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.

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.

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.

Agnathia-Otocephaly Complex Due to a De Novo Deletion in the OTX2 Gene.

Agnathia-otocephaly complex (AOC) is a rare and usually lethal malformation typically characterized by hypoplasia or the absence of the mandible, ventromedial and caudal displacement of the ears with or without the fusion of the ears, a small oral aperture with or without a tongue hypoplasia. Its incidence is reported as 1 in 70,000 births and its etiology has been attributed to both genetic and teratogenic causes. AOC is characterized by a wide severity clinical spectrum even when occurring within the same family, ranging from a mild mandibular defect to an extreme facial aberration incompatible with life. Most AOC cases are due to a de novo sporadic mutation. Given the genetic heterogeneity, many genes have been reported to be implicated in this disease but to date, the link to only two genes has been confirmed in the development of this complex: the orthodenticle homeobox 2 (OTX2) gene and the paired related homeobox 1 (PRRX1) gene. In this article, we report a case of a fetus with severe AOC, diagnosed in routine ultrasound scan in the first trimester of pregnancy. The genetic analysis showed a novel 10 bp deletion mutation c.766_775delTTGGGTTTTA in the OTX2 gene, which has never been reported before, together with a missense variant c.778T>C in cis conformation.

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.

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.

Prenatal Diagnosis of Combined Maternal 4q Interstitial Deletion and Paternal 15q Microduplication.

The 4q deletion syndrome is a well-known rare genetic condition caused by partial, terminal, or interstitial deletion in the long arm (q) of chromosome 4. The phenotype of this syndrome shows a broad spectrum of clinical manifestations due to the great variability in the size and location of the deletion. In the literature, the mostly terminal deletions of chromosome 4q and the relative phenotypes are described, while the interstitial deletions of the long arm of chromosome 4 are rarely cited. Here, we report on a female fetus presenting no abnormal ultrasound evidence but with multiple chromosome aberrations. Comparative genomic hybridization (aCGH) revealed an interstitial 10.09 Mb deletion at the chromosome at the region of 4q28, arr[hg19] 4q28.1q28.3 (124068262_134158728)x1 combined with a 386.81 Kb microduplication at chromosome 15q11.1, arr[hg19] 15.11 (20249932_20636742)x3. At birth, and after 11 months, the baby was confirmed healthy and normal. The identification of this case allows for a deeper understanding of 4q syndrome and provides an explanation for the wide genetic/phenotypic spectrum of this pathology. This report can provide a reference for prenatal diagnosis and genetic counseling in patients who have similar cytogenetic abnormalities, and underlines the importance of reporting unusual variant chromosomes for diagnostic genetic purposes.

Multi-analytical test based on serum miRNAs and proteins quantification for ovarian cancer early detection.

Advanced ovarian cancer is one of the most lethal gynecological tumor, mainly due to late diagnoses and acquired drug resistance. MicroRNAs (miRNAs) are small-non coding RNA acting as tumor suppressor/oncogenes differentially expressed in normal and epithelial ovarian cancer and has been recognized as a new class of tumor early detection biomarkers as they are released in blood fluids since tumor initiation process. Here, we evaluated by droplet digital PCR (ddPCR) circulating miRNAs in serum samples from healthy (N = 105) and untreated ovarian cancer patients (stages I to IV) (N = 72), grouped into a discovery/training and clinical validation set with the goal to identify the best classifier allowing the discrimination between earlier ovarian tumors from health controls women. The selection of 45 candidate miRNAs to be evaluated in the discovery set was based on miRNAs represented in ovarian cancer explorative commercial panels. We found six miRNAs showing increased levels in the blood of early or late-stage ovarian cancer groups compared to healthy controls. The serum levels of miR-320b and miR-141-3p were considered independent markers of malignancy in a multivariate logistic regression analysis. These markers were used to train diagnostic classifiers comprising miRNAs (miR-320b and miR-141-3p) and miRNAs combined with well-established ovarian cancer protein markers (miR-320b, miR-141-3p, CA-125 and HE4). The miRNA-based classifier was able to accurately discriminate early-stage ovarian cancer patients from health-controls in an independent sample set (Sensitivity = 80.0%, Specificity = 70.3%, AUC = 0.789). In addition, the integration of the serum proteins in the model markedly improved the performance (Sensitivity = 88.9%, Specificity = 100%, AUC = 1.000). A cross-study validation was carried out using four data series obtained from Gene Expression Omnibus (GEO), corroborating the performance of the miRNA-based classifier (AUCs ranging from 0.637 to 0.979). The clinical utility of the miRNA model should be validated in a prospective cohort in order to investigate their feasibility as an ovarian cancer early detection tool.

Mild Symptomatic SARS-CoV-2 P.1 (B.1.1.28) Infection in a Fully Vaccinated 83-Year-Old Man.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) and the associated coronavirus disease 2019 (COVID-19) continue to spread throughout the world, causing more than 120 million infections. Several variants of concern (VOCs) have emerged and spread with implications for vaccine efficacy, therapeutic antibody treatments, and possible reinfections. On 17 March 2021, several VOCs were detected, including lineage B.1.1.7, first identified in the UK, B.1.351 in South Africa, Lineage P.1 (B.1.1.28.1) in Brazil, and novel Sub-Lineage A (A.23.1), reported in Uganda, and B.1.525, reported in Nigeria. Here, we describe an 83-year-old man infected with the SARS-CoV-2 P.1 variant after two doses of the BNT162b2 mRNA COVID-19 vaccine.

Evaluation of SARS-CoV-2 viral RNA in fecal samples.

The need for timely establishment of a complete diagnostic protocol of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is demanded worldwide. We selected 15 positive novel coronavirus disease 19 (COVID-19) patients with mild or no symptom. Initially, fecal samples were negative in the 67% (10/15) of the cases, while 33% (5/10) of the cases were positive. After serial virus RNA testing, 73% (11/15) of the cases resulted positive to fecal specimens. In particular, 15 days after the first positive respiratory specimens test, 6 fecal specimens became positive for SARS-CoV-2 RNA, while 13 respiratory test returned negative result. In conclusion, qRT-PCR assays of fecal specimens, is an important step to control infection, suggesting that samples remained positive for SARS-CoV-2 RNA longer time then respiratory tract samples. Our results enhance the recent knowledge on this emerging infectious disease and offer suggestions for a more complete diagnostic strategy.

Quantification of circulating microRNAs by droplet digital PCR for cancer detection.

OBJECTIVE: Circulating cell-free microRNAs (miRNAs) which consist of short-sequence RNAs are released from cells into the blood stream and has emerged as new biomarkers in the clinical cancer diagnosis and treatment. For instance, ovarian cancer comprises one of the three major malignant tumor types in the female reproductive system. The mortality rate of this cancer is the highest among all gynecological tumors, with ovarian cancer metastasis constituting an important cause of death. Therefore, development of a diagnostic tool that enables the ovarian cancer diagnosis in earlier stages is urgent. RESULTS: We have described an efficient protocol for an accurate absolute quantification of circulating miRNAs in healthy and ovarian cancer serum samples. Our data showed that ddPCR methodology can accurately measure circulating miRNAs levels and that can be a useful tool in biomarkers discovery for ovarian cancer detection.

Cell-free DNA screening for aneuploidies in 7113 pregnancies: single Italian centre study.

INTRODUCTION: Non-invasive prenatal testing (NIPT) using cell-free foetal DNA has been widely accepted in recent years for detecting common foetal chromosome aneuploidies, such as trisomies 13, 18 and 21, and sex chromosome aneuploidies. In this study, the practical clinical performance of our foetal DNA testing was evaluated for analysing all chromosome aberrations among 7113 pregnancies in Italy. METHODS: This study was a retrospective analysis of collected NIPT data from the Ion S5 next-generation sequencing platform obtained from Altamedica Medical Centre in Rome, Italy. RESULTS: In this study, NIPT showed 100% sensitivity and 99.9% specificity for trisomies 13, 18 and 21. Out of the 7113 samples analysed, 74 cases (1%) were positive by NIPT testing; foetal karyotyping and follow-up results validated 2 trisomy 13 cases, 5 trisomy 18 cases, 58 trisomy 21 cases and 10 sex chromosome aneuploidy cases. There were no false-negative results. CONCLUSION: In our hands, NIPT had high sensitivity and specificity for common chromosomal aneuploidies such as trisomies 13, 18 and 21.

Cell-free DNA screening for sex chromosomal aneuploidies in 9985 pregnancies: Italian single experience.

OBJECTIVE: Non invasive prenatal testing (NIPT) using cell-free fetal DNA (cffDNA) has been widely accepted in recent years to detect common fetal autosomal chromosome aneuploidies and sex chromosome aneuploidies (SCAs). In this study, the clinical performance of our fetal DNA testing was investigated by analyzing the sex chromosome aneuploidy aberrations among 9985 pregnancies. The study was a retrospective analysis of collected NIPT data from the Ion S5 next-generation sequencing (NGS) platform obtained from Altamedica Medical Centre of Rome. RESULTS: NIPT analysis of 9985 pregnancies revealed 31 cases with abnormal SCA results (0.31%). Among the 31 positive NIPT cases, 22 women agreed to undergo fetal karyotyping, whereas 9 refused further analyses. Of the 22 women verified by karyotyping analysis, 77.3% (17/22) were confirmed to be true positive SCAs, whereas 22.7% (5/22) were false positive. Among the true positive cases, 53.0% (9/17) were positive for monosomy X, 17.6% (3/17) were positive for 47, XXX aneuploidy, 23.5% (4/17) were positive for 47, XXY aneuploidy, and 5.9% (1/17) were positive for 47, XYY aneuploidy. In conclusion, the present results confirm that NIPT is a potential method for SCA screening, although this technology needs to be further investigated to improve the test performance.

Cytogenetics and Molecular Investigations detect a Mosaic Variant of Turner Syndrome only Suspected by Non-Invasive Prenatal Testing: Two Case Reports with Negative Ultrasound Examinations.

Prenatal testing has been moving towards non-invasive methods to determine fetal risk for genetic disorders. Numerous studies have focused the attention on common trisomies; although the detection rate (DR) for trisomy 21 is high (over 95%), the accuracy regarding the DR for trisomies 13 and 18 has come under scrutiny. The testing has been applied to sex chromosome aneuploidies, but many studies have shown that it is not as effective as it is for common trisomies. Although non-invasive prenatal test (NIPT) has become a standard screening procedure for all pregnant women, invasive sampling procedures remain important in confirming NIPT-positive findings. In the present study, we report discordant results of Turner syndrome (TS) mosaicism between NIPT and karyotyping. A 35-year-old pregnant woman underwent NIPT, and a probable risk for Xp deletion was indicated. Subsequently, amniocentesis was performed. The karyotype was identified as mos 45,X [28]/46,X,i(X)(q1.0)[5]. In the second case, a 33-year-old woman underwent amniocentesis after a positive NIPT that indicated a probable risk for monosomy X. The result was mos 45,X [8]/46,XY[8]. Since NIPT is a screening test, the possibility of false-positive or false-negative results should always be considered. We underline the importance of pre/post detailed counseling. Furthermore, women with abnormal NIPT results should undergo immediate amniocentesis that remains the only tool for a correct diagnosis of sex chromosome aneuploidies.

Validation of Extensive Next-Generation Sequencing Method for Monogenic Disorder Analysis on Cell-Free Fetal DNA: Noninvasive Prenatal Diagnosis.

During pregnancy, a percentage of the cell-free DNA circulating in the maternal blood is represented by the cell-free fetal DNA (cffDNA), constituting an accessible source for noninvasive prenatal genetic screening. The coexistence of the maternal DNA, the dominant fraction of cell-free DNA, together with the cffDNA component and the scarcity of the cffDNA itself make applying traditional methods of genetics and molecular biology impossible. Next-generation sequencing methods are widely used to study fetal aneuploidies. However, in monogenic disorders, there have been relatively few studies that analyzed single mutations. We present a method for the analysis of an extended group of gene variants associated with recessive and dominant autosomal disorders using next-generation sequencing. The proposed test should allow a complete analysis of common genetic disorders and pathogen-associated variants for diagnostic use. The analysis of cffDNA for single gene disorders may replace invasive prenatal diagnosis methods, associated with the risk of spontaneous abortion and psychological stress for patients. The proposed test should assess reproductive risk for both genetic family disorders and de novo occurrences of the disease. The application of this method to a case of beta-thalassemia is also discussed.

A novel approach in the treatment of neonatal gastroschisis: a review of the literature and a single-center experience.

Gastroschisis is a congenital abdominal wall defect and its management remains an issue. We performed a review of the literature to summarize its evaluation, management and outcome and we describe a new type of surgical reduction performed in our center without anesthesia (GA), immediately after birth, in the delivery room. Between January 2002 and March 2013, we enrolled all live born infants with gastroschisis referred to the third-level Division of Obstetrics and Gynecology "San Camillo" of Rome. Two groups of infants were identified: group 1 in which gastroschis reduction was performed by the traditional technique and group 2 in which reduction was immediately performed after birth in the delivery room without GA. Twelve infants were enrolled in group 1, and seven infants in group 2. Statistical significance was observed between the groups regarding the hospital stay, for the duration of parenteral nutrition and full oral feeds (p = .004). Survival was similar between two groups. The reduction without GA performed immediately after birth in a delivery room encourages the relationship between the mother and her child and appears to be a safe and feasible technique in a selected group of patients with simple gastroschisis defect; for this reason, it could represent a valid alternative to traditional approach.