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Objective: The balance between benefits and risks of discordant outcomes makes the Genome-Wide Non-Invasive Prenatal Test (GW-NIPT) controversial. This study aims to evaluate performance and clinical utility in a wide cohort of unselected clinical cases from a single center when a standardized protocol is applied and integrated with a secondary algorithm for data interpretation., Method: In 2 years, over 70,000 pregnant patients underwent GW-NIPT for fetal common trisomies, sex chromosome aneuploidies, rare autosomal aneuploidies, segmental abnormalities (CNVs ≥ 7 Mb) and microdeletions (CNVs < 7 Mb). All samples were uniformly processed with Veriseq NIPT Solution v2 and analyzed using all data metrics along with a home-made algorithm for sequencing data analysis. Results were retrospectively reviewed for clinical outcomes., Results: Among 71,883 eligible cases including twin pregnancies, 1011 (1.4%) received a positive result and 781 were confirmed by invasive prenatal diagnosis. Clinical sensitivity ranged from 99.65% for common trisomy (T21, T18, T13) to 83.33% for microdeletions, while specificity remained high (99.98%) for each class of fetal abnormalities detected., Conclusions: Integrating a standardized protocol with an internal algorithm allowed discordant results to be reduced, yielding high accuracy. Observed reliability in detecting genome-wide chromosomal conditions reinforced the expanded NIPT utility in clinical practice., (© 2024 John Wiley & Sons Ltd.)

Objective: In this study, we expanded conventional cell-free fetal DNA (cfDNA)-based non-invasive prenatal testing (NIPT) to cover the entire genome. We aimed to compare the performance of the two tests in a large general population of pregnant women, in order to assess the clinical utility of the genome-wide screening., Method: Genome-wide cfDNA analysis was offered to 12 114 pregnant women undergoing NIPT for common fetal aneuploidy. Sequencing data were analyzed using an algorithm optimized to identify aneuploidies and subchromosomal aberrations., Results: Genome-wide screening allowed detection of 12 (7.4%) potentially viable clinically relevant chromosomal abnormalities, which would have remained overlooked if only conventional NIPT had been performed. This resulted in a statistically significant higher sensitivity (100% vs 92.64%, p < 0.001) than did standard screening. This was achieved without sacrificing the specificity of the test, which resulted similar to that obtained with standard cfDNA testing (99.87% vs 99.77%, p = 0.064)., Conclusion: Genome-wide cfDNA analysis represents an enhanced screening tool for prenatal detection of chromosomal abnormalities, allowing identification of clinically relevant imbalances that are not detectable by conventional cfDNA testing. The results of this study demonstrate the clinical utility of genome-wide cfDNA analysis. This level of screening provides a significant higher sensitivity compared to standard screening while maintaining a high specificity, with the potential to improve overall pregnancy management. © 2017 John Wiley & Sons, Ltd., (© 2017 John Wiley & Sons, Ltd.)

Distal hereditary motor neuropathies (dHMNs) are clinically and genetically heterogeneous neurological conditions characterized by degeneration of the lower motor neurons. So far, 18 dHMN genes have been identified, however, about 80% of dHMN cases remain without a molecular diagnosis. By a combination of autozygosity mapping, identity-by-descent segment detection and whole-exome sequencing approaches, we identified two novel homozygous mutations in the SIGMAR1 gene (p.E138Q and p.E150K) in two distinct Italian families affected by an autosomal recessive form of HMN. Functional analyses in several neuronal cell lines strongly support the pathogenicity of the mutations and provide insights into the underlying pathomechanisms involving the regulation of ER-mitochondria tethering, Ca 2+  homeostasis and autophagy. Indeed, in vitro, both mutations reduce cell viability, the formation of abnormal protein aggregates preventing the correct targeting of sigma-1R protein to the mitochondria-associated ER membrane (MAM) and thus impinging on the global Ca 2+  signalling. Our data definitively demonstrate the involvement of SIGMAR1 in motor neuron maintenance and survival by correlating, for the first time in the Caucasian population, mutations in this gene to distal motor dysfunction and highlight the chaperone activity of sigma-1R at the MAM as a critical aspect in dHMN pathology., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Several non-invasive prenatal testing (NIPT) methods, which analyze circulating fetal cell-free DNA (cfDNA) in maternal plasma, suggest a fetal fraction (FF) ≥ 4% for a reportable result, with the assumption that fetal aneuploidies may not be detectable at lower FF. This study determined the actual limit of detection (LOD) of a massively parallel sequencing-based NIPT method and evaluated its performance in testing samples with low FF., Method: An experimental model, involving the creation of artificial plasma mixtures with a final aneuploid FF ranging from 1% to 4%, simulated samples at different proportions of fetal cfDNA. We then analyzed 7103 blood samples, from pregnant women undergoing NIPT, to assess the impact of low FF on the performance of cfDNA testing., Results: Detection of common aneuploidies in samples with an FF as low as 2% is well within the ability of this technology. Of 105 pregnancies confirmed chromosomally abnormal, 25 (23.8%) involving a 2% <  FF  <  4% were consistently detected. These high-risk pregnancies would have not been identified using the suggested 4% FF cut-off., Conclusion: This study underscores the importance of determining the actual LOD for each specific NIPT methodology. It may reduce the incidence of test cancelations and shorten the time required for the diagnosis of aneuploidy. © 2016 John Wiley & Sons, Ltd., (© 2016 John Wiley & Sons, Ltd.)

The mode of inheritance of Alport syndrome (ATS) has long been controversial. In 1927, the disease was hypothesized as a dominant condition in which males were more severely affected than females. In 1990, it was considered an X-linked (XL) semidominant condition, due to COL4A5 mutations. Later on, a rare autosomal recessive (AR) form due to COL4A3/COL4A4 mutations was identified. An autosomal dominant (AD) form was testified more recently by the description of some large pedigrees but the real existence of this form is still questioned by many and its exact prevalence is unknown. The introduction of next generation sequencing (NGS) allowed us to perform an unbiased simultaneous COL4A3-COL4A4-COL4A5 analysis in 87 Italian families (273 individuals) with clinical suspicion of ATS. In 48 of them (55%), a mutation in one of the three genes was identified: the inheritance was XL semidominant in 65%, recessive in 4% and most interestingly AD in 31% (15 families). The AD form must therefore be seriously taken into account in all pedigrees with affected individuals in each generation. Furthermore, a high frequency of mutations (>50%) was shown in patients with only 1 or 2 clinical criteria, suggesting NGS as first-level analysis in cases with a clinical suspicion of ATS., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

LOX-1 (Lectin-like oxidized low-density lipoprotein receptor-1) is the primary endothelial receptor of oxidized LDL (oxLDL). Both in vitro and in vivo experiments have shown this protein to be important in the initiation of atherosclerosis and to be up-regulated by pro-atherogenic factors. Recently, it has been demonstrated that Olr1, the gene encoding Lox-1, is important for tumor growth and for maintaining the transformed state in different cancer cell lines, suggesting that it acts in a molecular pathway connecting cancer and atherosclerosis. Both diseases in humans are characterized by uncontrolled regulation of cellular growth and differentiation. We present evidence that Olr1 is expressed during mouse embryogenesis in developmental stages (from 7.5 to 9.5 dpc) in which cardiogenesis occurs. In addition, we identify two novel Olr1 isoform (hereafter referred to as D3D5Olr1 and D2D5Olr1) whose spatio-temporal expression pattern overlaps with Olr1 in vivo. In vitro, D3D5Olr1 localizes to the cell surface membrane as Olr1, in contrast with D2D5Olr1; these data suggest that D2D5Olr1 isoform translates a receptor that does not reach the plasma membrane. Accordingly, in silico transmembrane protein topology prediction analyses, show that D2D5Olr1 does not contain any transmembrane region. Finally, both isoforms can activate the same genetic pathways underlying Olr1 expression, such as, hypoxia and inflammation, even if with a different efficiency. All these data suggest a new functional involvement of Olr1, and probably of its spliceforms, in murine cardiogenesis and angiogenesis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Congenital heart defect (CHD) account for 25% of all human congenital abnormalities. However, very few CHD-causing genes have been identified so far. A promising approach for the identification of essential cardiac regulators whose mutations may be linked to human CHD, is the molecular and genetic analysis of heart development. With the use of a triple retinoic acid competitive antagonist (BMS189453) we previously developed a mouse model of congenital heart defects (81%), thymic abnormalities (98%) and neural tube defects (20%). D-TGA (D-transposition of great arteries) was the most prevalent cardiac defect observed (61%). Recently we were able to partially rescue this abnormal phenotype (CHD were reduced to 64.8%, p = 0.05), by oral administration of folic acid (FA). Now we have performed a microarray analysis in our mouse models to discover genes/transcripts potentially implicated in the pathogenesis of this CHD., Results: We analysed mouse embryos (8.5 dpc) treated with BMS189453 alone and with BMS189453 plus folic acid (FA) by microarray and qRT-PCR. By selecting a fold change (FC) ≥ ± 1.5, we detected 447 genes that were differentially expressed in BMS-treated embryos vs. untreated control embryos, while 239 genes were differentially expressed in BMS-treated embryos whose mothers had also received FA supplementation vs. BMS-treated embryos. On the basis of microarray and qRT-PCR results, we further analysed the Hif1α gene. In fact Hif1α is down-regulated in BMS-treated embryos vs. untreated controls (FCmicro = -1.79; FCqRT-PCR = -1.76; p = 0.005) and its expression level is increased in BMS+FA-treated embryos compared to BMS-treated embryos (FCmicro = +1.17; FCqRT-PCR = +1.28: p = 0.005). Immunofluorescence experiments confirmed the under-expression of Hif1α protein in BMS-treated embryos compared to untreated and BMS+FA-treated embryos and, moreover, we demonstrated that at 8.5 dpc, Hif1α is mainly expressed in the embryo heart region., Conclusions: We propose that Hif1α down-regulation in response to blocking retinoic acid binding may contribute to the development of cardiac defects in mouse newborns. In line with our hypothesis, when Hif1α expression level is restored (by supplementation of folic acid), a decrement of CHD is found. To the best of our knowledge, this is the first report that links retinoic acid metabolism to Hif1α regulation and the development of D-TGA.