Dysregulated placental microRNAs in Early and Late onset Preeclampsia

Copyright © 2017. Published by Elsevier Ltd.INTRODUCTION: To determine the miRNA expression profile in placentas complicated by Preeclampsia (PE) and compare it to uncomplicated pregnancies. METHODS: Sixteen placentas from women with PE, [11 with early onset PE (EOPE) and 5 with late onset PE (LOPE)], as well as 8 placentas from uncomplicated pregnancies were analyzed using miRNA microarrays. For statistical analyses the MATLAB® simulation environment was applied. The over-expression of miR-518a-5p was verified using Quantitative Real-Time Polymerase Chain Reaction. RESULTS: Forty four miRNAs were found dysregulated in PE complicated placentas. Statistical analysis revealed that miR-431, miR-518a-5p and miR-124* were over-expressed in EOPE complicated placentas as compared to controls, whereas miR-544 and miR-3942 were down-regulated in EOPE. When comparing the miRNA expression profile in cases with PE and PE-growth restricted fetuses (FGR), miR-431 and miR-518a-5p were found over-expressed in pregnancies complicated by FGR. DISCUSSION: Since specific miRNAs can differentiate EOPE and LOPE from uncomplicated placentas, they may be considered as putative PE-specific biomarkers. MiR-518a-5p emerged as a potential diagnostic indicator for EOPE cases as well as for PE-FGR complicated placentas, indicating a potential link to the severity of the disease.Peer reviewe

Prenatal diagnosis for CF using High Resolution Melting Analysis and simultaneous haplotype analysis through QF-PCR

AbstractBackgroundHigh Resolution Melting (HRM) Analysis is a validated, robust, low-cost, high throughput CF screening method. Here, we report the development and retrospective evaluation of the diagnostic value of a novel multiplex HRM, genotyping and haplotyping method for CF prenatal diagnosis (generic HRM/haplotyping).Methods80 study samples from 20 carrier couples referred for PND (whole blood in EDTA and CVS or amniotic fluid) were genotyped retrospectively using the suggested protocol.ResultsAll DNA samples (variable sources, extraction methods and unknown concentrations) were successfully amplified by the 1st and 2nd round PCR. The Se, Sp, NPV and PPV for the generic HRM/haplotyping method are calculated at 100%.ConclusionsThis generic protocol for PND using HRM, facilitates the simultaneous analysis of DNA samples from various sources in a fast, robust and efficient way. It can be easily adapted and applied for any genetic condition

Quantitative methods for the analysis of CFTR transcripts/splicing variants

AbstractIn cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs

The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis : toward an international consensus

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented

THE MOLECULAR BASIS OF NORMAL HBA2 (TYPE 2) Β-THALASSEMIA IN GREECE

ABOUT 4% OF THE B-THALASSEMIA HETEROZYGOTES IN THE GREEK POPULATION HAVE THE NORMAL A2 AN F, TYPE I OR TYPE II PHENOTYPES. HEMATOLOGICAL AND BIOSYNTHETIC STUDIES PRESUME THE PRESENCE OF GENETIC HETEROGENEITY AT THE MOLECULAR LEVEL INBOTH PHENOTYPES, WHICH TO DATE HAVE ONLY BEEN PARTIALLY DEFINED. THE MAIN OBJECTIVE OF THIS STUDY IS TO CHARACTERIZE THE MOLECULAR DEFECT ASSOCIATED WITH TYPE II NORMAL A2 B-THALASSEMIA. TO THIS END WE STUDIED 27 UNRELATED SIL II HETEROZYGOTES (NORMAL HBA2, TYPE II B-THAL) AND THEIR CLINICALLY AFFECTED OFFSPRING BY MAPPING THE B-GLOBIN GENE CLUSTER OR BY USING PCR TO AMPLIFY THE Β ORΔ GENES FOLLOWED BY SCREENING WITH SYNTHETIC OLIGOPROBE HYBRIDIZATION FOR Β AND Δ GLOBIN GENE MUTATIONS ALREADY CHARACTERIZED IN THE MEDITERRANEAN POPULATION. OF THE 27 SIL II CHROMOSOMAS 9 (33.33%) HAD THE CORFU ΔΒ-THAL MUTATION. OF THE REMAINING 18 SAMPLES 9 (33.33%) HAD THE Β+ IVSI-N6 MUTATION; 3 THE Β+ IVSI-N110; 1 THE Β FSC 6; 1 THE Β NS39; 1 THE Β IVSI-N1 AND 1 THE Β+ IVSII-N745.TWO ARE AS YET UNCHARACTERIZED FOR THEIR Β-THAL MUTATION. AMPLIFICATION OF THE Δ-GENE IN THE NON-CORFU SAMPLES AND SCREENING WITH Δ+27 AND Δ IVSI-N1 OLIGOPROBES DEMONSTRATED THAT ONE HAD THE Δ+27 MUTATION IN CIS TO THE Β-THAL MUTATION AND ONE IN TRANS. OUR FINDINGS SO FAR INDICATE THAT THE SIL II PHENOTYPE INGREECE IS VERY HETEROGENEOUS AND IS EITHER DUE TO MILD Β-THAL MUTATIONS OR CO-INHERITANCE OF Δ AND Β THALASSEMIA. (ABSTRACT TRUNCATED)ΕΠΙΔΗΜΙΟΛΟΓΙΚΕΣ ΜΕΛΕΤΕΣ ΕΔΕΙΞΑΝ ΟΤΙ 4% ΠΕΡΙΠΟΥ ΤΩΝ ΕΤΕΡΟΖΥΓΩΤΩΝ Β-ΜΕΣΟΓΕΙΑΚΗΣ ΑΝΑΙΜΙΑΣ (ΜΑ) ΕΧΟΥΝ ΦΥΣΙΟΛΟΓΙΚΑ ΕΠΙΠΕΔΑ HBA2 ΚΑΙ HBF. Ο ΑΙΜΑΤΟΛΟΓΙΚΟΣ ΑΥΤΟΣ ΦΑΙΝΟΤΥΠΟΣ ΓΝΩΣΤΟΣ ΚΑΙ ΣΑΝ ΣΙΩΠΗΛΟΣ (SILENT) ΔΙΑΧΩΡΙΖΕΤΑΙ ΣΕ ΔΥΟ ΤΥΠΟΥΣ, ΤΟΝ ΤΥΠΟ Ι (Β-SIL I MA) ΜΕ ΗΠΙΕΣ ΑΙΜΑΤΟΛΟΓΙΚΕΣ ΑΛΛΟΙΩΣΕΙΣ ΚΑΙ ΤΟΝ ΤΥΠΟ ΙΙ (Β-SIL II MA) ΜΕ ΕΚΣΕΣΗΜΑΣΜΕΝΕΣ ΑΛΛΟΙΩΣΕΙΣ ΤΩΝ ΕΡΥΘΡΩΝ. ΚΛΙΝΙΚΕΣ ΑΙΜΑΤΟΛΟΓΙΚΕΣ ΚΑΙ ΒΙΟΣΥΝΘΕΤΙΚΕΣ ΜΕΛΕΤΕΣ, ΥΠΟΔΗΛΟΥΝ ΤΗΝ ΠΑΡΟΥΣΙΑ ΓΕΝΕΤΙΚΗΣ ΕΤΕΡΟΓΕΝΕΙΑΣ ΚΑΙ ΣΤΟΥΣ ΔΥΟ ΦΑΙΝΟΤΥΠΟΥΣ. ΜΕΛΕΤΗΘΗΚΕ Η ΜΟΡΙΑΚΗ ΒΑΣΗ ΤΗΣ Β-SIL II MA ΣΕ 27 ΕΤΕΡΟΖΥΓΩΤΕΣ ΜΕ ΧΑΡΤΟΓΡΑΦΗΣΗ ΤΟΥ Β ΓΟΝΟΥ ΚΑΙ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ ΤΩΝ Β ΚΑΙ Δ ΓΟΝΩΝ (PCR) ΚΑΙ ΥΒΡΙΔΙΣΜΟ ΜΕ ΡΑΔΙΕΝΕΡΓΑ ΟΛΙΓΟΝΟΥΚΛΕΟΤΙΔΙΑ ΓΙΑ 9 ΜΕΤΑΛΛΑΞΕΙΣ ΤΟΥ Β ΓΟΝΟΥ ΚΑΙ 3 ΜΕΤΑΛΛΑΞΕΙΣ ΤΟΥ Δ ΓΟΝΟΥ. ΔΙΑΠΙΣΤΩΘΗΚΕ ΟΤΙ 9 ΑΠΟ ΤΑ 27 ΧΡΩΜΟΣΩΜΑΤΑ (33.33%) ΜΕ ΤΟΝ ΓΟΝΟ ΤΗΣ Β SIL II MA ΕΙΧΑΝ ΤΗΝ ΔΒ CORFU ΜΕΤΑΛΛΑΞΗ, 9 (33.3%) ΤΗΝ Β+IVSI-N6, 3 (11.1%) ΤΗΝ Β+ IVSI-N110 ΚΑΙ ΑΠΟ 1 ΧΡΩΜΟΣΩΜΑ ΜΕ ΤΙΣ ΜΕΤΑΛΛΑΞΕΙΣ Β FSC6, Β NS39,Β IVSI-N1 ΚΑΙ Β+ IVSII-N745, ΕΝΩ ΔΕΝ ΚΑΤΕΣΤΕΙ ΔΥΝΑΤΟΣ Ο ΚΑΘΟΡΙΣΜΟΣ ΤΗΣ ΓΕΝΕΤΙΚΗΣ ΒΛΑΒΗΣ ΣΕ ΔΥΟ ΧΡΩΜΟΣΩΜΑΤΑ. ΠΟΛΛΑΠΛΑΣΙΑΣΜΟΣ ΤΟΥ Δ ΓΟΝΟΥ ΚΑΙ ΥΒΡΙΔΙΣΜΟΣ ΜΕ ΟΛΙΓΟΝΟΥΚΛΕΟΤΙΔΙΑ ΓΙΑ ΤΟΝ ΕΛΕΓΧΟ ΤΩΝ ΜΕΤΑΛΛΑΞΕΩΝ Δ+27, Δ IVSI-N1 ΚΑΙ Δ+ 3'Δ+69BP ΤΟΥ POLY A SITE ΕΔΕΙΞΑΝ ΤΗΝ ΠΑΡΟΥΣΙΑ ΤΗΣ Δ+27 ΜΕΤΑΛΛΑΞΗΣ ΣΕ ΘΕΣΗ CIS ΤΟΥ Β ΓΟΝΟΥ ΣΕ ΕΝΑ ΑΤΟΜΟ ΚΑΙ ΣΕ ΘΕΣΗ TRANS ΣΕ ΑΛΛΟ. ΑΠΟ ΤΑ ΑΠΟΤΕΛΕΣΜΑΤΑ ΑΥΤΑ ΦΑΙΝΕΤΑΙ ΟΤΙ Ο ΦΑΙΝΟΤΥΠΟΣ Β SIL II, ΣΤΗΝ ΕΛΛΑΔΑ ΠΑΡΟΥΣΙΑΖΕΙ ΣΗΜΑΝΤΙΚΗ ΓΕΝΕΤΙΚΗ ΕΤΕΡΟΓΕΝΕΙΑ, ΟΦΕΙΛΕΤΑΙ ΚΥΡΙΩΣ ΣΕ ΗΠΙΕΣ ΜΕΤΑΛΛΑΞΕΙΣ ΤΟΥ Β ΓΟΝΟΥ (Β+ IVSI-N110) ΜΕ Η ΧΩΡΙΣ ΔΙΑΤΑΡΑΧΗ ΣΤΟΝ Δ ΓΟΝΟ, ΣΥΧΝΟΤΕΡΗ ΓΕΝΕΤΙΚΗ ΒΛΑΒΗ ΑΦΟΡΑ ΤΗΝ ΔΙΑΤΑΡΑΧΗ Δ Β+ CORFU

239-kb Microdeletion Spanning KMT2E in a Child with Developmental Delay: Further Delineation of the Phenotype

Pathogenic KMT2E variants underly O'Donnell-Luria-Rodan syndrome, a recently described neurodevelopmental disorder characterized by global developmental delay, variable degrees of intellectual disability, and subtle facial dysmorphism. Less common findings include autism, seizures, gastrointestinal (GI) problems, and abnormal head circumference. Occurrence of mostly truncating variants as well as the similar phenotype observed in individuals with deletions spanning KMT2E suggest haploinsufficiency of this gene as a common mechanism for the disorder, while a gain-of-function or dominant-negative effect cannot be ruled out for some missense variants. Deletions reported in the literature encompass several additional known or presumed haploinsufficient genes, thus leading to more complex phenotypes. Here, we describe a male with antenatal onset hydronephrosis, hypotonia, global developmental delay, prominent GI symptoms as well as facial dysmorphism. Chromosomal microarray revealed a 239-kb de novo microdeletion spanning KMT2E and LHFPL3. Clinical presentation of our proband, harboring one of the smallest deletions of the region confirms the core features of this disorder, suggests GI symptoms as a prominent finding in affected individuals while expanding the phenotypic spectrum to abnormalities of the urinary tract