Prenatal Diagnosis by Trio Clinical Exome Sequencing: Single Center Experience.

Fetal anomalies, characterized by structural or functional abnormalities occurring during intrauterine life, pose a significant medical challenge, with a notable prevalence, affecting approximately 2-3% of live births and 20% of spontaneous miscarriages. This study aims to identify the genetic cause of ultrasound anomalies through clinical exome sequencing (CES) analysis. The focus is on utilizing CES analysis in a trio setting, involving the fetuses and both parents. To achieve this objective, prenatal trio clinical exome sequencing was conducted in 51 fetuseses exhibiting ultrasound anomalies with previously negative results from chromosomal microarray (CMA) analysis. The study revealed pathogenic variants in 24% of the analyzed cases (12 out of 51). It is worth noting that the findings include de novo variants in 50% of cases and the transmission of causative variants from asymptomatic parents in 50% of cases. Trio clinical exome sequencing stands out as a crucial tool in advancing prenatal diagnostics, surpassing the effectiveness of relying solely on chromosomal microarray analysis. This underscores its potential to become a routine diagnostic standard in prenatal care, particularly for cases involving ultrasound anomalies.

Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial.

Chromosome imbalance (aneuploidy) is the major cause of pregnancy loss and congenital disorders in humans. Analyses of small biopsies from human embryos suggest that aneuploidy commonly originates during early divisions, resulting in mosaicism. However, the developmental potential of mosaic embryos remains unclear. We followed the distribution of aneuploid chromosomes across 73 unselected preimplantation embryos and 365 biopsies, sampled from four multifocal trophectoderm (TE) samples and the inner cell mass (ICM). When mosaicism impacted fewer than 50% of cells in one TE biopsy (low-medium mosaicism), only 1% of aneuploidies affected other portions of the embryo. A double-blinded prospective non-selection trial (NCT03673592) showed equivalent live-birth rates and miscarriage rates across 484 euploid, 282 low-grade mosaic, and 131 medium-grade mosaic embryos. No instances of mosaicism or uniparental disomy were detected in the ensuing pregnancies or newborns, and obstetrical and neonatal outcomes were similar between the study groups. Thus, low-medium mosaicism in the trophectoderm mostly arises after TE and ICM differentiation, and such embryos have equivalent developmental potential as fully euploid ones.

A Novel Nonsense Pathogenic TTN Variant Identified in a Patient with Severe Dilated Cardiomyopathy.

Both genetic and environmental factors contribute to the development of dilated cardiomyopathy. Among the genes involved, TTN mutations, including truncated variants, explain 25% of DCM cases. We performed genetic counseling and analysis on a 57-year-old woman diagnosed with severe DCM and presenting relevant acquired risk factors for DCM (hypertension, diabetes, smoking habit, and/or previous alcohol and cocaine abuse) and with a family history of both DCM and sudden cardiac death. The left ventricular systolic function, as assessed by standard echocardiography, was 20%. The genetic analysis performed using TruSight Cardio panel, including 174 genes related to cardiac genetic diseases, revealed a novel nonsense TTN variant (TTN:c.103591A > T, p.Lys34531*), falling within the M-band region of the titin protein. This region is known for its important role in maintaining the structure of the sarcomere and in promoting sarcomerogenesis. The identified variant was classified as likely pathogenic based on ACMG criteria. The current results support the need of genetic analysis in the presence of a family history, even when relevant acquired risk factors for DCM may have contributed to the severity of the disease.

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos.

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.

EPIGENETIC CLOCKS: IN AGING-RELATED AND COMPLEX DISEASES.

There is evidence that complex disease and mortality are associated with DNA methylation (DNAm) and age acceleration. Numerous epigenetic clocks, including Horvath, Hannum, DNA PhenoAge, DNA GrimAge, and DunedinPoAm continue to be developed in this young scientific field. The most well-known epigenetic clocks are presented here, along with information about how they relate to chronic disease. We examined all the literature until January 2023, investigating associations between measures of age acceleration and complex and age-related diseases. We focused on the scientific literature and researches that are most strongly associated with epigenetic clocks and that have shown promise as biomarkers for obesity, cardiovascular illness, type 2 diabetes, and neurodegenerative disease. Understanding the complex interactions between accelerated epigenetic clocks and chronic diseases may have significant effects on both the early diagnosis of disease and health promotion. Additionally, there is a lot of interest in developing treatment plans that can delay the onset of illnesses or, at the very least, alter the underlying causes of such disorders.

Whole-Exome and Transcriptome Sequencing Expands the Genotype of Majewski Osteodysplastic Primordial Dwarfism Type II.

Microcephalic Osteodysplastic Primordial Dwarfism type II (MOPDII) represents the most common form of primordial dwarfism. MOPD clinical features include severe prenatal and postnatal growth retardation, postnatal severe microcephaly, hypotonia, and an increased risk for cerebrovascular disease and insulin resistance. Autosomal recessive biallelic loss-of-function genomic variants in the centrosomal pericentrin (PCNT) gene on chromosome 21q22 cause MOPDII. Over the past decade, exome sequencing (ES) and massive RNA sequencing have been effectively employed for both the discovery of novel disease genes and to expand the genotypes of well-known diseases. In this paper we report the results both the RNA sequencing and ES of three patients affected by MOPDII with the aim of exploring whether differentially expressed genes and previously uncharacterized gene variants, in addition to PCNT pathogenic variants, could be associated with the complex phenotype of this disease. We discovered a downregulation of key factors involved in growth, such as IGF1R, IGF2R, and RAF1, in all three investigated patients. Moreover, ES identified a shortlist of genes associated with deleterious, rare variants in MOPDII patients. Our results suggest that Next Generation Sequencing (NGS) technologies can be successfully applied for the molecular characterization of the complex genotypic background of MOPDII.

Optimizing clinical exome design and parallel gene-testing for recessive genetic conditions in preconception carrier screening: Translational research genomic data from 14,125 exomes.

Limited translational genomic research data have been reported on the application of exome sequencing and parallel gene testing for preconception carrier screening (PCS). Here, we present individual-level data from a large PCS program in which exome sequencing was routinely performed on either gamete donors (5,845) or infertile patients (8,280) undergoing in vitro fertilization (IVF) treatment without any known family history of inheritable genetic conditions. Individual-level data on pathogenic variants were used to define conditions for PCS based on criteria for severity, penetrance, inheritance pattern, and age of onset. Fetal risk was defined based on actual carrier frequency data accounting for the specific inheritance pattern (fetal disease risk, FDR). In addition, large-scale application of exome sequencing for PCS allowed a deep investigation of the incidence of medically actionable secondary findings in this population. Exome sequencing achieved remarkable clinical sensitivity for reproductive risk of highly penetrant childhood-onset disorders (1/337 conceptions) through analysis of 114 selected gene-condition pairs. A significant contribution to fetal disease risk was observed for rare (carrier rate < 1:100) and X-linked conditions (16.7% and 41.2% of total FDR, respectively). Subgroup analysis of 776 IVF couples identified 37 at increased reproductive risk (4.8%; 95% CI = 3.4-6.5). Further, two additional couples had increased risk for very rare conditions when both members of a parental pair were treated as a unit and the search was extended to the entire exome. About 2.3% of participants showed at least one pathogenic variant for genes included in the updated American College of Medical Genetics and Genomics v2.0 list of secondary findings. Gamete donors and IVF couples showed similar carrier burden for both carrier screening and secondary findings, indicating no causal relationship to fertility. These translational research data will facilitate development of more effective PCS strategies that maximize clinical sensitivity with minimal counterproductive effects.

Recurrent herpes simplex virus-1 infection induces hallmarks of neurodegeneration and cognitive deficits in mice.

Herpes simplex virus type 1 (HSV-1) is a DNA neurotropic virus, usually establishing latent infections in the trigeminal ganglia followed by periodic reactivations. Although numerous findings suggested potential links between HSV-1 and Alzheimer's disease (AD), a causal relation has not been demonstrated yet. Hence, we set up a model of recurrent HSV-1 infection in mice undergoing repeated cycles of viral reactivation. By virological and molecular analyses we found: i) HSV-1 spreading and replication in different brain regions after thermal stress-induced virus reactivations; ii) accumulation of AD hallmarks including amyloid-ß protein, tau hyperphosphorylation, and neuroinflammation markers (astrogliosis, IL-1ß and IL-6). Remarkably, the progressive accumulation of AD molecular biomarkers in neocortex and hippocampus of HSV-1 infected mice, triggered by repeated virus reactivations, correlated with increasing cognitive deficits becoming irreversible after seven cycles of reactivation. Collectively, our findings provide evidence that mild and recurrent HSV-1 infections in the central nervous system produce an AD-like phenotype and suggest that they are a risk factor for AD.

Technical factors to consider when developing an Expanded Carrier Screening platform.

PURPOSE OF REVIEW: Expanded Carrier Screening (ECS) is a genetic test able to detect carriers for a large number of autosomal recessive and X-linked diseases. Its clinical utilization is increasing but some technical aspects for its implementation are still controversial. RECENT FINDINGS: In the current literature, several aspects of ECS panel implementation have been addressed. One of the most relevant topics involves which genes/pathologies should be included in an optimized ECS panel and which variants should be reported. SUMMARY: Here, we review the best practice criteria to refine and improve clinical utility and validity of an ECS panel. The criteria for optimal ECS panel implementation include the severity of pathologies, the prevalence of diseases in general population and a definitive or strong gene/disease association. Moreover, we discuss the main complications associated with the reporting of Variant of Uncertain Significance and the need for periodic reassessment.

Natural immune response and protection from SARS-CoV-2 reinfection.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the associated disease Coronavirus disease 2019 (COVID-19) continues to spread throughout the world, causing millions of infections and dead. One major question in predicting the course of the COVID-19 pandemic is how well and how long the immune response protects the host from reinfection. Although more studies are needed, evidence suggests that virus-specific B cell response in people with SARS-CoV-2 infection is rapidly generated and seems to be more reliable marker of long-lasting humoral responses than serum antibodies. Here we reviewed all related major studies of immune response to SARS-CoV-2 virus to better understand the natural protection against the virus, and the risk of reinfection. The ability of our community to eradicate this virus will mostly depend on our knowledge of the immune response, critical not only for vaccine development and distribution but also for therapeutic options. Keywords: SARS-CoV-2 virus reinfection; humoral immune response; SARS-CoV-2 virus variants; vaccination.

Experimental Data Based Machine Learning Classification Models with Predictive Ability to Select in Vitro Active Antiviral and Non-Toxic Essential Oils.

In the last decade essential oils have attracted scientists with a constant increase rate of more than 7% as witnessed by almost 5000 articles. Among the prominent studies essential oils are investigated as antibacterial agents alone or in combination with known drugs. Minor studies involved essential oil inspection as potential anticancer and antiviral natural remedies. In line with the authors previous reports the investigation of an in-house library of extracted essential oils as a potential blocker of HSV-1 infection is reported herein. A subset of essential oils was experimentally tested in an in vitro model of HSV-1 infection and the determined IC50s and CC50s values were used in conjunction with the results obtained by gas-chromatography/mass spectrometry chemical analysis to derive machine learning based classification models trained with the partial least square discriminant analysis algorithm. The internally validated models were thus applied on untested essential oils to assess their effective predictive ability in selecting both active and low toxic samples. Five essential oils were selected among a list of 52 and readily assayed for IC50 and CC50 determination. Interestingly, four out of the five selected samples, compared with the potencies of the training set, returned to be highly active and endowed with low toxicity. In particular, sample CJM1 from Calaminta nepeta was the most potent tested essential oil with the highest selectivity index (IC50 = 0.063 mg/mL, SI > 47.5). In conclusion, it was herein demonstrated how multidisciplinary applications involving machine learning could represent a valuable tool in predicting the bioactivity of complex mixtures and in the near future to enable the design of blended essential oil possibly endowed with higher potency and lower toxicity.

Preimplantation Genetic Testing for Aneuploidy Improves Clinical, Gestational, and Neonatal Outcomes in Advanced Maternal Age Patients Without Compromising Cumulative Live-Birth Rate.

PURPOSE: To report the effects of blastocyst stage aneuploidy testing on clinical, gestational, and neonatal outcomes for patients of advanced maternal age undergoing IVF. METHODS: This is a single-center observational-cohort study with 2 years follow-up. The study includes a total of 2538 couples undergoing 2905 egg collections (control group), 308 (PGT-A), and 106 (drop-out group, consenting for PGT-A but withdrawing due to poor embryological outcome) RESULTS: Compared with control group, PGT-A showed improved clinical outcomes (live-birth rate per transferred embryo, LBR 40.3% vs 11.0%) and reduced multiple pregnancy rate (MPR, 0% vs 11.1%) and pregnancy loss (PL, 3.6% vs 22.6%). Drop-out group showed the worst clinical outcomes suggesting that abandoning PGT-A due to poor response to ovarian stimulation is not a favorable option. Cytogenetic analysis of product of conceptions and CVS/amniocentesis showed higher aneuploid pregnancy rates for control group regardless of embryo transfer strategy (0%, 17.9%, and 19.9%, for PGT-A, control day 5 and day 3, respectively). Multivariate analysis showed no negative impact of PGT-A-related interventions on cumulative delivery rate (26.3%, 95% CI 21.5-31.6 vs 24.0%, 95% CI 22.5-25.6 for PGT-A and control, respectively) and on neonatal outcomes. CONCLUSION: PGT-A improves clinical outcomes, particularly by reducing pregnancy loss and chromosomally abnormal pregnancy for patients of advanced maternal age, with no major impact on cumulative live-birth rate (CLBR) per egg retrieval.

Using listener-based perceptual features as intermediate representations in music information retrieval.

The notion of perceptual features is introduced for describing general music properties based on human perception. This is an attempt at rethinking the concept of features, aiming to approach the underlying human perception mechanisms. Instead of using concepts from music theory such as tones, pitches, and chords, a set of nine features describing overall properties of the music was selected. They were chosen from qualitative measures used in psychology studies and motivated from an ecological approach. The perceptual features were rated in two listening experiments using two different data sets. They were modeled both from symbolic and audio data using different sets of computational features. Ratings of emotional expression were predicted using the perceptual features. The results indicate that (1) at least some of the perceptual features are reliable estimates; (2) emotion ratings could be predicted by a small combination of perceptual features with an explained variance from 75% to 93% for the emotional dimensions activity and valence; (3) the perceptual features could only to a limited extent be modeled using existing audio features. Results clearly indicated that a small number of dedicated features were superior to a "brute force" model using a large number of general audio features.

Digenic Origin of Difference of Sex Development in a Patient Harbouring DHX37 and MAMLD1 Variants.

Background: The diagnostic process for identifying variations in sex development (DSD) remains challenging due to the limited availability of evidence pertaining to the association between phenotype and genotype. DSD incidence is reported as 2 in 10,000 births, and the etiology has been attributed to genetic causes. Case Presentation. The present study investigated genetic causes implicated in a case of a 15-year-old 46, XY patient, raised as a girl. Genetic analysis by clinical exome sequencing (CES) showed a digenic inheritance due to two known pathogenic mutations in the DHX37 gene and the MAMLD1 gene, while we excluded variants with pathogenic significance in 209 DSD-related genes. Conclusions: Based on our literature review, this is the first case with the combined presence of pathogenic mutations in the MAMLD1 gene and DHX37 gene in a patient with gonadal dysgenesis.

Dynamics of SARS-CoV-2-Specific B Cell Memory Responses in Infected and Vaccinated Individuals.

Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly resulted in a pandemic constituting a global health emergency. As an indicator of long-term immune protection from reinfection with the SARS-CoV-2 virus, the presence of memory B cells (MBCs) should be evaluated. Since the beginning of COVID-19 pandemic, several variants of concerns have been detected, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1/B.1.1.28.1), Delta (B.1.617.2), and Omicron (BA.1) variants with several different mutations, causing serious concern regarding the increased frequency of reinfection, and limiting the effectiveness of the vaccine response. At this regard, we investigated SARS-CoV-2-specific cellular immune responses in four different cohorts: COVID-19, COVID-19 infected and vaccinated, vaccinated, and negative subjects. We found that MBC response to SARS-CoV-2 at more than 11 months postinfection was higher in the peripheral blood of all COVID-19 infected and vaccinated subjects respect to all the other groups. Moreover, to better characterize the differences of SARS-CoV-2 variants immune responses, we genotyped SARS-CoV-2-positive samples from the patients' cohort. We found a higher level of immunoglobulin M+ (IgM+) and IgG+ spike MBCs in SARS-CoV-2-positive patients (5-8 months after symptoms onset) infected with the SARS-CoV-2-Delta variant compared with the SARS-CoV-2-Omicron variant implying a higher immune memory response. Our findings showed that MBCs persist more than 11 months after primary infection indicating a different involvement of the immune system according to the different SARS-CoV-2 variant that infected the host.

De novo 3q13.13q21.2 interstitial deletion and paternal 12p13.3 microdeletion in a fetus with dysplasia of the corpus callosum and ventriculomegaly: A case report.

Chromosome 3q syndrome is a well-known genetic condition caused by interstitial deletion in the long arm of chromosome 3. The phenotype of this syndrome is variable and the great variability in the extent of these deletions leads to a wide spectrum of clinical manifestations. Terminal 12p deletion represents one of the rarest subtelomeric imbalances; patients with distal monosomy 12p present different phenotypes ranging from muscular hypotonia to autism spectrum disorders. The present study reported a prenatal diagnosis of a male fetus presenting ultrasound evidence of corpus callosum dysplasia and ventriculomegaly showing a 3q13q21.2 deletion and a 12p13.33 microdeletion paternally inherited. Among several features previously attributed to the terminal deletion of 3q, corpus callosum dysplasia and ventriculomegaly have rarely been reported together. As the 12p13.33 microdeletion in the father was associated only with muscular hypotonia and joint laxity, the involvement of terminal 12p deletions in the clinical features of the fetus was not possible to verify during the prenatal period. The present case report may provide a reference for prenatal diagnosis and genetic counseling in patients who present 3q13q21.2 deletions and 12p13.33 microdeletion.

Long QTc in hypertrophic cardiomyopathy: A consequence of structural myocardial damage or a distinct genetic disease?

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease, characterized by the presence of unexplained left ventricular hypertrophy. This condition is often associated with electrocardiographic abnormalities including QTc prolongation occurring in 13% of patients. The main explanation for prolonged QTc in HCM is myocardial hypertrophy and the related structural damage. However, other mechanisms, including long QT syndrome (LQTS) genes mutations, may be involved. In the present study we explored the hypothesis of a distinct genetic basis underlying QTc prolongation in HCM by investigating the potential co-inheritance of pathogenic gene variants associated with LQTS and HCM. For this purpose, starting from a cohort of 150 HCM patients carrying pathogenic variants in sarcomere genes, we selected 25 patients carrying a QTc prolongation unexplained by any other cause. The QTc was considered prolonged if greater than 450 ms in males and greater than 470 ms in females. The NGS analysis was performed with Illumina TrueSight Cardio panel genes on Illumina MiniSeq platform. We identified pathogenic/likely pathogenic variants in the KCNQ1 in two patients (c.1781G > A, p. Arg594Gln; c.532G > A, p. Ala178Thr) (8%). Variants of uncertain significance were identified in SCN5A, KCNJ5, AKAP9 and ANK2 in four patients (16%). Although the results are limited by the small number of patients included in the study, they highlight a minor contribution of LQTS genes for QTc prolongation in HCM patients. The screening for ion channel genes mutations may be considered in HCM patients with prolonged QTc unexplained by any other cause. This in-depth molecular diagnosis may contribute to improve risk stratification and treatment planning.

The role of genetic testing in suspected fulminant myocarditis: A case report.

ACM is a rare hereditary heart disease characterized by a progressive fibro-fatty replacement of the myocardium that can affect either the right or the left ventricle or both. It is mainly caused by variants in the desmosome genes with autosomal dominant transmission and incomplete penetrance. The disease shows a wide spectrum of clinical manifestations, including ventricular arrhythmias, HF and myocarditis. The latter is considered a 'hot phase' in the natural history of the disease and must therefore be distinguished from the isolated AM, which is frequently due to viral infections. Our case report is an example of how an AM, as the first manifestation of the disease, helped to reach a diagnosis of ACM through the genetic analysis. In fact, the multi-parametric investigation, which also included CMR and EMB, revealed controversial aspects that led us to perform the genetic test. The latter revealed a heterozygous pathogenic variant in the PKP2 that was considered definitive proof of ACM.

A Rapid and Consistent Method to Identify Four SARS-CoV-2 Variants during the First Half of 2021 by RT-PCR.

Since 2020, the COVID-19 pandemic has spread worldwide, causing health, economic, and social distress. Containment strategies rely on rapid and consistent methodology for molecular detection and characterization. Emerging variants of concern (VOCs) are currently associated with increased infectivity and immune escape (natural defence mechanisms and vaccine). Several VOCs have been detected, including Alpha variant (B.1.1.7), Beta variant (B.1.351), Gamma variant (P.1/B.1.1.28.1) and Delta variant (B.1.617.2), first identified in the UK, South Africa, Brazil and India, respectively. Here, a rapid and low-cost technique was validated to distinguish the Alpha, Beta, Gamma, and Delta SARS-CoV-2 variants by detecting spike gene mutations using a real-time reverse transcription polymerase chain reaction methodology (RT-PCR). A total of 132 positive patients affected by coronavirus disease-19 (COVID-19) were analysed by employing RT-PCR to target single-nucleotide polymorphisms (SNPs) to screen spike protein mutations. All data were validated by the next-generation sequencing (NGS) methodology and using sequences from a public database. Among 132 COVID-19-positive samples, we were able to discriminate all of the investigated SARS-CoV-2 variants with 100% concordance when compared with the NGS method. RT-PCR -based assays for identifying circulating VOCs of SARS-CoV-2 resulted in a rapid method used to identify specific SARS-CoV-2 variants, allowing for a better survey of the spread of the virus and its transmissibility in the pandemic phase.