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.

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.

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.

Predictive value of pregnancy-associated plasma protein-A (PAPP-A) and free beta-hCG on fetal growth restriction: results of a prospective study.

PURPOSE: Low levels of plasmatic pregnancy-associated plasma protein-A (PAPP-A) and high levels of free-beta human chorionic gonadotropin (beta-hCG) could influence the outcome of pregnancy. The objective of this study is to assess the correlation between PAPP-A and free beta-hCG and birth weight. MATERIALS AND METHODS: Prospective follow-up study performed on 3332 patients in the first trimester of pregnancy who were subjected to a screening test focused on evaluation of fetal aneuploidy (SCA-TEST). The values of PAPP-A and free beta-hCG were both analyzed as raw values and subsequently converted to a multiple of the median (MoM). Statistical analysis was performed using SPSS version 17.0.1 (SPSS Inc., Chicago, USA). RESULTS: The incidence of "small for gestational age" in patients with PAPP-A MoM <1st and <5th ‰ was statistically significant (12 and 9.8 %; p < 0.0001). Also statistically significant data have been highlighted about free beta MoM > 95th ‰ (7 %; p = 0.03). The values of PAPP-A MoM > 99th ‰ are significantly correlated with an increased risk of "large for gestational age" (16.7 %; p < 0.0001). CONCLUSION: Our study demonstrates that specific values of PAPP-A and free beta-hCG could identify the risk of low or high birth weight since the first trimester of pregnancy.

Next Generation Sequencing Approach in a Prenatal Case of Cardio-Facio-Cutaneus Syndrome.

Cardiofaciocutaneous syndrome (CFCS) belongs to a group of developmental disorders due to defects in the Ras/Mitogen-Activated Protein Kinase (RAS/MAPK) signaling pathway named RASophaties. While postnatal presentation of these disorders is well known, the prenatal and neonatal characteristics are less recognized. Noonan syndrome, Costello syndrome, and CFCS diagnosis should be considered in pregnancies with a normal karyotype and in the case of ultrasound findings such as increased nuchal translucency, polyhydramnios, macrosomia and cardiac defect. Because all the RASopathies share similar clinical features, their molecular characterization is complex, time consuming and expensive. Here we report a case of CFCS prenatally diagnosed through Next Generation Prenatal Diagnosis (NGPD), a new targeted approach that allows us to concurrently investigate all the genes involved in the RASophaties.

Effects of exogenous progesterone on fetal nuchal translucency: an observational prospective study.

OBJECTIVE: Nuchal translucency (NT) seen ultrasonographically at 11-14 weeks' gestation is a sensitive marker for Down syndrome. Despite its important role for Down syndrome screening, its use is still considered controversial due to high false-positive rates. We speculated that progesterone could lead to abnormal blood flow patterns and, subsequently, to increased NT. Our primary endpoint was to evaluate the effects of exogenous progesterone on NT thickness compared to controls. The secondary endpoint was to evaluate these effects in a subgroup at low risk for fetal aneuploidies, identifying the strongest factors influencing NT variation. The tertiary endpoint was to evaluate, within the treatment group, if there is any difference in NT according to the type of progesterone administered, route of administration, and dose regimen. STUDY DESIGN: All women who came to measure NT at 11-14 weeks' gestation (crown-rump length between 45-84 mm) were considered eligible. We divided patients into 2 groups: women receiving exogenous progesterone and controls. Afterwards, 3 NT scans were performed for each case, and the largest value, accurate to 2 decimal points, was recorded. RESULTS: In all, 3716 women were enrolled and analyzed. In a crude analysis, NT (P < .05) increased in the exogenous progesterone group. The same results were obtained in the low-risk group (P < .05). The factorial analysis of variance model confirmed a correlation between altered NT and gestational age (P < .0001) and progesterone exposure (P < .05). The characteristics of treatment (route, formulation, dose) were examined separately and no statistically significant differences among the subgroups were observed. CONCLUSION: Exogenous progesterone increases NT.

What Is the Rate of Incomplete Fetal Anatomic Surveys During a Second-Trimester Scan? Retrospective Observational Study of 4000 Nonobese Pregnant Women.

OBJECTIVES: The purpose of this study was to estimate the rate of incomplete fetal anatomic surveys during a second-trimester scan due to an unfavorable fetal position in a nonobese population. METHODS: All pregnant women who came to the Altamedica Fetal-Maternal Medical Center, a specialized center for prenatal diagnosis, for a routine second-trimester scan between January 2012 and April 2013 were retrospectively included in the analysis. Patients with a body mass index higher than 30.0 kg/m(2) or anterior fibroids larger than 5 cm were not included in the study. RESULTS: Of 4000 pregnant women admitted for a second-trimester scan, 169 (4.2%) came back within 2 weeks to complete the examination because of an unfavorable fetal position. In particular, 104 (2.6%) needed visualization of only 1 view, and 65 (1.6%) needed more than 1 view. The most difficult organ to visualize was the corpus callosum, in 73 cases (1.8%); the face was not visualized in 69 cases (1.7%); the cerebellar vermis was not seen in 47 fetuses (1.1%); and the heart could not be completely examined in 40 fetuses (1.0%). Of the 4000 women, 169 (4.2%) had a nonexhaustive scan; 149 (3.7%) needed a second scan to complete the second-trimester survey; 14 (0.35%) needed a third scan; and 2 (0.05%) remained with a not completely exhaustive scan. CONCLUSIONS: There is always a small percentage of incomplete fetal anatomic surveys during a second-trimester scan, which cannot be modified by the sonographer's skill or by technical sonographic innovations.

Reference charts for fetal corpus callosum length: a prospective cross-sectional study of 2950 fetuses.

OBJECTIVES: The purpose of this study was to establish reference charts for fetal corpus callosum length in a convenience sample. METHODS: A prospective cross-sectional study was conducted at the Artemisia Fetal-Maternal Medical Center between December 2008 and January 2012. Among 16,975 fetal biometric measurements between 19 weeks and 37 weeks 6 days' gestation, 3438 measurements of the corpus callosum (20.3%) were available. After excluding 488 measurements (14.2%), a total of 2950 fetuses (85.8%) were considered and analyzed only once. Parametric and nonparametric quantile regression models were used for the statistical analysis. To evaluate the robustness of the proposed reference charts with respect to various distributional assumptions on the sonographic measurements at hand, we compared the gestational age (GA)-specific reference curves produced by the statistical methods used. RESULTS: The mean corpus callosum length was 26.18 mm (SD, 4.5 mm; 95% confidence interval, 26.01-26.34 mm). The linear regression equation expressing the length of the corpus callosum as a function of GA was length (mm) = -11.17 + 1.62 × GA. The correlation between the dimension and gestation was expressed by the coefficient r = 0.83. Normal mean lengths according the parametric and nonparametric methods were defined for each week of gestation. CONCLUSIONS: This work provides new quantile-based reference charts for corpus callosum length measurements that may be useful for diagnosis of congenital corpus callosum anomalies in fetal life.

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.

Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies.

Physiologic concentration in amniotic fluid (AF) of several metabolites has not been established with certainty. In this study, we initially assayed purines, pyrimidines, and amino compounds in 1,257 AF withdrawn between the 15th and the 20th week of gestation from actually normal pregnancies (normal gestations, normal offspring). Results allowed to determine physiologic reference intervals for 45 compounds. In these AF, not all purines and pyrimidines were detectable and uric acid (238.35±76.31 µmol/l) had the highest concentration. All amino compounds were measurable, with alanine having the highest concentration (401.10±88.47 µmol/l). In the second part of the study, we performed a blind metabolic screening of AF to evaluate the utility of this biochemical analysis as an additional test in amniocenteses. In 1,295 additional AF from normal pregnancies, all metabolites fell within the confidence intervals determined in the first part of the study. In 24 additional AF from women carrying Down's syndrome-affected fetuses, glutamate, glutamine, glycine, taurine, valine, isoleucine, leucine, ornithine, and lysine were different from physiologic reference values. One AF sample showed phenylalanine level of 375.54 µmol/l (mean value in normal AF=65.07 µmol/l) and was from a woman with unreported phenylketonuria with mild hyperphenylalaninemia (serum phenylalanine=360.88 µmol/l), carrying the IVS 4+5 G-T and D394A mutations. The fetus was heterozygote for the maternal D394A mutation. An appropriate diet maintained the mother phenylalanine in the range of normality during pregnancy, avoiding serious damage in fetal and neonatal development. These results suggest that the metabolic screening of AF might be considered as an additional biochemical test in amniocenteses useful to highlight anomalies potentially related to IEM.

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.

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.

Uterine fibroids and risk for complications following second-trimester amniocentesis.

OBJECTIVE: To compare the abortion rate and preterm premature rupture of membranes (PPROM) after amniocentesis in women who have undergone antibiotic prophylaxis with uterine fibroids and control. STUDY DESIGN: Retrospective study using the Antibiotic Prophylaxis before Second-Trimester Genetic Amniocentesis trial database carried out between January 1999 and December 2005 at the Artemisia Fetal-Maternal Medical Center (Rome, Italy). All women underwent antibiotic prophylaxis before amniocentesis. A follow-up within 4 weeks from the procedure was available. RESULTS: A total of 2,497 of 21,219 (11.8%) women with uterine fibroids were identified. The rate of abortion was 2 of 2,497 (0.08%) in women with fibroids and 4 of 18,722 (0.03%) in women without fibroids (p = 0.42). The rate of PPROM was 4 of 2,497 (0.16%) in women with fibroids and 10 of 18,722 (0.05%) in women without fibroids (p = 0.12). CONCLUSION: The risk for abortion and PPROM does not increase in the presence of uterine fibroids in women who have undergone antibiotic prophylaxis.