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.

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.

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.

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.

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.

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.

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 new case of interstitial 1q 25.3-32.1 deletion: cytogenetic analysis molecular characterization and ultrasound findings.

INTRODUCTION: deletion of long arm of chromosome 1(1q-) is a rare condition. Clinical features include Dwarfism, severe mental retardation, microcephaly and short neck delineating the "intermediate 1q deletion syndrome". CASE REPORT: we report a new case of interstitial deletion of the long arm of chromosome 1, diagnosed in a 22+3 weeks gestation fetus in which cytogenetic analysis localized a loss of genetic materials of 18Mb in the 1q25.3-32.1. Fetal ultrasound showed neurodegenerative defects resembling Dandy-Walker's syndrome and bilateral clubfoot. CONCLUSIONS: clinical characteristics of our case are markedly mild. This suggests that the type and the extension of the mutation obtained through cytogenetic studies, CGH array and ultrasound evaluation should be taken into account for prognostic evaluation and management of these patients.

Six consecutive false positive cases from cell-free fetal DNA testing in a single referring centre.

INTRODUCTION: recent studies have proposed the introduction of cell-free fetal DNA testing (NIPT-Non Invasive Prenatal Testing) in routine clinical practice emphasizing its high sensibility and specificity. In any case, false positive and false negative findings may result from placental mosaicism, because cell-free fetal DNA originates mainly from placenta. CASE: WE REPORT SIX CASES OF WOMEN WHO UNDERWENT CHORIONIC VILLUS SAMPLING (CVS) OR AMNIOCENTESIS TO CONFIRM THE RESULTS FROM NIPT: two Turner syndromes, two Triple X, one Patau syndrome, one Edward syndrome. RESULTS: using classic cytogenetic analysis and, also, Array - Comparative Genomic Hybridization (Array CGH) the karyotype of all 5 fetuses was found to be normal. CONCLUSION: results from NIPT must always be confirmed by invasive prenatal diagnosis. It is mandatory to inform the patient that the CVS and amniocentesis still represent the only form of prenatal diagnostic test available.

Retrospective study evaluating the performance of a first-trimester combined screening for trisomy 21 in an Italian unselected population.

OBJECTIVES: to assess the performance of a combined first-trimester screening for trisomy 21 in an unselected Italian population referred to a specialized private center for prenatal medicine. METHODS: a retrospective validation of first-trimester screening algorithms [risk calculation based on maternal age and nuchal translucency (NT) alone, maternal age and serum parameters (free ß-hCG and PAPP-A) alone and a combination of both] for fetal aneuploidies evaluated in an unselected Italian population at Artemisia Fetal-Maternal Medical Centre in Rome. All measurements were performed between 11(+0) and 13(+6) weeks of gestation, between April 2007 and December 2008. RESULTS: of 3,610 single fetuses included in the study, we had a complete follow-up on 2,984. Fourteen of 17 cases of trisomy 21 were detected when a cut-off of 1:300 was applied [detection rate (DR) 82.4%, 95% confidence interval (CI) 64.2-100; false-positive rate (FPR) 4.7%, 95% CI 3.9-5.4; false-negative rate (FNR) 17.6%, 95% CI 0-35.8%]. CONCLUSION: in our study population the detection rate for trisomy 21, using the combined risk calculation based on maternal age, fetal NT, maternal PAPP-A and free ß-hCG levels, was superior to the application of either parameter alone. The algorithm has been validated for first trimester screening in the Italian population.

The use of DHPLC (Denaturing High Performance Liquid Chromatography) in II level screening of the CFTR gene in Prenatal Diagnosis.

OBJECTIVE: The aim of the study is to evaluate the role of Denaturing High Performance Liquid Chromatography (DHPLC) in the second level screening of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. METHODS: A 9-month prospective study, between June 2008 and March 2009 at Artemisia Fetal Medical Centre, included 3829 samples of amniotic fluid collected from women undergoing mid-trimester amniocentesis.The genetic diagnosis of CF was based on research of the main mutations of the CFTR gene on fetal DNA extracted from the amniocytes, (first level screening) using different commercial diagnostic systems. A second level screening using DHPLC, on the amniotic fluid and on a blood sample from the couple, was offered in case of fetuses heterozygous at first level screening. RESULTS: Of 3829 fetuses, 134 were found to be positive, 129 heterozygous and 5 affected. Of the 129 couples, following appropriate genetic counselling, 53 requested a second level screening. Through the use of DHPLC, 44 couples were found to be negative, and in nine couples, nine rare mutations were identified. CONCLUSIONS: The first level screening can be useful to evidence up to 75% of the CF mutations. The second level screening can identify a further 10% of mutant alleles. DHPLC was found to be a reliable and specific method for the rapid identification of the rare CFTR mutations which were not revealed in initial first level screening.

Prenatal screening of Cystic Fibrosis: a single centre experience.

OBJECTIVE: The gene responsible for the pathogenesis of cystic fibrosis has been known for over 15 years and represent the most common autosomal recessive disease in the european population. We aimed to investigate the incidence of this condition during fetal life. METHODS: In the past 10 years we examined in our centre 25393 fetuses of women underwent to amniocentesis. We carried out the examination of the most frequent mutations which enable, according to the literature data, the identification of almost 80% of the affected alleles. RESULT: We identified 922 heterozygous and 9 homozygous for the mutation. The frequency of heterozygousin the examined sample was 1/27,5 while that of the affected was 1/2821. CONCLUSION: We encourage new thoughts regarding the diagnostic validity of the most frequent panel of mutations among the italian population in order to exclude never encountered mutations and the insertion of other more significant mutations.