Maternal plasma mRNA species in fetal heart defects: a potential for molecular screening.

OBJECTIVE: To verify the hypothesis that aberrant placental mRNA genes related to cardiogenesis can be detected in maternal plasma at the second trimester of pregnancy. METHODS: NanoString technology was used to identify aberrant genes, comparing 39 women carrying a fetus with a congenital heart defect (CHD) to 31 controls at 19-24 weeks of gestation. The genes with differential expression were subsequently tested using real time polymerase chain reaction. Linear discriminant analysis (LDA) was used to combine all the mRNA species with discriminant ability for CHD. A multivariable receiver operating characteristic (ROC) curve having the estimated discriminant score as an explanatory variable was generated. RESULTS: Six genes with differential expression, namely FALZ, PAPP-A, PRKACB, SAV1, STK4 and TNXB2, were found. The ROC curve yielded a detection rate of 66.7% at a false positive rate of 10%. A higher discriminant score (>75(th) centile) was reached for 14 CHD cases (82.4%) and only 1 control (5.8%). Two cases (11.8%) of heart rhythm disorders also yielded a discriminant score value >75(th) centile. CONCLUSION: These data represent a step forward in the screening of CHDs. Additional studies are needed to detect more mRNAs with discriminant ability and to move the first trimester screening.

An Unclassified Deletion Involving the Proximal Short Arm of Chromosome 10: A New Syndrome?

To date, only 13 studies have described patients with large overlapping deletions of 10p11.2-p12. These individuals shared a common phenotype characterized by intellectual disability, developmental delay, distinct facial dysmorphic features, abnormal behaviour, visual impairment, cardiac malformation, and cryptorchidism in males. Molecular cytogenetic analysis revealed that the deletion in this chromosomal region shares a common smallest region of overlap (SRO) of 80 kb, which contains only the WAC gene (WW-domain-containing adaptor with coiled coil). In this clinical case report, we report a 5-year-old girl, born from non-consanguineous parents, with a 10p11.22p11.21 microdeletion. She presents clinical features that overlap with other patients described in the literature, such as dysmorphic traits, speech delay, and behavioural abnormalities (hyperactivity), even though the WAC gene is not involved in the microdeletion. Our results are the first to highlight that the deletion described here represents a contiguous gene syndrome that is enough to explain the distinct phenotype but partially overlaps with the previous cases reported in the literature, even though the same genes are not involved. In particular, in this study, we speculate about the role of the WAC gene that seems to be associated with normal motor development. In fact, we found that our patient is the only one described in the literature with a large deletion in the 10p11.22p11.21 region without the involvement of the WAC gene deletion, and, interestingly, the patient did not have motor delay.

Urinary miRNAs as a Diagnostic Tool for Bladder Cancer: A Systematic Review.

Bladder cancer is the 10th most common cancer type worldwide. Cystoscopy represents the gold standard for bladder cancer diagnosis, but this procedure is invasive and painful, hence the need to identify new biomarkers through noninvasive procedures. microRNAs (miRNAs) are considered to be promising diagnostic molecules, because they are very stable in biological fluids (including urine) and easily detectable. This systematic review analyses the power of urine miRNAs as bladder cancer diagnostic markers. We conducted this systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. A total of 293 records related to miRNAs and their diagnostic significance in BC were retrieved from the PubMed and Embase databases. A systematic search of the literature was performed, and a total of 25 articles (N = 4054 participants) were identified and reviewed. Although many of the selected studies were of high scientific quality, the results proved to be quite heterogeneous, because we did not identify a univocal consensus for a specific miRNA signature but only isolated the signatures. We did not identify a univocal consensus for a specific diagnostic miRNA signature but only isolated the signatures, some of them with better diagnostic power compared to the others.

The need to perform α-thalassemia genetic testing in Italian patients with ß-thalassemia trait: A case report.

Here, we describe a case report of a Sardinian woman diagnosed as pure beta-thalassemia carrier for her anemia who underwent to alpha-thalassemia genetic testing that revealed she was heterozygous for both thalssemias. This allowed to reach a conclusive diagnosis useful for family counseling and for assess the reproductive risk.

Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR.

Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2-specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulates monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations.

Abnormal Circulating Maternal miRNA Expression Is Associated with a Low (<4%) Cell-Free DNA Fetal Fraction.

The present pilot study investigates whether an abnormal miRNA profile in NIPT plasma samples can explain the finding of a low cell-free DNA (cfDNA) fetal fraction (cfDNAff) in euploid fetuses and non-obese women. Twelve women who underwent neoBona® NIPT with a normal fetal karyotype were studied. Six with a cfDNAff < 4% were matched with a control group with normal levels of cfDNAff > 4%. Samples were processed using the nanostring nCounter® platform with a panel of 800 miRNAs. Four of the maternal miRNAs, miR-579, miR-612, miR-3144 and miR-6721, had a significant abnormal expression in patients. A data filtering analysis showed that miR-579, miR-612, miR-3144 and miR-6721 targeted 169, 1, 48 and 136 placenta-specific genes, respectively. miR-579, miR-3144 and miR-6721 shared placenta-specific targeted genes involved in trophoblast invasion and migration pathways (IGF2R, PTCD2, SATB2, PLAC8). Moreover, the miRNA target genes encoded proteins localized in the placenta and involved in the pathogenesis of pre-eclampsia, including chorion-specific transcription factor GCMa, PRG2, Lin-28 Homolog B and IGFBP1. In conclusion, aberrant maternal miRNA expression in circulating plasma could be a source of dysregulating trophoblast invasion and migration and could represent a novel cause of a low cfDNAff in the sera of pregnant women at the time of NIPT analysis.

Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue.

The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.

Levels of Circulating mRNA for the Tenascin-X (TNXB) Gene in Maternal Plasma at the Second Trimester in Pregnancies with Isolated Congenital Ventricular Septal Defects.

OBJECTIVE: Maternal plasma is a source of circulating placental nucleic acids. In this study, we validated previous observations on abnormal levels of circulating messenger RNA (mRNA) for the tenascin-X gene in pregnancies with ventricular septal defects in the second trimester of pregnancy. METHODS: This was a bicentric retrospective study conducted from March 2016 to July 2017. Real-time polymerase chain reaction was used to identify abnormally expressed genes, comparing ten women carrying a euploid fetus with ventricular septal defects to 30 controls at 19-24 weeks of gestation. The univariable analysis was used to determine whether the mean mRNA for the tenascin-X gene values would differ from the expected values for the controls. RESULTS: mRNA for tenascin-X gene values was higher in ventricular septal defects, 4.38 ± 3.01 versus 1.00 ± 0.80. The result was still significant even after adjustment for gestational age. CONCLUSIONS: These data confirm previous studies on the specific association of mRNA species and type of congenital heart defect and confirm that ventricular septal defects are associated with abnormal mRNA for the tenascin-X gene. The positive predictive value of this molecular marker in the general population should be assessed through prospective studies.

Cell-Free DNA (cfDNA) Fetal Fraction in Early- and Late-Onset Fetal Growth Restriction.

OBJECTIVE: Our objective was to retrospectively evaluate whether the levels of cell-free DNA (cfDNA) fetal fraction differed in the first trimester of pregnancies between controls and those who subsequently developed early- or late-onset fetal growth restriction (FGR). METHODS: This was a case-control study conducted between May 2015 and May 2018 in 231 low-risk women who had received first trimester screening for major fetal aneuploidies (Panorama, Natera, San Carlos, CA, USA). Early- and late-onset FGR developed in 5 and 16 women, respectively, according to Delphi criteria. Multiples of median (MoM) were used to evaluate the differences in cfDNA fetal fraction between cases and controls. cfDNA fetal fraction was adjusted for gestational age (from 10 + 0 to 13 + 6 gestational weeks) and maternal weight (43-96 kg). RESULTS: The median cfDNA fetal fractions for controls and early- and late-onset FGR were 1.00 (interquartile range [IQR] 0.89-1.12), 0.69 (IQR 0.44-0.84) and 0.93 (IQR 0.83-1.03) MoM, respectively. Statistically lower cfDNA fetal fraction MoM values were observed only in patients with early-onset FGR (Kruskal-Wallis test with Dunn post hoc test). In a 1:35 ratio (one case of early-onset FGR: 35 controls), the mean observed rank of 2.00 ± 2.23 in the cases was significantly lower than the expected 18.97 ± 10.17 (p < 0.001). CONCLUSIONS: Low-risk pregnancies that developed early-onset FGR had lower cfDNA fetal fractions than did the matched controls. This result is consistent with the placental dysfunction typical of early-onset FGR. For possible clinical use, the cfDNA fetal fraction would yield a better predictive value if adjusted for maternal weight, since maternal weight affects both cfDNA fetal fraction and the occurrence of FGR.

Workload measurement for molecular genetics laboratory: A survey study.

Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory.

Circulating mRNA in Maternal Plasma at the Second Trimester of Pregnancy: A Possible Screening Tool for Cardiac Conotruncal and Left Ventricular Outflow Tract Abnormalities.

OBJECTIVE: Maternal plasma is a source of circulating placental nucleic acids. This study was designed to detect aberrantly expressed placental mRNA genes circulating in the maternal plasma of pregnancies affected with fetal conotruncal anomalies (CNTRA) and left-ventricular outflow tract (LVOT) obstruction in the second trimester of pregnancy. METHODS: This was a retrospective monocentric study conducted from 1 Jan 2016 to 31 Dec 2016. NanoString technology was used to identify aberrantly expressed genes, comparing 36 women carrying a fetus with CNTRA or LVOT obstruction to 42 controls at 19-24 weeks of gestation. The genes with differential expression were subsequently tested using real-time polymerase chain reaction. Linear discriminant analysis was used to combine all the mRNA species with discriminant ability for CNTRA and LVOT obstruction. A multivariable receiver operating characteristic (ROC) curve having the estimated discriminant score as an explanatory variable was generated for the two affected groups versus controls. RESULTS: Three genes with differential expression, namely MAPK1, IQGAP1 and Visfatin were found. The ROC curves yielded detection rates of 60 and 62.5% at a false-positive rate of 5% for CNTRA and LVOT, respectively. CONCLUSIONS: These data suggested that molecular screening of CNTRA and LVOT obstruction in the second trimester is feasible. Prospective studies are needed to test the discriminant ability of these genes and to calculate the predictive positive value in the general population.

A real-time PCR approach to evaluate adipogenic potential of amniotic fluid-derived human mesenchymal stem cells.

Regulation of adipocyte differentiation is an important process in the control of adipose tissue development. So far, adipogenesis has been investigated through the use of various experimental models. In this work, we used human mesenchymal stem cells (hMSCs) obtained from amniotic fluid (AF) as an alternative model more representative of what naturally happens in vivo. In our opinion, these hMSCs are still not influenced by differentiation stimuli and could act in a way more correspondent to the physiological process of adipogenesis, representing also an ethically acceptable alternative to totipotent human embryonic stem cells (ES). Adipocyte differentiation was monitorated following the expressions of key genes. We measured the expression levels of PPARgamma2, PPARgamma-C1alpha, UCP-1, adipsin, and leptin genes using quantitative real-time PCR. We tested our experimental model with two different media. Understanding in vivo adipogenesis mechanisms will shed light on the pathophysiology of many diseases.

Study of the adenosinergic system in the brain of HPRT knockout mouse (Lesch-Nyhan disease).

BACKGROUND: Lesch-Nyhan disease (LND), an X-linked genetic disease caused by complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), is characterized by hyperuricemia and psychiatric disturbance, mainly self-aggressiveness. Literature dates support the hypothesis that dopaminergic deficit and serotonergic excess in the circuit of basal ganglia are responsible for the aggressive behavior. Altered adenosine transport across the membrane of HPRT-deficient lymphocytes has been reported, suggesting adenosine involvement in LND. METHODS: The expression of several genes related to the adenosinergic system (ADORA1A, ADORA2A, ADORA2B) were studied in the brain of the murine model of LND by real time PCR. Nucleotide levels and enzyme activities possibly involved in adenosine release were also measured. RESULTS: Studies performed by real time PCR showed 95% increase in ADORA1A expression, 15% decrease in ADORA2A expression, and no change in ADORA2B expression in knockout mice compared to controls. No significant differences were found in the level of nucleotides or enzyme activities between control and mutant mice. CONCLUSIONS: Our results suggest that adenosine neurotransmission might be involved in the specific neurobehavioral features of LND by increased expression of adenosine A1 receptors.

An innovative test for non-invasive Kell genotyping on circulating fetal DNA by means of the allelic discrimination of K1 and K2 antigens.

OBJECTIVE: The aim of this study was to present a new method for fetal Kell genotyping by means of the allelic discrimination of K1 and K2 in real-time polymerase chain reaction (PCR). METHODS: Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL. RESULTS: By means of this method, the K1/K2 genotype was able to be determined in all blood samples analyzed. Results using cell-free fetal DNA (cffDNA) from two Kell-negative pregnant women confirmed the Kell-positive genotype of fetuses. The real-time PCR analysis also allowed the determination of the fetal fraction using the quantification of Kell-positive DNA. CONCLUSION: An efficient and reliable strategy for Kell genotyping is herein presented. The method was optimized on cffDNA to create a non-invasive prenatal test which could be routinely used for the prevention of hemolytic disease of the fetus and the newborn (HDFN).

The First Case Report in Italy of Di George Syndrome Detected by Noninvasive Prenatal Testing.

Panorama Plus (Natera), a single-nucleotide polymorphism- (SNP-) based approach that relies on the identification of maternal and fetal allele distributions, allows the detection of common aneuploidies and also incorporates a panel of 5 microdeletions including Di George syndrome. We report here the first case of Di George syndrome detected by NIPT in Italy; blood was drawn at 12 weeks' gestation. The patient had an amniocentesis to confirm the diagnosis by MLPA (multiplex ligation-dependent probe amplification) and an ultrasound aimed to detect the features associated with the syndrome. A right aortic arch and suspect of thymus atrophy were detected, but not other severe malformations typical of the disease. The patient terminated the pregnancy at 17 weeks. NIPT allowed an early screening of Di George syndrome. As the patient was at low risk, it is likely that an ultrasound would have missed the condition.

Differentially expressed genes in multidrug resistant variants of U-2 OS human osteosarcoma cells.

Multidrug resistance (MDR) to anticancer agents is a major barrier to the successful treatment of human osteosarcomas. Current understanding of the genes that contribute to the features of MDR is limited, and the mechanisms remain unclear. Here we applied differential display reverse transcription-polymerase chain reaction (DDRT-PCR) to parental and MDR-variants of U-2 OS human osteosarcoma cells, to clarify the genes involved in the MDR cells, and identified five candidate genes. These are BCRP (breast cancer resistance protein) encoding a transmembrane efflux pump; RB1CC1 (RB1-inducible coiled-coil 1), a tumor suppressor regulating RB1 (retinoblastoma 1) expression; a novel transcriptional variant of dUTPase; SSR2 (beta-signal sequence receptor), which is associated with protein translocation across ER membrane; and HSP105 encoding high molecular mass heat shock proteins. Molecular and biological characterization of these genes will yield further insight into the features between MDR and tumor progression in human osteosarcomas.

Real-time PCR and linkage studies to identify carriers presenting HPRT deleted gene.

Lesch-Nyhan syndrome (LNS) is an X-linked genetic disorder resulting in hyperuricemia, choreoathetosis, mental retardation, and self-injurious behavior. It is caused by loss of activity of the ubiquitous enzyme hypoxanthine-guanine-phosphoribosyltransferase (HPRT). The biochemical analysis of residual HPRT activity in patients' red blood cells is the first step in LNS diagnosis, and it precedes molecular study to discover the specific mutation. Unfortunately, biochemical diagnosis of healthy carriers is difficult because HPRT enzymatic activity in blood cells is similar in LNS carriers and in healthy people; genetic tests can help reveal mutations at the genomic or cDNA level, whereas gross deletions involving the first or last exons of HPRT gene are not detectable. Until now, a test based on 6-thioguanine-resistant phenotype of HPRT mutant cells from LNS patients is the only method accepted for the diagnosis of any kind of mutation in carriers. In this work, we introduce a new approach to identify carriers of large deletions in HPRT gene using real-time PCR. Results were validated in a blinded manner with a linkage study and with results obtained in Italian families previously analyzed with selective medium test. Real-time PCR analysis clearly confirmed the results obtained by selective medium; linkage data strengthened real time results, allowing us to follow the allele with the mutated HPRT through the family pedigree. We hope that the real-time PCR approach will provide a useful and reliable method to diagnose LNS carriers of large deletions in HPRT gene.