20%) experienced MVA, consisting of appropriate ICD shocks in all of the cases. In particular, the occurrence of MVA in LGE+ vs. LGE− groups was 8/25 vs. 0/16 (P = 0.014). Of note, no significant differences between LGE+ and LGE− patients were found in currently recognized risk factors for sudden cardiac death (male gender, non-missense mutations, baseline LVEF 0.05. Conclusions In LMNA-CMP patients, LGE at baseline CMR is significantly associated with MVA. In particular, as suggested by this preliminary experience, the absence of LGE allowed to rule-out MVA at 10 years mean FU.

In this case report, we characterize a novel inherited frameshift mutation c.4700_4701del (p.Phe1567Cysfs*221) in a single copy of the SCN5A gene and its association with Brugada syndrome (BrS). The proband experienced a life-threatening ventricular arrhythmia successfully treated with DC-shock and he also suffered from supraventricular tachycardia. Ajmaline test confirmed the BrS diagnosis. No other mutation nor low frequency variants in the other 23 analyzed genes were detected. The same mutation was found in the father and sister, who were both diagnosed with BrS. We hypothesize that this mutation could be responsible for BrS and potentially linked to supraventricular tachycardias. Further studies are needed to confirm this observation and to assess the clinical relevance of this mutation, in terms of risk-stratification.

Brugada syndrome (BrS) is a known cause of sudden cardiac death. The genetic basis of BrS is not well understood, and no one single gene is linked to even a majority of BrS cases. However, mutations in the gene SCN5A are the most common, although the high amount of phenotypic variability prevents a clear correlation between genotype and phenotype. Research techniques are limited, as most BrS cases still remain without a genetic diagnosis, thus impairing the implementation of experimental models representative of a general pathogenetic mechanism. In the present study, we report the largest family to-date with the segregation of the heterozygous variant NM_198056:c.4894C>T (p.Arg1632Cys) in the SCN5A gene. The genotype-phenotype relationship observed suggests a likely pathogenic effect of this variant. Functional studies to better understand the molecular effects of this variant are warranted.

Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5′-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense mutation in the GTPase domain of DRP1 (NM_012062.3:c.436G>A, NP_036192.2: p.D146N variant in DNM1L). Patient’s fibroblasts show hyperfused/balloon-like giant mitochondria, highlighting the importance of D146 residue for DRP1 function. This dramatic mitochondrial rearrangement phenocopies what observed overexpressing DRP1-K38A, a well-known experimental dominant negative version of DRP1. In addition, we demonstrated that p.D146N mutation has great impact on peroxisomal shape and function. The p.D146N mutation compromises the GTPase activity without perturbing DRP1 recruitment or assembly, causing decreased mitochondrial and peroxisomal turnover. In conclusion, our findings highlight the importance of sensory neuropathy in the clinical spectrum of DRP1 variants and, for the first time, the impact of DRP1 mutations on mitochondrial turnover and peroxisomal functionality.

Aims The Brugada syndrome (BrS) is an inherited disease associated with an increased risk of sudden cardiac death. Often, the genetic cause remains undetected. Perhaps due at least in part because the NaV1.8 protein is expressed more in both the central and peripheral nervous systems than in the heart, the SCN10A gene is not included in diagnostic arrhythmia/sudden death panels in the vast majority of cardiogenetics centres. Methods and results Clinical characteristics were assessed in patients harboring either SCN5A or novel SCN10A variants. Genetic testing was performed using Next Generation Sequencing on genomic DNA. Clinical characteristics, including the arrhythmogenic substrate, in BrS patients harboring novel SCN10A variants and SCN5A variants are comparable. Clinical characteristics, including gender, age, personal history of cardiac arrest/syncope, spontaneous BrS electrocardiogram pattern, family history of sudden death, and arrhythmic substrate are not significantly different between probands harboring SCN10A or SCN5A variants. Conclusion Future studies are warranted to further characterize the role of these specific SCN10A variants.

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.

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.

Background Mutations in the LMNA (lamin A/C) gene have been associated with neuromuscular and cardiac manifestations, but the clinical implications of these signs are not well understood. Objective To learn more about the natural history of LMNA-related disease. Design Observational study. Setting 13 clinical centers in Italy from 2000 through 2018. Patients 164 carriers of an LMNA mutation. Measurements Detailed cardiologic and neurologic evaluation at study enrollment and for a median of 10 years of follow-up. Results The median age at enrollment was 38 years, and 51% of participants were female. Neuromuscular manifestations preceded cardiac signs by a median of 11 years, but by the end of follow-up, 90% of the patients had electrical heart disease followed by structural heart disease. Overall, 10 patients (6%) died, 14 (9%) received a heart transplant, and 32 (20%) had malignant ventricular arrhythmias. Fifteen patients had gait loss, and 6 had respiratory failure. Atrial fibrillation and second- and third-degree atrioventricular block were observed, respectively, in 56% and 51% of patients with combined cardiac and neuromuscular manifestations and 37% and 33% of those with heart disease only. Limitations Some of the data were collected retrospectively. Neuromuscular manifestations were more frequent in this analysis than in previous studies. Conclusion Many patients with an LMNA mutation have neurologic symptoms by their 30s and develop progressive cardiac manifestations during the next decade. A substantial proportion of these patients will have life-threatening neurologic or cardiologic conditions. Primary funding source None.

Brugada syndrome is an inherited arrhythmogenic disorder leading to sudden death predominantly in the 3–4 decade. To date the only reliable treatment is the implantation of a cardioverter defibrillator; however, better criteria for risk stratification are needed, especially for asymptomatic subjects. Brugada syndrome genetic bases have been only partially understood, accounting for

AIMS: The Na(+) channel mutation (p.S216L), previously associated with type 3 long-QT syndrome (LQT3) phenotype, and a common polymorphism (p.H558R) were detected in a patient with an intermittent Brugada syndrome (BS) ECG pattern. The study was aimed to assess the p.S216L electrical phenotype, its modulation by p.H558R, and to identify abnormalities compatible with a mixed BS-LQT3 phenotype. METHODS AND RESULTS: The mutation was expressed alone (S216L channels), or in combination with the polymorphism (S216L-H558R channels), in a mammalian cell line (TSA201). Functional analysis included standard voltage clamp and dynamic clamp with endo- and epicardial action potential waveforms. Expression of S216L channels was associated with a 60% reduction in maximum Na(+) current (I(Na)) density, attributable to protein misfolding (rescued by mexiletine pretreatment) and moderate slowing of inactivation. I(Na) density partially recovered in S216L-H558R channels, but I(Na) inactivation and its recovery were further delayed. The persistent component of I(Na) (I(NaL)) was unchanged. Under dynamic clamp conditions, I(Na) decreased in S216L channels and displayed a 'resurgent' component during late repolarization. In S216L-H558R channels, I(Na) density partially recovered and did not display a resurgent component. I(Na) changes during dynamic clamp were interpreted by numerical modelling. CONCLUSION: The BS pattern of p.S216L might result from a decrease in I(Na) density, which masked gating abnormalities that might otherwise result in a LQT phenotype. The p.H558R polymorphism decreased p.S216L expressivity, partly by lessening p.S216L effects and partly through the induction of further gating abnormalities suitable to blunt p.S216L effects during repolarization.

Carbamazepine (5H-dibenz[b,f]azepine-5-carboxamide) and oxcarbazepine (10,11-dihydro-10-oxo-5H-dibenz[b,f]azepine-5-carboxamide) are widely used for the treatment of partial epilepsy. Recent work indicates that these drugs, in addition to targeting voltage-gated Na(+) channels, can modulate ligand-gated channels. These compounds appear to be particularly effective for treatment of nocturnal frontal lobe epilepsy, which can be caused by mutant neuronal nicotinic receptors. We compared the effects of carbamazepine and oxcarbazepine on heteromeric nicotinic receptors to better understand the underlying mechanism of the effect of these drugs in epileptic patients. Receptors were expressed in cell lines and studied by patch-clamp methods at -60 mV. For alpha2beta4 receptors activated with 100 microM nicotine, IC(50) for carbamazepine was 49 microM. Receptors in which alpha2 was substituted with alpha2-I279 N, linked to autosomal dominant nocturnal frontal lobe epilepsy, had an IC(50) of 21 microM. For oxcarbazepine, the IC(50) was larger than 500 microM for wild-type receptors and approximately 100 microM for mutant receptors. A similar inhibition was observed in the presence of 10 microM nicotine, indicating a non-competitive mechanism. The monohydroxy derivative (MHD) of oxcarbazepine, clinically the most relevant compound, was tested on both alpha2beta4 and alpha4beta2 receptors, to obtain a broader view of its possible physiological effects. At the typical concentration present in blood (100 microM), MHD produced an approximate 40% channel block on alpha4beta2, but no significant effect on alpha2beta4 receptors. Oxcarbazepine and MHD retarded the channel deactivation, suggesting that these compounds produce open channel block. These results may explain the particular efficacy of these drugs in nocturnal frontal lobe epilepsy.

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the α4 and β2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor α2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic α2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior. © 2006 by The American Society of Human Genetics. All rights reserved.

In contrast to population-based medical decision making, which emphasizes the use of evidence-based treatment strategies for groups of patients, personalized medicine is based on optimizing treatment at the level of the individual patient. The creation of molecular profiles of individual patients was made possible by the advent of "omics" technologies, based on high throughput instrumental techniques in combination with biostatistics tools and artificial intelligence. The goal of personalized laboratory medicine is to use advanced technologies in the process of preventive, curative or palliative patient management. Personalized medicine does not rely on changes in concentration of a single molecular marker to make a therapeutic decision, but rather on changes of a profile of markers characterizing an individual patient's status, taking into account not only the expected response to treatment of the disease but also the expected response of the patient. Such medical approach promises a more effective diagnostics with more effective and safer treatment, as well as faster recovery and restoration of health and improved cost effectiveness. The laboratory medicine profession is aware of its key role in personalized medicine, but to empower the laboratories, at least an enhancement in cooperation between disciplines within laboratory medicine will be necessary. ispartof: CLINICAL CHEMISTRY AND LABORATORY MEDICINE vol:56 issue:12 pages:1981-1991 ispartof: location:Germany status: published